WO2011007350A2

WO2011007350A2 - Immunomodulatory agents and a tissue-targeted delivery system thereof for the treatment of immune-related disorders

Info

Publication number: WO2011007350A2
Application number: PCT/IL2010/000560
Authority: WO
Inventors: Ilan Rosenshine
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2009-07-13
Filing date: 2010-07-13
Publication date: 2011-01-20
Also published as: WO2011007350A3

Abstract

The present invention relates to a composition and to a tissue-targeted delivery system of an immunomodulatory protein comprising at least one of NleE, NleC, NleD and NleB, and to a nutraceutical composition comprising the same. The invention further provides pharmaceutical compositions, methods and uses thereof. More particularly, the invention relates to the compositions, tissue-targeted delivery systems and nutraceutical compositions for preventing, treating, or ameliorating an immune-related disorder, preferably by inhibition of at least one of NF- kB, JNK and p38- mediated signal transduction pathways, thereby leading to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a cell of said treated subject.

Description

IMMUNOMODULATORY AGENTS AND A TISSUE-TARGETED DELIVERY SYSTEM THEREOF FOR THE TREATMENT OF IMMUNE-RELATED DISORDERS

FIELD OF THE INVENTION

The invention relates to immunomodulatory agents for treating immune-related disorders and a tissue-targeted delivery system thereof. More particularly, the invention relates to the immunomodulatory NIeE, NIeC, NIeD and NIeB bacterial proteins, compositions, uses, methods of treatment and attenuated TTSS-bacteria expressing at least one of said proteins for tissue targeted delivery thereof for the treatment of inflammatory conditions.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Escherichia coli (E. coli) are Gram-negative, rod-shaped bacteria belonging the family Enter obacteriaceae. There are five known classes of enterovirulent E. coli (collectively referred to as the EEC group) that cause gastroenteritis in humans, including: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC). Each class falls within a serological subgroup and manifests distinct features in pathogenesis. EPEC are defined as E. coli belonging to serogroups epidemiologically implicated as pathogens but whose virulence mechanism is unrelated to the excretion of typical E. coli enterotoxins. Source(s) and prevalence of EPEC are controversial because food borne outbreaks are sporadic. EPEC shows strong species specificity and accordingly, EPEC isolated from humans are human-specific. E. coli are present in the normal gut flora of these mammals. The proportion of pathogenic to nonpathogenic strains, although the subject of intense research is unknown.

EPEC and EHEC belong to a group of pathogens defined by then" ability to form "attaching and effacing" (AE) histopathology on intestinal epithelia of human and animal hosts, leading to diarrhea. AE pathogens employ their type III protein secretion system (TTSS) to inject (translocate) toxic proteins (effectors) into the host cell. This secretion system is distinguished from at least five other secretion systems found in Gram-negative bacteria. A large number of bacterial species, many of them pathogenic, possess a TTSS. The TTSS is comprised in each species of approximately 30 different proteins and shares many similarities with bacterial flagella-long, extracellular structures used for motility. Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus that is used for protein injection into eukaryotic cells.

The injected effectors subvert normal host cell functions to benefit the bacteria [Dean, P. and Kenny, B., Curr Opin Microbiol 12(1): 101-109 (2009)]. Different EPEC and EHEC isolates carry different numbers of effector proteins ranging from 21 to 50 different effectors. Seven of them are encoded in the enterocyte effacement (LEE) region, a pathogenicity island that also encodes the TTSS structural genes, whereas the other effector genes are distributed in regions termed pathogenicity islands including prophages and insertion elements. TTSS effectors manipulate host cells in several ways, for example, the promoting of uptake of the bacterium by the host cell. TTSS effectors have also been shown to tamper with the host's cell cycle and some of them are able to induce apoptosis. One of the most researched TTSS effectors, IpaB from Shigella flexneri, serves in a double role, both as a translocator, creating a pore in the host cell membrane, and as an effector, exerting multiple detrimental effects on the host cell, such as apoptosis in macrophages, by interacting with caspase 1. Another important role for TTSS effectors during infection is the suppression of significant inflammatory response to the infection.

Upon infection, bacterial pathogen-associated molecular patterns (PAMPs) including LPS, flagellin, lipoproteins, and CpG DNA stimulate host cell Toll-like receptors (TLRs), leading to a formidable immune response via the activation of NF -KB and AP-I transcription factor family members [Kawai, T. and Akira, S., Cell Death Differ 13(5):816-825 (2006)]. The NF-κB family comprises closely related transcription factors that play a key role in the expression of genes involved in inflammation, immune, and stress responses. NF-κB is a collective term used for homo- and heterodimeric complexes formed by the Rel/NF-κB proteins. In mammals, five of such proteins are known: ReIA (p65), ReIB, c-Rel, p50 (NF-κBl), and p52 (NF-κB2). Under non-stimulating conditions, NF-κB is retained in the cytoplasm through its association with inhibitory proteins (IKBS). A variety of signaling pathways activate IKB kinases (IKK) to phosphorylate IKB, leading to its ubiquitination and degradation by the proteasome. This allows translocation of NF- KB to the nucleus, activation of NF-κB-regulated promoters, and establishment of an inflammatory response [Karin, M. and Ben-Neriah, Y., Annu. Rev. Immunol. 18(621-663 (2000)].

The c-Jun N-terminal kinases (JNKs) are serine/threonine kinases that belong to MAP kinases. JNK which are activated by a plethora of extracellular signals and consequently represent essential mediators of signal transduction. JNK proteins phosphorylate the proto-oncoprotein c-Jun, which belongs to the AP-I group of transcription factors that is a crucial regulator of cellular proliferation, apoptosis inflammation and tumorigenesis [Shaulian, E. and Karin, M., Oncogene 20(19):2390-2400 (2001)]. The JNK family consists of three genes JNKl, JNK2 and JNK3. JNK1/2 are expressed in most of the tissues and JNK3 is expressed mainly in the brain. Notably, each of these genes produces several isoforms. JNK activation involves its phosphorylation on threonine- 183 and tyrosine-185, located on a region termed the "activation loop". JNK activation can be mediated by several MAPKK4/7, which are activated by TAKl upon stimulation of TLRs, ILlR or TNFR [Kawaiand Akira (2006) ibid.]. Upon activation JNKs phosphorylate the proto-oncoprotein c-Jun, a key member of the AP-I group of transcription factors that regulate cellular proliferation, apoptosis, inflammation and tumorigenesis [Shaulian and Karin (2001) ibid.].

The capacity of EPEC to induce and repress inflammatory responses including activation and repression of NF-κB and MAPK (including JNK and p38) was studied extensively. Taken together, these reports suggest that EPEC have the capacity for both eliciting NF- KB activation by a TTSS-independent mechanism and repression of NF-κB by a TTSS- dependent mechanism [Dean and Kenny (2009) ibid.]. Although a recent publication indicated that NIeHl inhibits the translocation of the NF-κB cofactor, RPS3, to the nucleus [Gao, X., et al PLoS Pathog. PLoS Pathog. 5(12):el000708 (2009)], it has been speculated that additional effectors must be involved in blocking the inflammatory response. The major arms of the inflammatory response involve the NF-κB, JNK and p38 pathways. The inventors show here that enteropathogenic E. coli use its type III secretion system to inject the host cells with effector proteins NIeE, NIeC, NIeD and NIeB that block the NF- KB, JNK and p38 signaling pathways. More specifically, the present invention now shows that NIeE blocks IKKβ-activation, NIeB blocks the TNFα-mediated NF-κB- activation, upstream to IKB degradation, and NIeD and NIeC, which are Zn- metalloproteases, specifically cleave and inactivate JNK, p38 and the p65 subunit of NF- KB, respectively. The invention discloses the manner in which a combination of the injected factors NIeE, NIeC and NIeB inhibit the NF-κB arm while NIeD inhibits the JNK and p38 signaling arms, together halting the expression of pro-inflammatory genes and modulating apoptotic processes.

Thus, one object of the invention is providing compositions comprising bacterial immuno- , modulatory proteins, method and uses thereof which exert an anti-inflammatory effect and modulate apoptotic processes.

More specific object of the invention is to provide a modular tissue-targeting delivery system providing different combinations of the anti-inflammatory and apoptotic- modulatory proteins of the invention to a specific tissue of a subject suffering of an inflammatory disorder.

These and other objects of the invention will become apparent as the description proceeds.

SUMMARY OF THE INVENTION

In the first aspect, the present invention is directed to a composition comprising as an active ingredient at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. The invention further provides pharmaceutical compositions, methods and uses of the composition of the invention in the treatment of immune-related disorders.

In another aspect, the present invention relates to a tissue-targeted delivery system of an immunomodulatory protein comprising a non-virulent/attenuated Type-Three Secretion System (TTSS)-expressing microorganism, comprising nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. The nucleic acid sequences comprised in the delivery system are operably linked to TTSS secretion signal sequences. The invention further provides pharmaceutical compositions, methods and uses of the tissue-targeted delivery system of the invention in the treatment of immune- related disorders.

A further aspect of the invention discloses a nutraceutical composition comprising as an active ingredient an effective amount of attenuated EPEC or EHEC pro-biotic bacteria expressing intact Tir (translocated intimin receptor) and intimin encoding genes, wherein the attenuation is caused by deletion or inactivation of at least one of: (a) at least one gene encoding at least one of type IV pilli and type I pilli; (b) a gene encoding the effector Map (Mitochondrial-associated protein), and, optionally, any further effector; and (c) genes encoding exotoxins selected from the group shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins, hemolysin and EspC, EspP and LifA. The nutraceutical composition optionally further comprises a pharmaceutically acceptable carrier.

These and other aspects of the invention will become apparent by the hand of the following figures. BRIEF DESCRIPTION OF THE FIGURES

Figure 1A-1D. EPEC inhibit TNFa-induced IKB degradation andNF-κB translocation to the nucleus in a TTSS-dependent manner

Fig. IA. HeLa cells were infected with wild-type EPEC (WT), a TTSS mutant

{escN::kaή) or remained uninfected (N/I). After 3h, cells were washed and treated with 10 ng/ml TNFα. At the indicated time post TNFα treatment, cells were extracted and subjected to western blot analysis with anti-IκB antibodies and anti-tubulin (loading control).

Fig. IB. HeLa cells were infected with wild-type EPEC or with an EPEC TTSS mutant

(escV::kan) or remained uninfected, washed and treated with TNFα. At 30 minutes post

TNFα induction, cells were harvested and fractionated into nuclear and cytoplasmic fractions. The presence of the p65 in the different fractions was analyzed by Western blot using anti-p65 antibody.

Fig. 1C. IKB degradation assay was conducted (as described for (Fig. IA) using uninfected cells, cells infected with EPEC wild-type strain and with an EPEC mutant deleted of its two nleH alleles (nleH mutant).

Abbreviations: Cont. (control); Nuc. (nuclear); Cytop. (cytoplasmic); Mut. (mutant); WT

(wild type); Min. (minutes).

Figure 2 A-2C. NIeE is required for EPEC inhibition of TNFa-induced IKB

degradation

Fig. 2 A. A schematic illustration of the region within IE6 containing the nleBE genes and the chromosomal deletions used for the analysis. Black lines represent the chromosomal

DNA and gray dashed lines represent the deleted regions. The corresponding names of the deleted strains are indicated on the right side.

Fig. 2B. HeLa cells were either uninfected (N/I) or infected with wild-type EPEC (WT) or with the different mutants shown in Fig. 2A, as indicated. After 3h, cells were washed, treated with TNFα for 40 minutes and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin (Tub.; loading control) antibodies.

Fig. 2C. HeLa cells were infected with a strain (SC3518) carrying a chromosomal deletion of the nleBE region, which was complemented or not complemented with plasmids expressing nleB, nleE, or nleBE (indicated as B, E, and BE, respectively). After 3h, cells were washed, treated with TNFα for 30 minutes and extracted. The extracts were analyzed by western blot using anti-IκB and anti-tubulin antibodies.

Abbreviations: WT (wild type); Plas. (plasmid); Tub. (Tubulin).

Figure 3. Comparison between different NIeE genes of different EPEC and EHEC strains

The EPEC E2348 NIeE_IE6 is indicated as NLEE2 and NIeEi_E2 as NLEEl (also denoted as SEQ ID NOs.: 90 and 89, respectively). Other NIeE proteins are those of Citrobacter rodentium (also denoted as SEQ ID NO.: 91), two EPEC strains (0111 B171 and 0103 E22, also denoted as SEQ ID NO.: 92, since the two sequences are identical) and two EHEC 0157 strains (SAKAI and EDL933, also denoted as SEQ ID NO.: 105, identical as well).

Figure 4. Comparison between different NIeB genes of different bacterial strains

The EPEC (E. coli O127:H6 str. Ε2348/69) NIeB peptide sequence is indicated as EPEC, also denoted as SEQ ID NO.: 8). Other examples of NIeB homologues are those of EPEC strain 0103 E22, also denoted as SEQ ID NO.: 101), EHEC 0157 strain EDL933 (also denoted as SEQ ID NO.: 102), Citrobacter rodentium (also denoted as SEQ ID NO.: 103) and Salmonella enterica subsp. arizonae serovar 62:z4,z23:— (also denoted as SEQ ID NO.: 104).

Figure 5A-5C. NIeE ofIE2 is deficient in translocation and inhibition of IKB degradation

Fig. 5A. HeLa cells were either uninfected (N/I) or infected with a strain deleted of nleE_IE6 (SC3680) (see Fig. 2A), complemented or not complemented with plasmids expressing NIeE_IE6 or NIeEi_E6 (indicated as E2 and E6, respectively). After 3h, cells were washed, treated with TNFα for 30 minutes and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin antibodies.

Fig. 5B and Fig. 5C. HeLa cells were infected with wild-type EPEC harboring plasmids expressing nleEi_E2 or nleE_IE6 fused to the blaM reporter gene (indicated as pnleE2 and pnleE₆, respectively). As a negative control, cells were infected with EPEC harboring the parental vector pCX341 (Vector). The β-lactamase activity in the infecting bacteria, reflecting the expression levels of the fusion proteins (Fig. 5B), and the β-lactamase activity in the infected HeLa cells, reflecting the levels of translocation of the fusion proteins into the HeLa cells (Fig. 5C) were determined. The experiment was done twice in triplicates and typical results are shown. Standard errors are indicated by bars. Abbreviations: Plas.(plasmid); Exp. Arb. Uni. (expression (arbitrary units)); Trans. Lev. Arb. Uni.(translocation levels (arbitrary units)); Vec.(vector); Tub. (Tubulin).

Figure 6A-6B. NleE is required to block IL-8 secretion by EPEC

HeLa cells were infected with the parental strain deleted of the IE2 region (strain

EM3327, indicated as WTΔIE2) or with several isognic mutants including a mutant deleted of nleE (strain SC3908, indicated as ΔnIeE), and a mutant deleted of nleE and complemented with a plasmid expressing nleE (pSC3982), indicated as ΔnleE/pE. As positive and negative controls the inventors used cells, which were either uninfected (N/I) or infected with wild type EPEC (WT) or with a TTSS mutant (strain SNl 961, indicated as AescV).

Figs. 6A. After 3 hours infection, cells were washed and treated with TNFα to induce NF-

KB and with gentamicin to kill the bacteria. After additional 3 hours incubation the cells were harvested and RNA was extracted and analyzed for the amount of IL-8 transcripts.

The amount of IL-8 mRNA in each strain is shown as a percentage of the level relatively to the transcript levels in the Aesc V mutant.

Figs. 6B. After 3 hours infection, cells were washed and treated with gentamicin to kill the bacteria. After additional 3 hours incubation the cells were harvested and RNA was extracted and analyzed for the amount of IL-8 transcripts, with no addition of TNFα. The amount of IL-8 mRNA in each strain is shown as a percentage of the level relatively to the transcript levels in the Aesc V mutant.

The experiment was done twice in duplicates and typical results are shown. Bars indicate standard errors.

Abbreviations: log ReI. Uni.(log relative units); WT (wild type). Figure 7. A mutant bearing a deletion ofnleBE is deficient in repressing self-activated, or TNFa-induced, IL8 repression

HeLa cells were infected with the indicated strains and analyzed as described in Fig. 6A and Fig. 6B. To terminate the infection and induce IL8 expression, the medium was replaced with fresh DMEM supplemented with 2% FCS, lOOμg/μl gentamicin and with or without 10 ng/ml TNFα and incubated for additional 3 h. Cells were than washed with 2 ml of cold TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), scraped with 1 ml of cold TBS, collected and centrifuged, (800 g, 2 min, 4°C). RNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (EPICENTRE Biotechnologies) and used to synthesize cDNA with the Verso cDNA kit (Thermo Scientific). hHPRT transcript levels were used to normalize total RNA levels in samples. Real time analysis was than conducted using Absolute Blue QPCR SYBR Green (Thermo Scientific) in a real-time cycler (Rotor-Gene 6000, Corbett). The amount of IL-8 mRNA in each strain is shown as a percentage of the level relatively to the transcript levels in the Aesc V mutant. The experiment was done twice in duplicates and typical results are shown. Bars indicate standard errors.

Abbreviations: ReI. IL-8 mRNA Lev. (relative IL-8 mRNA levels); WT (wild type).

Figure 8. NIeE is involved in blocking IL8 secretion

HeLa cells (8><10⁴ per well, seeded in 24-well plate) were infected for 3h with different EPEC strains as indicated or remained uninfected (N/I). After 3.5 h, supernatants were replaced with 300 μl DMEM, 2% FCS, and 50 μg/ml gentamycin with or without 10 ng/ml TNFα. After 16 h, 100 μl of cleared supernatant taken from each well was used for IL-8 measurements using Human CXCL8/IL-8 Quantikine immunoassay assay (R & D), according to the manufacturer's instructions. PBS and IL-8 were used as negative and positive controls for the detection assay. The relative amounts of IL-8 are shown. The experiment was done twice in duplicates and typical results are shown. Standard errors are indicated by bars. In the case of ΔescV (indicated by a striped bar), the signal was above the upper limit of the detection levels. Untreated and uninfected cells secreted -300 units of IL-8, whereas TNFα treatment induced a 10-fold increase in IL-8 secretion (-3000 units) (Fig. 6A). In contrast, pre-infection with wild-type EPEC or with the ΔIE2 mutant (WTΔIE2), reduced IL-8 secretion below the detection levels, even upon TNFα treatment. Furthermore, the TTSS esc V mutant was completely deficient in blocking IL-8 secretion. In conclusion, EPEC strongly reduces IL-8 secretion by a TTSS-dependent mechanism. Importantly, the inventors found that the AnIeE or AnIeEB mutants were strongly deficient in blocking IL-8 secretion but not as deficient as the TTSS mutant (escV). These results show that (i) NIeE is required for full inhibition of IL-8 secretion and (ii) other putative TTSS effector(s) might function in parallel to NIeE to inhibit IL-8 expression and/or secretion.

Abbreviations: IL-8 Arb. Uni. (IL-8 arbitrary units); WT (wild type).

Figure 9A-9D. NIeE and NIeB are sufficient to block translocation of NF-κB to the nucleus

Fig. 9A. HeLa cells were treated with TNFα for 1 h, or remained untreated, after which they were fixed and stained with anti-p65 (green) (bar represents 20 μm).

Fig. 9B. HeLa cells transfected with plasmid expressing mCherry or mCherry-NleE (red) were treated with TNFα for 1 hr, or remained untreated, after which they were fixed and stained with anti-p65 (green). Yellow arrows indicate cells exhibiting p65 translocation to the nucleus and white arrows indicate cells where the p65 remained cytoplasmic (bar represents 20 μm).

Fig. 9C. To quantify the results shown in Fig. 9B, the percentage of red cells (expressing mCherry or mCherry-NleE) containing nuclear p65 was determined. The number of cells quantified is indicated and standard errors are indicated by bars.

Fig. 9D. To compare the capacity of NIeE to block NF-κB to that of NIeB, HeLa cells were transfected with plasmids expressing only mCherry (indicated as mCherry), or mCherry fused to NIeE (NIeE) or NIeB (NIeB). The transfected cells were treated with TNFα and analyzed by microscopy as described in 8B and 8C. The percentage of cells were translocation of p65 to the nucleus is indicated (n = ~50 cells). Shown are the results of one typical experiment out of three.

Abbreviations: Phas. Cont. (phase contrast); Nuc. Trans % (nucleus translocation (%)); α- p65 (anti-p65 antibody). Figure 10. NleE[_E2 is deficient in blocking TNF-induced translocation of p65 to the nucleus

HeLa cells were transected with plasmids expressing mCherry, mCherry-NleE_IE2 or mCherry-NleE_IE6 . The expressing cells were treated with TNFα for 1 hr, or remained untreated, after which they were fixed and stained with anti-p65. The slides were analyzed by fluorescent microscopy and the percentage of red cells (expressing mCherry or mCherry fused to NIeE₁E₆ or NIeE_1E2) containing nuclear p65 was determined. The number of cells quantified is indicated and standard errors are indicated by bars. The results show that while NIeE_1E6 inhibited p65 translocation, NIeE_IE2 failed to do so. Abbreviations: Nuc. Trans % (nucleus translocation (%)).

Figure 1 IA-I IE. NIeE inhibits IKB and IKKβ phosphorylation

Fig. HA. HeLa cells were infected for 3 hours with different EPEC strains, as indicated followed by TNFα treatment in the absence or presence of proteasome inhibitor (MG 132) or IPTG (0.01 mM) as indicated. Proteins were extracted at 0, 20, and 40 minutes post

TNFα treatment. The blots were developed with anti-IκB (IKB), anti-phospho IKB (P-

IKB), or anti-tubulin antibodies (loading control). The strains used are indicated above the lanes. The parental strain deleted of the IE2 region, strain EM3327 is indicated as

WTΔIE2. The band-densities of IKB and P-IKB at 40 minutes post TNFα treatment were measured and the relative amounts of the unphosphorylated IKB were calculated

(indicated as "unphos. IKB [-%]") and shown below the corresponding lanes.

Fig. HB. HeLa cells were infected as in Fig. HA and proteins were extracted at 40 minutes post TNFα treatment. The blots were developed and the ~% unphos. IKB was determined as in Fig. 1 IA.

Fig. HC. Schematic illustration of the signaling pathways initiated upon activation of the

TNF and ILl receptors (TNF-R and ILl-R, respectively).

Fig. HD-HE. HeLa cells were infected with different strains as indicated or not (N/I) and treated with TNFα or ILl β as indicated. Proteins were then extracted and the levels of IKB

(Fig. HD and Fig. HE), phospho-IκB (P-IKB), IKKβ and phospho-IKKβ (P-IKK) (Fig.

HE), were determined by immunoblot analysis with anti-IκB, anti-IKKβ and anti- phospho-IKK respectively.

Abbreviations: Cont. (control); Ind. (induction); Unphos. (unphosphorylated); min.

(minutes); WT (wild type); P-IKB (phospho-IκB). Figure 12. Schematic illustration of inhibition ofNF-κB signaling by EPEC

Signals (indicated as lightning) sensed by different receptors elicit signal transduction cascades that converge at the level of TAKl activation and IKK phosphorylation, which subsequently leads to IKB phosphorylation. This step is inhibited by NIeBE. NIeE inhibits IKKβ phosphorylation, but the exact target of NIeE and NIeB is yet to be defined. The phosphorylated IKB is subjected to ubiquitination and proteasome-mediated degradation, allowing NF-κB translocation to the nucleus and activation of expression of target genes including IL-8. The present results predicate that in addition to NIeBE, a putative non- TTSS factor and a putative TTSS effector might inhibit NF-κB translocation to the nucleus and IL-8 expression, respectively (Fig. 6).

Abbreviations: Put. n-TTSS Fac. (putative non-TTSS factor); Put. TTSS Eff. (putative TTSS effector); Exp. (expression).

Figure 13 A-13F. NIeD is a Zn metalloprotease that specifically clips JNK

Fig. 13 A. NIeD is required for JNK degradation. HeLa cells were infected with one of the following EPEC: wild type (WT), nleD deletion mutant (AnIeD), nleD deletion mutant complemented with a plasmid expressing wild type nleD (pKB4345, indicated as pNleD) or nleD deletion mutant complemented with a plasmid expressing mutated nleD (pLG4457, indicated as pNleD-E143A). After 3 hours, proteins were extracted and subjected to Western blot analysis using anti-JNK antibody. JNK and its degradation fragments are indicated. Cells infected with a TTSS deficient mutant (ΔescV) were used as negative control.

Fig. 13B. Activity kinetics of injected NleD. HeLa cells were infected with EPEC for the indicated periods before proteins were extracted and subjected to Western blot analysis using anti-JNK antibody. JNK and its degradation fragments are indicated. Non-infected cells (NI) and cells infected with nleD deletion mutant (AnIeD) served as controls.

Fig. 13C. Ectopically expressed NleD induces JNK degradation. HEK293 cells were transfected with one of the following plasmids: mCherry-NleD, mCherry-NleD-E143A or mCherry (pLG4419, pLG4477 and pSC4141, respectively). 24 h later proteins were extracted and subjected to Western blot analysis using anti-JNK antibody. JNK and its degradation fragments are indicated. Fig. 13D. Ectopically expressed NIeD inhibits JNK activity. HEK293 cells transfected with plasmids expression mCherry, mCherry-NleD or mCherry-NleD-E143A, were irradiated with 30J/m2 of UV and harvested 3 h later. The levels of phospho c-Jun and total c-Jun were determined by Western analysis using anti c-Jun and anti-phosphpho-c- Jun antibodies.

Fig. 13E. NIeD clips ectopic JNK in E. coli cytoplasm. E. coli BL21 were co-transformed with plasmid expressing JNK2 and either vector only (pCX341) or plasmid expressing nleD (pEM3654). Co-expression of JNK2 and NIeD was induced for 2 h by IPTG, before proteins were extracted and subjected to Western blot analysis using anti- JNK antibody. JNK and its degradation fragments are indicated.

Fig. 13F. NIeD clips JNK in vitro. Purified JNK2 and NIeD were incubated in a reaction mixture at a molar ratio of 40:1, in the presence or absence of the Zn protease inhibitor phenanthroline. The reaction was stopped by addition of SDS loading buffer and proteins separated by PAGE-SDS. Finally, proteins were visualized by coomassie blue staining. JNK2 and its degradation fragments are indicated. Of note, NIeD does not appear in this gel as its concentration is below detection levels.

Abbreviations: WT (wild type); Frag, (fragment); Vec. (vector); min. (minutes); KDa (kilo dalton);P-c-Jun (phosphorylated c-Jun).

Fig 14A-14D. JNK degradation by NleD

Fig. 14A. EPEC deleted of the PP2 chromosomal region is deficient in inducing JNK degradation. HeLa cells were infected for 3 h with different EPEC strains containing deletion of large chromosomal regions (the deleted regions are indicated above the lanes).

Proteins were then extracted and subjected to Western blot analysis with anti- JNK antibody. The locations of the intact and fragmented JNK proteins are indicated. Mutant with inactivated TTSS (ΔescV) was used as negative control.

Fig. 14B. EPEC is not inducing ERK degradation. HeLa cells were infected for 3 hours with EPEC WT and AescV strains and subjected to Western blot analysis with anti-ERK antibody.

Fig. 14C. Complete JNK degradation by EPEC. HeLa cells were infected with EPEC for different periods with wild type EPEC (WT) or nleD mutant (AnIeD), as indicated above the lanes. Proteins were then extracted and subjected to Western blot analysis with anti-

JNK antibody. The locations of the intact and fragmented JNK proteins are indicated. Fig. 14D. Citrobacter rodentium NIeDs function similarly to the EPEC NIeD. All EPEC and EHEC strains appear to encode one copy of NIeD, which is virtually identical to that of EPEC 0127 (Fig. 15). The closely related pathogen Citrobacter rodentium (CR) encodes two NIeD proteins, which are -70% similar to that of EPEC, as shown in Figure 15. These proteins exhibit identical amino acid sequence but differ in their DNA sequence. To test whether the CR NIeDl and NleD2 function similarly to the EPEC NIeD, both alleles were cloned in an expression vector and the resulting plasmids were used to complement the EPEC nleD mutant. HeLa cells were infected for 3 h with EPEC nleD mutant (AnIeD) complemented with different plasmids including vector (pSAlO), and plasmids expressing the EPEC nleD (pKB4345), the CR nleDl (pKB4505) or the CR nleD2 (pKB4506) as indicated above the lanes. Proteins were then extracted and subjected to Western blot analysis with anti-JNK antibody. The locations of the intact and fragmented JNK proteins are indicated. Both of the CR nleD alleles effectively restored the capacity of EPEC to induced clipping of JNK. These results indicate that the EPEC/EHEC NIeDs and the CR NIeDs function similarly to clip JNK.

Abbreviations: WT (wild type); Frag, (fragment); Vec. (vector); min. (minutes).

Figure 15. Multiple alignment of NleD homologes

The NleD protein sequences of EPEC (Escherichia coli O127:H6 str. E2348/69 also denoted as SEQ ID NO.: 6), EHEC (Escherichia coli O157:H7 EDL933 also denoted as SEQ ID NO.: 93), CR (Citrobacter rodentium ICC 168 - has two copies of the nleD gene, encoding for an identical protein, also denoted as SEQ ID NO.: 94), SE (Salmonella enterica subsp. arizonae serovar 62:z4,z23:~ also denoted as SEQ ID NO.: 95) and HD (Candidatus Hamiltonella defensa 5AT, also denoted as SEQ ID NO.: 96) were aligned using ClustalW2. The HEXXH Zn-metalloprotease motif (also denoted as SEQ ID NO.: 9) is framed. Figure 16A-16F. NIeD clips JNK at its activation loop

Fig. 16A. Schematic diagram of JNK2. The location of the HA and anti-JNK antibody epitopes, activation loop (A loop), T and Y phosphorylation sites and NIeD cleavage site are indicated.

Figs 16B and 16C. NIeD cleaves JNK2 in vivo. HeLa cells were transfected with plasmids expressing HA-tagged JNK2. 25 h post transfection the cells were infected with wild type (WT) or nleD deletion mutant (AnIeD) EPEC. After 2.5 h proteins were extracted from the infected HeLa cells, immunoprecipitated using anti-HA antibody and subjected to Western blot analysis using anti-HA (Fig. 16B) or anti-JNK (Fig. 16C) antibodies. JNK2 and its degradation fragments are indicated.

Figs 16D and 16E. NIeD cleaves JNK2 in vitro. Purified, N-terminally tagged, 6xHis

JNK2 was incubated with purified NIeD or NIeD-E 143 A. After 60 min. the reaction was stopped with SDS loading buffer. To estimate the size of the N-terminal fragment of the clipped JNK2, the reaction mixture was subjected to Western blot analysis using anti-

6xHis antibody (Fig. 16D). Intact JNK2 and its N-terminal fragment are indicated. To determine the size of the two JNK2 fragments, the reaction mixture was analyzed also by

SDS-PAGE followed by comassie blue staining (Fig. 16E). JNK2 and its degradation fragments are indicated. The framed C-terminal band was excised from the gel and subjected to mass spectrometry analysis.

Figure 16F. A model of JNK structure. The N-terminal portion, activation loop and C- terminal region are indicated. The mass spectrometry analysis revealed that NIeD cuts

JNK between residues Alal73 and Argl74 (indicated). This JNK2 model was created using PyMOL (www.pymol.org).

Abbreviations: WT (wild type); Epit. (epitope); A.L. (activation loop); N-term. or N' (N- terminus); C-term. or C (C-terminus); Term, (terminus); Term. Frag, (terminal fragment); α-JNK (anti-JNK antibody); KDa (kilo dalton); α-HA (anti-HA (hemagglutinin) antibody). Figure 17A-17C. NIeD cuts JNKl in vivo at its activation loop

Fig. 17A. Schematic diagram of JNKl. The location of the HA and anti-JNK antibody epitopes, activation loop (A loop), T and Y phosphorylation sites and NIeD cleavage site are indicated.

Figs 17B and 17C. HEK293 cells were transfected with a plasmids expressing JNKl, N- terminally tagged with the HA epitope. After 25 h, the cells were infected with wild type EPEC, or with EPEC nleD mutant and after 2.5 h infection the proteins were extracted from the infected cells and HA-JNK was immunoprecipitated with anti-HA antibodies. The precipitated HA-tagged JNK and JNK fragments were analyzed using Western blot with either anti-HA (Fig. 17B), or anti-JNK (Fig. 17C) antibodies. Analysis with the anti- HA and anti-JNK antibodies identified N-terminal fragment of -23 kD in size, and C- terminal fragments of ~24 kD in size. The latter is shorter from the JNK2 C-terminus fragment seen in Fig. 16C, since JNKl C-terminus region is shorter then that of JNK2. These results indicate that the C-terminal portion of the clipped HA-JNK remained in complex, and co-precipitated, with the JNK N-terminus fragment. Based on the predicted size of the C- and N- terminal fragments generated by NIeD digestion the inventors estimated that, like in the case of JNK2, NIeD cut JNKl within the activation loop (Fig 16A and l7A).

Abbreviations: WT (wild type); Epit. (epitope); A.L. (activation loop); N-term. (N- terminus); C-term. (C-terminus); Frag, (fragment); α-JNK (anti-JNK antibody); α-HA (anti-HA (hemagglutinin) antibody).

Figure 18. NIeD cleaves different isoforms ofp38

HEK 293T cells were transfected with vectors expressing either JNK2, p38α, p38β, p38γ or p38δ with N-terminal HA tag. After 24 hours the transfected cells were infected with either wild type (indicated as WT) or ΔnleD (indicated as ΔD) strains for 3 hours. Cells were than extracted for total protein and the extracts subjected to a western blot analysis using a monoclonal anti-HA antibody. The cleaved N-terminal products are indicated by asterisks. Figure 19A-19C. NIeC is a metalloprotea.se that inhibits the NF-κB pathway

Fig. 19A. Deletion analysis to identify the EPEC gene that represses IL-8 induction. HeLa cells were infected with one of the following EPEC: EPEC with a deleted IE6 region (ΔIE6), EPEC with deleted IE6 and PP4 regions (ΔIE6, ΔPP4), or the latter complemented with plasmids expressing either wild type NIeD, NIeG or NIeC (pnleD, pnleG, pnleC, respectively). Cells infected with TTSS mutant (ΔescV) and wild type EPEC (WT) served as negative and positive controls, respectively. HeLa cells were infected with the relevant EPEC for 2 h to allow injection of effectors before stimulation with TNFα for 3 h. Then RNA was extracted from the HeLa cells and RT-PCR performed to quantify IL-8 mRNA levels. Error bars indicate standard deviation.

Fig. 19B. NIeC is required for inhibition of TNFα-induced IL-8 expression. HeLa cells were infected with nleC mutant, or this mutant complemented with plasmids expressing wild type NIeC (pnleQ or NIeC-El 84A mutant (pnleC-El 84A). TTSS mutant (AescV) or wild type EPEC (WT) served as negative and positive controls, respectively. HeLa cells were infected with the relevant EPEC for 2 h to allow injection of effectors before stimulation with TNFα for 3 h. Then RNA was extracted from the HeLa cells and RT- PCR performed to quantify IL-8 mRNA levels. The indicated values are relative to IL-8 RNA levels in cells infected with ΛescV mutant. Error bars indicate the standard deviation.

Fig. 19C. NIeC reduces p65 levels in vivo. HeLa cells transfected with plasmid expressing mCherry, mCherry-NleC or mCherry-NleC-E184A were treated with TNFα for 30 min., after which they were fixed and visualized using anti-p65 antibody. Arrows indicate cells expressing mCherry proteins. Bar represents 20 μm.

Abbreviations: Vec. OnI. (vector only); WT (wild type); ReI. IL8 mRNA Exp. (relative IL8 mRNA expression); Phas. (phase).

Figure 20. Multiple alignment of NIeC homologs

The NIeC protein sequences of EPEC (Escherichia coli O127:H6 str. E2348/69, SEQ ID NO.: 4), EHEC (Escherichia coli O157:H7 EDL933, SEQ ID NO.: 97), CR (Citrobacter rodentium ICC168, SEQ ID NO.: 98), YA (Yersinia Aldovae, SEQ ID NO.: 99) and SE (Salmonella enterica subsp. enterica serovar Javian, SEQ ID NO.: 100) were aligned using ClustalW2. The HEXXH Zn-metalloprotease motif (SEQ ID NO.: 9) is framed. Figure 21 A-21C. NIeC clips p6S leading to reduced nuclear p65 levels

Fig. 21A. NIeC cleaves p65 in vivo. HeLa cells were infected for 3 hours with the ΔIE2

AnIeBE, AnIeC mutant EPEC that was complemented, or not, with plasmids expressing

NIeC or mutated NIeC (NIeC-E 184A), as indicated. Proteins were extracted from the infected HeLa cells, separated into cytosolic and nuclear fractions and subjected to

Western blot analysis using anti-p65 antibody. An EPEC mutant deficient in TTSS activity (AescV) and EPEC deleted of the IE2 region (ΔIE2) served as negative and positive controls, respectively, the latter was used here as wild type. Intact and clipped p65 are indicated.

Fig. 21B. NIeC clips the N-terminal end of p65. HeLa cells were infected for 3 hours with the AnIeC mutant EPEC that was complemented with plasmids expressing NIeC or vector only, as indicated. Proteins were extracted from the infected HeLa cells and subjected to

Western blot analysis using anti-N-terminus of p65 or anti-C-terminus of p65 antibodies.

Intact and clipped p65 are indicated.

Fig. 21C. NIeC clips p65 in vitro. The cytosolic fraction of HeLa extracts was combined with NIeC, NIeC-El 84 A, or buffer alone in the presence or absence of phenanthroline, a

Zn metalloprotease inhibitor, as indicated. The incubation time is indicated above each lane. Reaction products were visualized by Western blot analysis using anti-p65 antibody.

Intact and clipped p65 are indicated.

Abbreviations: Clip, (clipped); Nuc. Frac. (nuclear fraction); Cytop. Frac. (cytoplasmatic fraction); Vec. (vector); α-N.term (anti- N terminus); α-C.term (anti- C terminus); min.

(minutes).

Figure 22. NIeC and NIeBE cooperate to achieve full inhibition of TNFa-mediated NF- _kB activation

HeLa cells were infected with ΔIE2 mutant EPEC, which was used here as wild type, or with a triple mutant AnIeBE, AnIeC (AnIeBEC), or with the triple mutant complemented with plasmids expressing NIeC, or NIeBE or vector (pnleC, pnleBE, and Vector, respectively). Uninfected and untreated cells served as the positive control (no IL-8 secretion - full repression) and uninfected cells stimulated with TNFα as the negative control (high IL-8 secretion-no repression), respectively. HeLa cells were infected with the relevant EPEC for 3 hours to allow injection of effectors before stimulation with TNFα for 16 hours. Finally, the growth media was harvested and the amount of secreted

IL-8 determined using the ELISA assay.

Abbreviations: IL8 Sec. Arb. Un. (IL8 secretion, arbitrary units); Vec. (vector).

Figure 23. A model of the anti-inflammatory activity of NIeBCDE and NIeHl

Schematic diagram of the signaling cascades activated by PAMPs, ILl and TNF are shown. The different EPEC effectors (orange boxes) interact with this signaling network at multiple points. NIeB inhibits the TNFR signaling upstream to the TAKl complex, NIeE inhibits IKK activation, NIeC cleaves and inactivates cytoplasmic and nuclear p65, and NIeHl inhibits the interaction of NF-κB with some promoters. Finally, NIeD cleaves and inactivates JNK. These effectors function in concert to modulate the inflammation levels and apoptosis rate.

Abbreviations: CeI. Memb. (cell membrane); Nuc. Memb. (nuclear membrane); Apopt. (apoptosis); Infl.(inflammation); Prot. Degrad. (proteasomal degradation); Eff. (effectors); Comp.(complex).

Figure 24. NIeE gene and protein sequences as appearing in NCBI

Peptide sequence of EPEC NIeE, (Accession No.: YP 002330704.1), encoded by nleE (GenelD: 7062602), also denoted as SEQ ID NOs.: 2 and 1, respectively.

Figure 25. NIeC gene and protein sequences as appearing in NCBI

Peptide sequence of EPEC NIeC, (Accession No.: YP 002328603.1), encoded by nleC (GenelD: 7061096), also denoted as SEQ ID NOs.: 4 and 3, respectively.

Figure 26. NIeD gene and protein sequences as appearing in NCBI

Peptide sequence of EPEC NIeD, (Accession No.: YP_002328604.1), encoded by nleD, (GenelD: 7064968), also denoted as SEQ ID NOs.: 6 and 5, respectively.

Figure 27. NIeB gene and protein sequences as appearing in NCBI

Peptide sequence of EPEC NIeB, (Accession No.: YP_002330703.1), encoded by nleB, (GenelD: 7061121), also denoted as SEQ ID NOs.: 8 and 7, respectively. DETAILED DESCRIPTION OF THE INVENTION

Enteropathogenic Escherichia coli (EPEC) induce a severe watery diarrhea responsible for several hundred thousand infant deaths each year by a process correlated with the loss (effacement) of absorptive microvilli. Effacement is linked to the locus of enterocyte effacement pathogenicity island that encodes an "injection system" (Type III Secretion System, or TTSS), "effector" proteins, and the Intimin outer membrane protein. EPEC use the TTSS to inject effector proteins into target cells, the epithelium lining the intestinal lumen, and manipulate their intracellular signaling for their benefit.

The inventors have discovered several such effector proteins and revealed their novel mechanism of action. The effectors, NIeE, NIeC, NIeD and NIeB, are capable of modulating the major arms of the inflammatory response; the NF-κB, JNK and p38 pathways. More specifically, the inventor showed that NIeE inhibits NF-kB activation by preventing activation of IKKβ and consequently the degradation of the NF-kB inhibitor, IkB. NIeB also functions to prevent NF-κB activation and IKB degradation as shown by Figure 9D. The inventors further showed that two other effector proteins, NIeC and NIeD are Zn dependent endopeptidases that specifically clip and inactivate the p65 subunit of NF-κB, JNK and p38, respectively.

More specifically, The inventors showed that an nleE deletion mutant is deficient in blocking IKB phosphorylation and in preventing its degradation. Moreover, an nleE mutant was attenuated in blocking TNFα-induced NF-κB migration to the nucleus as well as in IL-8 expression and secretion. These abilities were restored to the mutant upon complementation with a plasmid expressing the wild-type nleE allele. Importantly, the inventors demonstrated that NleE expressed in HeLa cells blocks NF-κB translocation to the nucleus upon TNFα treatment. Taken together, the presented findings indicate that NleE is sufficient to inhibit NF-κB signaling by blocking IKB phosphorylation. Further analysis shows that NleE blocks the NF-κB signaling upstream to IKB phosphorylation, possibly by directly or indirectly blocking IKKβ activation. NleE was found to be an effective inhibitor of IKKβ activation regardless of the signaling input, as it blocked the NF-κB activation mediated by infecting EPEC, presumably via stimulation of TLR5 by flagellin, or by TNFα, or IL lβ, as illustrated by Figure 23. NIeB is required for full inhibition of NF-κB and specifically inhibits the TNFα signaling pathway, as shown by Figure 23.

Importantly, NIeE was found to be required for full virulence of C. rodentium upon infection of wild-type mice, but this requirement was diminished upon infection of mice deficient in TLR4. The presented examples revealed the rationale behind this intriguing phenomenon. The inventors assert that NleE-mediated NF-κB repression is no longer needed if the host itself is deficient in TLR4/LPS-induced NF-κB signaling.

The inventors also show that NIeB enhances NIeE activity. The nleE mutant still exhibited residual inhibition of IKB degradation, which was eliminated upon further deletion of nleB. Moreover, complementation of the nleBE double mutant with a plasmid expressing nleBE was more efficient than a plasmid expressing only nleE. These results suggest that NIeB plays a role in IKB stabilization. Similar results were obtained when expression of IL-8 was used as a readout for inhibition of the NF-κB signaling. The mechanism underlying NIeB function is not yet apparent. Nevertheless, the notion that NIeB and NleE function together is supported by the facts that nleE form a putative bicistronic operon with nleB and that nleE is consistently associated with nleB in natural isolates of diarrheagenic EPEC. Other isolates of EPEC, EHEC, and C. rodentium carry nleB and nleE alleles almost identical to

A multiple sequence alignment of NleE in different EPEC and EHEC strains is presented, for illustration, by Figure 3. Alignment of NleB from different strains is presented by Figure 4. The inventors thus predict that all NleBE proteins function similarly. The discovery that NleE and NleB repress NF-κB activation is surprising in view of previous reports suggesting exactly the opposite, i.e. that NleE and OspZ, an NleE-homolog encoded by Shigella, activate NF-κB signaling. The discrepancy between the present invention and previous observations might result from the use of different cell lines. Activation of NF-κB, rather than the presently shown inhibition thereof, may have been the result of the use of cell lines that activate NF-κB upon sensing of either TNFα or bacterial PAMPs including LPS.

The phenomenon of effectors functioning in parallel is common in EPEC. Interestingly, the TTSS mutants (esc V) and AnIeBE were only partially deficient in blocking the TNFα- induced migration of NF-κB to the nucleus, suggesting that an additional TTSS- independent mechanism might function in parallel to NleBE to inhibit translocation of NF-κB to the nucleus. Furthermore, the TTSS escV mutant was completely deficient in inhibition of IL8 expression, while the nleBE mutant was only partially deficient in this function. The inventors therefore predicted that EPEC encode additional putative effector(s) that function in parallel to NleBE by blocking IL-8 expression (Fig. 8). Indeed, as discussed later, such effectors were eventually identified and characterized.

Like EPEC, Yersinia also employs an injected effector, YopJ, to block IKB phosphorylation. YopJ is an acetyl transferase that acetylates critical IKKβ residues and thus prevents its activation. Although both NIeE and YopJ block IKKβ phosphorylation, they are very different in sequence, which probably reflects functional differences. The inventors investigated the NleE's mode of action. Other effectors that interfere with NF- KB function include the Salmonella SspH, and Shigella IpaH9.8, which are targeted to the host cell nucleus and inhibit NF-κB-dependent transcription. Shigella also uses OspG, which inhibits ubiquitination of phospho-IκB. Interestingly, EPEC encode an OspG homolog, NIeH. However, the inventors found that an EPEC strain, mutated in its two nleH alleles, still inhibit IKB degradation.

As suggested by Figure 8, additional TTSS-injected effector(s) inhibits) NF-κB signaling, and the inventors accordingly set out to identify the remaining effectors. The inventors identified NIeC and NIeD as additional anti-inflammatory effectors.

The inventors found that NIeC is a Zn-endopeptidase that specifically targets and inactivates p65. The term "Zn-endopeptidase", also referred to as "endopeptidase" or "endoproteinase" relates to proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule). For this reason, endopeptidases cannot break down peptides into monomers. Zn-endopeptidase is a metalloprotease, classified by the nature of the most prominent functional group in its active site. Metalloproteases are proteolytic enzymes whose catalytic mechanism involves a metal. The Zn ion is coordinated to the protein via three histadine imidazole ligands. The fourth coordination position is taken up by a labile water molecule. Zn endopeptidases share a HEXXH motif (also denoted as SEQ ID NO.: 9) in their catalytic site. Upon its injection, NIeC snips off a p65 N-terminus region, resulting in its inactivation. Upon long incubations, NIeC leads to reduced levels of p65 in the expressing cells presumably by compromising the stability of the clipped p65 peptides. Notably, the native injection levels of both NIeC are very low, and thus it only partially inhibits TNFα- induced IL-8 expression. However, NIeC overexpression in EPEC leading to increased injection resulted in almost complete inhibition of IL-8 expression. It thus appears that the bacteria use endogenous NIeC in relatively low doses. NIeD is also a Zn- endopeptidase and, similarly to NIeC, the bacteria inject it in small amounts. NIeD specifically cleaves and inactivates JNK. It is the first endopeptidase toxin shown to directly target JNK by cleaving it in its unstructured activation loop, leading to blocking of c-Jun activation. The natively injected NIeD was sufficient to clip most of the JNK proteins but the inventors also frequently observed remains of intact JNK. In contrast, EPEC that overexpressed NIeD exhibited increased JNK digestion. This implied that although the bacteria could, by increasing NIeD expression, completely block the JNK signaling, it did not to do so in effect. As shown by Example 14, the inventors further surprisingly found that NIeD also specifically cleaves different isoforms of p38, and therefore may also block p38 mediated signaling pathways.

The bacterial effectors, NIeE, NIeC, NIeD and NIeB that were shown by the invention as capable of modulating the major arms of the inflammatory response, the NF-κB, c-Jun and p38 pathways, may be used in different combinations thereof as powerful immunomodulatory agents targeting different arms of the immune response. Thus, according to a first aspect, the present invention relates to a composition comprising as an active ingredient at least one immuno-modulatory protein, specifically, a bacterial immunomodulatory protein, more specifically, at least one of the immuno-modulatory NIeE, NIeC, NIeD and NIeB bacterial proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. It should be noted that in certain embodiments, the composition of the invention may further comprise at least one additional therapeutic agent and a pharmaceutically acceptable carrier or excipient.

According to certain embodiments, the composition of the invention comprises at least one of isolated and purified NIeE, NIeC, NIeD and NIeB proteins. The term "isolated", "purified" or "substantially purified", when applied to a nucleic acid or protein, such as any one of the NIeE, NIeC, NIeD and NIeB molecules, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state, although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified.

It should be noted that the isolated and purified NIeE, NIeC, NIeD and NIeB proteins or any fragment thereof used by the composition of the invention, may be provided as any one of a purified recombinant protein, and a cell lysate or membrane preparation of a transformed host cell expressing the NIeE, NIeC, NIeD and NIeB molecules. The terms fragments and functional fragments used herein mean the NIeE, NIeC, NIeD and NIeB molecules or any fragment, variant, homolog or derivative thereof, with any insertions, deletions, substitutions and modifications, that is capable of inducing specific modulation of at least one of the NF-κB, c-Jun and p38 signal transduction pathways, as reflected by any one of nuclear localization of NF-κB, phosphorylation of IKB, secretion of an inflammatory cytokine such as IL-8, induction of apoptosis, phosphorylation of c-Jun, phsphorylation of MEF-2 or ELK-I and p38/JNK-dependent survival assay using the yeast model system. More specifically, functional fragment, variant, homolog or derivative of NIeE or NIeB should retain the ability to stabilize IKB and thereby inhibit nuclear translocation of NF-κB and the signaling mediated by these pathways. Functional fragment, variant, homolog or derivative of NIeC should retain the ability to cleave p65 and thereby to inhibit NF-κB signaling. Functional fragment, variant, homolog or derivative of NIeD exhibit specific cleavage of at least one of JNK and p38.

It should be appreciated that according to certain embodiments used herein in the specification and in the claim section below, the NIeE, NIeC, NIeD and NIeB proteins refer to a protein having the amino acid sequence of bacterial, specifically, EPEC NIeE, NIeC, NIeD and NIeB, or any fragment, variant homolog or derivative thereof. An example for EPEC NIeE molecule is a protein comprising the amino acid sequence as denoted by GeneBank Accession No. YP_002330704.1, shown by Figures 24A-24B and also denoted as SEQ ID NO.: 2 or 90, encoded by the EPEC NIeE gene as shown by GenBank Accession No. 7062602, as denoted by SEQ ID NO. 1.

An example for EPEC NIeC molecule is a protein comprising the amino acid sequence as denoted by GeneBank Accession No. YP 002328603.1, shown by Figures 25A-25B and also denoted as SEQ ID NO.: 4, encoded by the EPEC NIeC gene as shown by GenBank Accession No. 7061096, also denoted by SEQ ID NO. 3.

The EPEC NIeD molecule is a protein comprising the amino acid sequence as denoted by GeneBank Accession No. YP_002328604.1, shown by Figures 26A-26B and also denoted as SEQ ID NO.: 6, encoded by the EPEC NIeD gene as shown by GenBank Accession No. 7064968, as denoted by SEQ ID NO. 5. In yet another embodiment, the EPEC NIeB molecule is a protein comprising the amino acid sequence as denoted by GeneBank Accession No. YP 002330703.1, shown by Figures 27A-27B and also denoted as SEQ ID NO.: 8, encoded by the EPEC NIeB gene as shown by GenBank Accession No. 7061121, as denoted by SEQ ID NO. 7.

With respect to amino acid sequences, for example, the amino acid sequence of the any one of NIeE, NIeC, NIeD and NIeB proteins, specifically, the EPEC NIeE, NIeC, NIeD and NIeB proteins, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention.

For example, substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M).

It should be noted that the term "Amino acid(s)" as used herein refer to all naturally occurring L-amino acids, e.g. and including D-amino acids. The amino acids are identified by either the well known single-letter or three-letter designations.

The term "derivative" is used to define amino acid sequence variants, and covalent modifications of a polypeptide made use of in the present invention, e.g. of a specified sequence. The functional derivatives of a any one of NIeE, NIeC, NIeD and NIeB polypeptides utilized according to the present invention, e.g. of a specified sequence of any one of the immuno-modulatory NIeE, NIeC, NIeD and NIeB polypeptides, preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology, identity or similarity with the amino acid sequence of any one of NIeE, NIeC, NIeD and NIeB polypeptides as structurally defined above, e.g. of a specified sequence, more specifically, an amino acid sequence any one of NIeE, NIeC, NIeD and NIeB polypeptides as denoted by SEQ ID NOs. 2 or 90, 4, 6 and 8, respectively.

"Homology" with respect to a native any one of NIeE, NIeC, NIeD and NIeB polypeptides and its functional derivatives is defined herein as the percentage of amino acid residues in the candidate sequence that are identical or similar with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions or deletions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. It should be appreciated that by the terms "insertions" or "deletions", as used herein it is meant any addition or deletion, respectively, of amino acid residues to any one of NIeE, NIeC, NIeD and NIeB polypeptide molecules used by the invention, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues and specifically, between 1 to 10 amino acid residues. More particularly, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be recognized that insertions or deletions may be additions or reduction of amino acid residues from the N-terminal, the C-terminal end of the molecule or within the molecule re any combinations thereof.

The terms "identical", "substantial identity", "substantial homology" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region or over the entire molecule.

Specific examples for homology are provided by homologues of NIeE shown by Figure 3. More specifically, the Figure presents some homologues of E. coli E2348 NLEE2, including E. coli E2348 NLEEl (SEQ ID NOs.: 90 or 2 and 89, respectively; 66% homology), Citrobacter rodentium NIeE (SEQ ID NO.: 91; 85% homology), E. coli B171 and E22 strains (identical sequences; SEQ ID NO.: 92, respectively; 100% homology), EHEC O157 strain SAKAI (identical sequences; SEQ ID NO.: 105, respectively; 99% homology). Specific examples for homology are provided by homologues of NIeB shown by Figure 4. More specifically, the Figure presents some homologues of E. coli E2348 (EPEC) NIeB, including E. coli E22 NIeB (SEQ ID NOs.: 8 and 101, respectively; 99% homology), EHEC 0157 strain EDL933 NIeB (SEQ ID NO.: 102; 97% homology), Citrobacter rodentium NIeB (SEQ ID NO.: 103; 88% homology) and Salmonella enterica subsp. arizonae serovar 62:z4,z23:— NIeB (SEQ ID NO.: 104; 61% homology). Specific examples for homology are provided by homologues of NIeD shown by Figure 15. More specifically, the Figure presents some homologues of E. coli E2348 (EPEC) NIeD, including EHEC {Escherichia coli O157:H7 EDL933) NIeD (SEQ ID NOs.: 6 and 93, respectively; 98% homology), Citrobacter rodentium ICC 168 NIeD (SEQ ID NO.: 94; 76% homology), Salmonella enterica subsp. arizonae serovar 62:z4,z23:~ NIeD (SEQ ID NO.: 95; 76% homology) and Candidatus Hamiltonella defensa 5AT NIeD (SEQ ID NO.: 96; 68% homology). Yet other specific examples for homology are provided by homologues of NIeC shown by Figure 20. The examples include some homologues of Escherichia coli O127:H6 str. E2348/69 (EPEC) NIeC, including Escherichia coli O157:H7 EDL933 (EHEC) NIeC (SEQ ID NOs.: 4 and 97, respectively; 99% homology), Citrobacter rodentium ICC 168 NIeC (SEQ ID NO.: 98; 90% homology), Yersinia Aldovae NIeC (SEQ ID NO.: 99; 83% homology) and Salmonella enterica subsp. enterica serovar Javian NIeC (SEQ ID NO.: 100; 70% homology).

As shown by the present invention, NIeE, NIeC, NIeD and NIeB are effector proteins which modulate the host NF-κB, JNK and p38 mediated signal transduction pathways, thereby leading to at least one of an anti-inflammatory response, effect or reaction, an anti-apoptotic effect or a pro-apoptotic effect in cells. Thus, NIeE, NIeC, NIeD and NIeB may modulate the outcome of an inflammatory response or an immune condition, for example. Upon infection, bacterial Pathogen-Associated Molecular Patterns (PAMPs) stimulate host cell Toll-like receptors (TLRs), leading to an inflammatory response. TLR activation unleashes a complex signaling network that culminates in the activation of two central groups of transcription factors: the NF-κBs and AP-I factors including c-Jun, as illustrated in Figure 23. Together, the activated c-Jun and NF-κB modify the transcription patterns of hundreds of genes. The ultimate outcome of NF-κB and c-Jun activation is complex and varied according to the specific cell type and other signaling inputs that coincide with the TLR activation. Schematically however, both NF-κB and c-Jun are thought to function synergistically to induce inflammation. On the other hand, while c-Jun can also stimulate apoptosis, NF-κB induces the expression of anti-apoptotic factors. p38 is also involved in signaling mediating inflammatory responses, apoptosis and cell proliferation.

NF-kB (nuclear factor kappa beta) as used herein is a transcription factor that plays important roles in the immune system. NF-kB regulates the expression of cytokines, inducible nitric oxide synthase (iNOS), cyclo-oxgenase 2 (COX-2), growth factors, inhibitors of apoptosis and effector enzymes in response to ligation of many receptors involved in immunity including T-cell receptors (TCRs), B-cell receptors (BCRs) and members of the Toll-like receptor/IL-1 receptor super family. NF-kB also plays a role in the development and the activity of a number of tissues including the central nervous system. Moreover, pathological dysregulation of NF-kB is linked to inflammatory and autoimmune diseases as well as cancer. In mammals, the NF-kB family is composed of five related transcription factors: p50, p52, ReIA (p65), c-Rel and ReIB. These transcription factors are related through an N-terminal, 300 amino acid, DNA binding/dimerization domain, called the ReI homology domain (RHD), through which they can form homodimers and heterodimers that bind to 9-10 base pair DNA sites, known as kB sites, in the promoters and enhancer regions of genes, thereby modulating gene expression. NF-kB is not synthesized de novo; therefore its transcriptional activity is silenced by interactions with inhibitory IkB proteins present in the cytoplasm. There are two signaling pathways leading to the activation of NF-kB known as the canonical pathway (or classical) and the non-canonical pathway (or alternative pathway). The common regulatory step in both of these cascades is activation of an IkB kinase (IKK) complex. Activation of NF-kB dimers is due to IKK-mediated phosphorylation-induced proteasomal degradation of the IkB inhibitor enabling the active NF-kB transcription factor subunits to translocate to the nucleus and induce target gene expression. NF-kB activation leads to the expression of the IkBa gene, which consequently sequesters NF-kB subunits and terminates transcriptional activity unless a persistent activation signal is present.

As shown by the invention, NIeE and NIeB inhibit degradation of IkB and thereby block nuclear translocation of NF-kB and the signaling mediated by said pathway. NIeC was also shown by the invention as inhibitor of NF-kB by cleaving the p65. The invention further demonstrated the involvement of an additional effector, the NIeD, in other cellular signaling pathways. More specifically, NIeD was demonstrated by the invention as specifically cleaving JNK, and thereby attenuating or inhibiting the c-Jun signaling.

Stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) are members of the MAPK family and are activated by a variety of environmental stresses, inflammatory cytokines, growth factors and GPCR agonists. Stress signals are delivered to this cascade by small GTPases of the Rho family. The membrane proximal kinase is a MAPKKK, typically MEKKl-4, or a member of the mixed lineage kinases (MLK) that phosphorylates and activates MKK4 (SEK) or MKK7, the SAPK/JNK kinases. Alternatively, MKK4/7 can be activated by a member of the germinal center kinase (GCK) family in a GTPase-independent manner. SAPK/JNK translocates to the nucleus where it can regulate the activity of multiple transcription factors. As with all MAPKs, the JNKs are part of a three kinase module. The TXY motif in the activation loop of each JNK is dually phosphorylated by specific MAPK kinases (MKKs). MKK4 and MKK7 phosphorylate the threonine and tyrosine within the activation loop TXY motif resulting in JNK activation. JNK/SAPKs have been characterized to be involved in proliferation, apoptosis, motility, metabolism and DNA repair. Dysregulated JNK signaling is now believed to contribute to many diseases involving neurodegeneration, chronic inflammation, birth defects, cancer and ischemia/reperfusion injury. AP-I transcription factors are heterodimers composed of Jun, Fos, Maf and ATF subunits. c-Jun, ATF2 and ATF3 are substrates for phosphorylation by JNKs, which enhances AP-I transcriptional control of specific gene expression. There are three JNK genes (JNKl, JNK2, JNK3). JNKl and JNK2 are ubiquitously expressed, while JNK3 is restricted to brain, heart and testes.

The invention further demonstrates that the NIeD protein specifically cleaves different isoforms of p38, and thereby may block p38 mediated signaling. p38 mitogen-activated protein kinases as used herein, are a class of mitogen-activated protein kinases which are responsive to stress stimuli, such as inflammatory cytokines, lipopoly saccharides (LPS), ultraviolet irradiation, heat shock, osmotic shock and growth factors, and are involved in cell differentiation and apoptosis.

Four isoforms of p38 MAP kinase (MAPK, also called RK or CSBP): p38-α(MAPK14), - β(MAPKl 1), -γ (MAPK12 or ERK6) and -δ(MAPK13 or SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAP kinase is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), Ultraviolet light and growth factors. Stress signals are delivered to this cascade by members of small GTPases of the Rho family (Rac, Rho, Cdc42). As with other MAPK cascades, the membrane-proximal component is a MAPKKK, typically a MEKK or a mixed lineage kinase (MLK). The MAPKKK phosphorylates and activated MKK3/5, the p38 MAPK kinase (MKK3 and SEK activate p38 MAP kinase by phosphorylation at Thrl80 and Tyrl82). MKK3/6 can also be activated directly by ASKl, which is stimulated by apoptotic stimuli. P38 MAK is involved in regulation of Hsp27 and MAPKAP-2 and several transcription factors including ATF2, STATl, THE Max/Myc complex, MEF-2, ELK-I and indirectly CREB via activation of MSKl.

More specifically, a strong link has been established between the p38 pathway and inflammation. Rheumatoid arthritis, Alzheimer's disease and inflammatory bowel disease are all postulated to be regulated in part by the p38 pathway. The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines (IL-I, TNFα- and IL-6), induction of enzymes such as COX-2 which controls connective tissue remodeling in pathological conditions; expression of intracellular enzymes such as iNOS, a regulator of oxidation; induction of VCAM-I and other adherent proteins along with other inflammatory related molecules. In addition, a regulatory role for p38 in the proliferation and differentiation of immune system cells such as GM-CSF, EPO, CSF and CD-40 has been established.

Abundant evidence for p38 involvement in apoptosis exists to date and is based on concomitant activation of p38 and apoptosis induced by a variety of agents such as NGF withdrawal and Fas ligation. Cysteine proteases (caspases) are central to the apoptotic pathway and are expressed as inactive zymogens. Caspase inhibitors then can block p38 activation through Fas cross-linking, suggesting p38 functions downstream of caspase activation. However, overexpression of dominant active MKK6b can also induce caspase activity and cell death thus implying that p38 may function both upstream and downstream of caspases in apoptosis. It must be mentioned that the role of p38 in apoptosis is cell type and stimulus dependent. While p38 signaling has been shown to promote cell death in some cell lines, in different cell lines p38 has been shown to enhance survival, cell growth, and differentiation. As indicated by the invention blocking of p38 signaling by the NIeD molecule of the invention may be used for modulation of inflammatory and apoptotic responses.

The invention therefore also contemplates compositions that inhibit, or lead to the inhibition of, at least one of an NF-κB, JNK and p38 mediated signal transduction pathway, thereby leading to at least one of an anti-inflammatory response, effect or reaction, an anti-apoptotic effect or a pro-apoptotic effect in a target cell. More specifically, as mentioned above and shown by the following Examples, NIeE and NIeB inhibit IKB phosphorylation, via inhibition of IKKβ activation, and therefore inhibit nuclear translocation of NF-κB. NIeC was shown as a p65-specific Zn metalloprotease, and NIeD is a JNK-specific and p38-specific Zn metalloprotease, and thus inhibits JNK and p38 mediated signal transduction.

The term "inhibition" as referred to herein, relates to the retardation, attenuation, retraining or reduction of a process. More specifically, according to certain embodiments the compositions and also to any of the methods of the invention specifically inhibit the NF-κB or the c-Jun signaling pathways by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

Since the bacterial, specifically, the EPEC effector proteins were shown by the invention as exhibiting an immuno-modulatory, and specifically, an anti-inflammatory effect, the invention thus contemplates a pharmaceutical composition comprising at least one of NIeE, NIeD and NIeB proteins and, optionally, NIeC protein and any combinations thereof, for preventing, treating, or ameliorating an immune-related disorder. According to certain embodiments, an immune-related disorder may be an inflammatory disease, an autoimmune disease and a malignant or a non-malignant proliferative disorder. It should be indicated that these disorders will be specifically described in connection with the methods of treatment using these compositions of the invention.

As indicated before, in certain embodiments, the invention contemplates compositions and pharmaceutical compositions comprising various combinations of the proteins of the invention, i.e., NIeE, NIeC, NIeB and NIeD, optionally also comprising another therapeutic agent or protein and a pharmaceutically acceptable carrier or excipient. The additional therapeutic agent enhances the therapeutic effect of the immunomodulatory proteins of the invention.

The term "therapeutic agent" as referred to herein may be any one of therapeutic protein, small molecules or any other anti-inflammatory drugs. For treating cancer or any other proliferative disorder, induction of apoptosis may be beneficial. In order to enhanc the pro-apoptotic effect of the effector proteins of the invention, it may be desired to add as an additional therapeutic agent, a pro-apoptotic agent, a cytotoxic agent or a cytostatic agent.

Example of cytotoxic proteins may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins and hemolysin, EspC and EspP toxins. Examples of cytostatic proteins are p21, p27, p53, p53175P, p57, pl5, pl6, pl8, pl9, ρ73, GADD45, APCl, ρ73RBl, WTl, NFl, and VHL. Example of apoptotic proteins are those belonging to the BC12 family, and various caspases. Examples of other immunomodulatory proteins comprise NIeHl, YopJ and IpaB.

The optional therapeutic agent of the compositions of the invention may be a small molecule, which is a low molecular weight organic compound that is not a polymer. The term "small molecule", especially within the field of pharmacology, is usually restricted to a molecule that also binds with high affinity to a biopolymer such as protein, nucleic acid, or polysaccharide and in addition alters the activity or function of the biopolymer. The upper molecular weight limit for a small molecule is approximately 800 Daltons which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action. These compounds can be natural (such as secondary metabolites) or artificial (such as antiviral drugs). Non-limiting examples of small molecules include: Alkaloids, Glycosides, Lipids, Flavonoids, Nonribosomal peptides such as actinomycin-D, Phenazines, Phenols, Polyketide, Terpenes, including steroids and Tetrapyrroles.

As will be discussed in detail herein after, the present invention provides a modular platform for customized compositions, specifically suitable for treating different disorders. The invention therefore encompasses compositions comprising any combination of the NIeE, NIeC, NIeD, and NIeB immuno-modulatory proteins of the invention and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. More specifically, a particular embodiment of the invention relates to the composition of the invention comprising NIeE, NIeC, NIeB and optionally, NIeD proteins, also denoted by SEQ ID NOs.: 2 or 90, 4, 6 and 8, respectively, and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof.

In some embodiments, the composition of the invention comprises NIeE (as denoted by SEQ ID NO. 2 or 90, and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof), the composition optionally further comprising at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE and at least one of NIeC, NIeD and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent.

In other embodiments, the composition of the invention comprises NIeC (as denoted by SEQ ID NO. 4, and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof), the composition optionally further comprising at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeC and at least one of NIeE, NIeD and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent.

In certain embodiments, the composition of the invention comprises NIeD (as denoted by SEQ ID NO. 6, and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof), the composition optionally further comprising at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeD and at least one of NIeC, NIeE and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent.

In further embodiments, the composition of the invention comprises NIeB (as denoted by SEQ ID NO. 8, and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof), the composition optionally further comprising at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeB and at least one of NIeC, NIeD and NIeE proteins, the composition optionally further comprises at least one additional therapeutic agent. In still other embodiments, the composition of the invention comprises NIeE and NIeD proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE and NIeD and at least one of NIeC and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeE and NIeC proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE and NIeC and at least one of NIeD and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeE and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE and NIeB and at least one of NIeC and NIeD proteins, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeC and NIeD proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeC and NIeD and at least one of NIeE and NIeB proteins, the composition optionally further comprises at least one additional therapeutic agent. hi still other embodiments, the composition of the invention comprises NIeB and NIeD proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeB and NIeD and at least one of NIeC and NIeE proteins, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeB and NIeC proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprises at least one additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeC and NIeB and at least one of NIeD and NIeE proteins, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeE, NIeC and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprising an additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE, NIeC and NIeB proteins and, optionally, NIeD protein, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeE, NIeC and NIeD proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprising an additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE, NIeC and NIeD proteins and, optionally, NIeB protein, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeB, NIeC and NIeD proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprising an additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeB, NIeC and NIeD proteins and, optionally, NIeE protein, the composition optionally further comprises at least one additional therapeutic agent.

In still other embodiments, the composition of the invention comprises NIeE, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, the composition optionally further comprising an additional therapeutic agent. According to another embodiment, the composition of the invention comprises NIeE, NIeD and NIeB proteins and, optionally, NIeC protein, the composition optionally further comprises at least one additional therapeutic agent.

It should be further appreciated that the invention further provide compositions comprising as an active ingredient, nucleic acid sequences encoding at least one of the NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins of the invention. Non-limiting examples for such nucleic acid sequences are denoted by any one of SEQ ID NO. 1, 3, 5 and 7, respectively. The invention therefore further encompasses compositions and uses of expression vectors comprising such nucleic acid sequences, as well as host cells expressing the same.

All compositions of the invention described herein and herein after, may comprise pharmaceutically acceptable carrier or excipient. As used herein "pharmaceutically acceptable carrier or excipient" includes any and all solvents, dispersion media, coatings and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except any conventional media or agent incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

Pharmaceutically acceptable salts, for example, refer to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid. Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, trifluoroacetate and the like.

Pharmaceutically acceptable acid addition salt are those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

Pharmaceutically acceptable esters are those esters which retain, upon hydrolysis of the ester bond, the biological effectiveness and properties of the carboxylic acid or alcohol and are not biologically or otherwise undesirable. These esters are typically formed from the corresponding carboxylic acid and an alcohol. Generally, ester formation can be accomplished via conventional synthetic techniques. The alcohol component of the ester will generally comprise (i) a C..sub.2~C..sub.l2. aliphatic alcohol that can or can not contain one or more double bonds and can or can not contain branched carbon chains or (ii) a C..sub.7~C..sub.l2 aromatic or heteroaromatic alcohols. This invention also contemplates the use of those compositions which are both esters as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.

Pharmaceutically acceptable amides are those amides which retain, upon hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine and are not biologically or otherwise undesirable. These amides are typically formed from the corresponding carboxylic acid and an amine. Generally, amide formation can be accomplished via conventional synthetic techniques. This invention also contemplates the use of those compositions which are both amides as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.

"Pharmaceutically or therapeutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.

In some particular embodiments, the composition of the invention is specifically suitable for oral administration, however it should be noted that the administration may include intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, transdermal, intranasal, mucosal, topical or subcutaneous administration, or any combination thereof. Any composition of the invention may be administered orally. The immunomodulatory agents employed in the instant therapy can be administered in various oral forms including, but not limited to, tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. It is contemplated that the immuno-modulatory proteins of the invention can be delivered by any pharmaceutically acceptable route and in any pharmaceutically acceptable dosage form. These include, but are not limited to the use of oral conventional rapid-release, time controlled-release, and delayed-release pharmaceutical dosage forms. The immunomodulatory proteins of the invention can be administered in a mixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected to with respect to the intended form of administration. As indicated, it is contemplated that oral administration can be effectively employed. Thus, tablets, capsules, syrups, and the like as well as other modalities consistent with conventional pharmaceutical practices can be employed.

In instances in which oral administration is in the form of a tablet or capsule, the active components that are the immuno-modulatory proteins of the invention, can be combined with a non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, modified sugars, modified starches, methylcellulose and its derivatives, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, and other reducing and non- reducing sugars, magnesium stearate, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate and the like. For oral administration in liquid form, the immunomodulatory agents can be combined with non-toxic pharmaceutically acceptable inert carriers such as ethanol, glycerol, water and the like. When desired or required, suitable binders, lubricants, disintegrating agents and coloring and flavoring agents can also be incorporated into the mixture. Stabilizing agents such as antioxidants, propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7- hydroxycoumarin can also be added to stabilize the dosage forms. Other suitable compounds can include gelatin, sweeteners, natural and synthetic gums such as acacia, tragacanth, or alginates, carboxymethylcellulose, polyethylene, glycol, waxes and the like. Alternatively, the compositions of the invention may also be administered in controlled release formulations such as a slow release or a fast release formulation. Such controlled release formulations may be prepared using methods well known to those skilled in the art. The method of administration will be determined by the attendant physician or other person skilled in the art after an evaluation of the subject's conditions and requirements.

For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art.

As indicated herein above, the NIeE, NIeC, NIeD and NIeB immuno-modulatory bacterial proteins of the invention effectively modulate major signaling pathways in target mammalian cells, modulating immune response, specifically, attenuating proinflammatory response and modulating apoptosis. The invention thus further contemplates as a second aspect, a method for preventing, treating, or ameliorating an immune-related disorder in a subject in need thereof. According to certain embodiments, the method of the invention comprises the step of administering to the treated subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologies, variants, fragments, derivatives, mixtures, any combinations thereof or any compositions comprising the same.

According to one specific embodiment, the method of the invention involves the administration of at least one additional therapeutic agent. Such method therefore comprises the step of administering to the treated subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins of the invention and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, and optionally at least one additional therapeutic agent or any compositions or combined compositions comprising the same.

The invention further provides methods using nucleic acid sequences encoding at least one of said irnmuno-modulatory proteins, expression vectors comprising said sequences and host cells expressing at least one of said proteins.

Furthermore, in some embodiments, the method of treatment of the invention leads to inhibition of at least one of an NF-κB, c-Jun (by inhibiting JNK) and p38 mediated signal transduction pathways, thereby leading to at least one of an anti-inflammatory response, effect or reaction, an anti-apoptotic effect or a pro-apoptotic effect in a cell of said treated subject

Anti-inflammatory effects of the compositions and methods of the invention as used herein refer to a decrease or reduction in the amount or expression of pro-inflammatory cytokines such as IL-8. It should be however appreciated that such anti-inflammatory effect may also involve reduction in any other pro-inflammatory cytokine, for example, IL-2, IL- 17, IL-23, IFN-γ, IL-6 and TNFα. Such a decrease or reduction according to the invention may be a reduction of about 5% to 99%, specifically, a reduction of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% as compared to untreated control. The anti-inflammatory effects may also refer to an increase in the amount or expression of anti-inflammatory cytokines such as TGF-β, IL-10, IL-4, IL-5, IL-9 and IL-13. Such an increase according to the invention may be an increase of about 5% to 99%, specifically, an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% as compared to untreated control.

In other embodiments, the compositions and methods of the invention may also lead to an increase, induction or elevation in apoptosis of treated cells, said increase, induction or elevation of apoptosis may be an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% as compared to untreated control, hi still other embodiments, the composition and the methods of the invention may also lead to a decrease, inhibition or reduction in apoptosis of treated cells, said decrease, inhibition or reduction of apoptosis may be a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% as compared to untreated control.

The anti-inflammatory effects of the compositions and methods of the invention are applicable for conditions where an anti-inflammatory response is beneficial and desired. Thus, in particular embodiments, the method of the invention is effective for treatment of an immune-related disorder. An "Immune-related disorder" is a condition that is associated with the immune system of a subject, either through activation or inhibition of the immune system, or that can be treated, prevented or diagnosed by targeting a certain component of the immune response in a subject, such as the adaptive or innate immune response. Such disorder may be any one of an inflammatory disease, an autoimmune disease and a malignant or non-malignant proliferative disorder.

According to one specific embodiment, the method of the invention may be specifically suitable for treating an inflammatory disease or an inflammatory-associated condition. The terms "inflammatory disease" or "inflammatory-associated condition" refers to any disease or pathologically condition which can benefit from the reduction of at least one inflammatory parameter. The condition may be caused (primarily) from inflammation, or inflammation may be one of the manifestations of the diseases caused by another physiological cause.

Examples of immune-related disorders include, but are not limited to, Ulcerative Colitis, Crohn's Disease, Irritable Bowel Disease (IBD), Alopecia Areata, Lupus, Ankylosing Spondylitis, Meniere's Disease, Antiphospholipid Syndrome, Mixed Connective Tissue Disease, Autoimmune Addison's Disease, Multiple Sclerosis, Autoimmune Hemolytic Anemia, Myasthenia Gravis, Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet's Disease, Pernicious Anemia, Bullous Pemphigoid, Polyarthritis Nodosa, Cardiomyopathy, Polychondritis, Celiac Sprue-Dermatitis, Polyglandular Syndromes, Chronic Fatigue Syndrome (CFIDS), Polymyalgia Rheumatica, Chronic Inflammatory Demyelinating, Polymyositis and Dermatomyositis, Chronic Inflammatory Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST Syndrome, Raynaud's Phenomenon, Cold Agglutinin Disease, Reiter's Syndrome, Rheumatic Fever, Discoid Lupus, Rheumatoid Arthritis, Essential Mixed, Cryoglobulinemia Sarcoidosis, Fibromyalgia, Scleroderma, Grave's Disease, Sjogren's Syndrome, Guillain-Barre, Stiff- Man Syndrome, Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I), Vasculitis, Lichen Planus, and Vitiligo. The compositions and delivery systems described herein can be administered to a subject to treat or prevent disorders associated with an abnormal or unwanted immune response associated the above diseases.

As indicated above, the term "immune-related disorder" is also meant to embrace proliferative disorders, such as cancer. As used herein to describe the present invention, "cancer", "tumor" and "malignancy" all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the compositions and methods of the present invention may be used in the treatment of non-solid and solid tumors. It should be appreciated that the therapeutic proteins of the invention modulate signaling pathways that control both inflammatory response and, importantly, apoptosis. Specifically, NIeE, NIeC and NIeB inhibit NF-κB signaling which inhibits apoptosis, whereas NIeD modulates JNK and p38 signaling which promotes apoptosis. The use of different combinations and dosages of the compositions of the invention comprising different combinations of said therapeutic proteins may be employed for the effective treatment of proliferative diseases, modulating cellular apoptosis rates.

Malignancy, as contemplated in the present invention may be selected from the group consisting of carcinomas, melanomas, lymphomas, myeloma, leukemia and sarcomas. Malignancies that may find utility in the present invention can comprise but are not limited to solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Karposi) and hematological malignancies (including leukemia, lymphoma and myeloproliferative disorders), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic). More particularly, the malignant disorder may be GI tract and colon cancers.

As mentioned above, in connection with the compositions of the invention, the present invention provides a modular platform for creation of customized methods of treating immune-related disorders. It should be noted that the methods of the invention comprise the step of administering any combinations of the NIeE, NIeC, NIeB and NIeD proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof or any compositions comprising any possible combinations thereof. Specifically, any combination described for the composition of the invention herein before should be considered as applicable for any of the methods and uses of the invention. Optionally, the methods of the invention involve the use of combinations of at least one of the bacteria immunomodulatory proteins of the invention, also with at least one additional therapeutic agent. Such additional therapeutic agent may be any therapeutic protein, agent, drug or a small molecule as described for the compositions of the invention herein before.

In specific embodiments, the method of the invention comprises the step of administering to the subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeB and optionally, NIeD proteins (also denoted by SEQ ID NOs.: 2 or 90, 4, 8 and 6, respectively) and any functional homologues, variants, fragments, derivatives and mixtures thereof and optionally at least one additional therapeutic agent, and any compositions comprising the same.

In another specific embodiment, the invention involves administration of a therapeutically effective amount of NIeE, NIeC and NIeB immuno-modulatory proteins, or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. It yet another embodiment, the method of the invention involves administration of the NIeE, NIeC, NIeB and NIeD immuno-modulatory proteins, or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof.

It should be further appreciated that any of the possible combinations described herein above for any of the compositions of the invention are also applicable in any of the methods of the invention. It should be further noted that as such, the methods and compositions of the invention provide modular and versatile system enabling creation and design of specific compositions suitable for treating a particular disorder.

As indicated above, the invention described herein encompasses methods for the treatment of subjects in need thereof. The term "treatment" concerns improvement of at least one undesired manifestation of the disease such as: increase in disease free periods, decrease in acute disease periods (in time and severely), decrease in severity of the disease, improvement in life quality, decreased mortality, decrease in the rate of disease progression as well as prophylactic treatment before disease occurs.

The term "subject in need thereof' relates to a mammalian subject, such as human, bovine, equine, murine, feline, canine or other, suffering from an immune-related disorder as described, the treatment of which with any of the therapeutic immuno-modulatory proteins of the invention, combinations and compositions thereof according to the invention, would ameliorate or decrease in acute disease periods (in time and severely), decrease in severity of the disease, or even prevent.

As used herein, the term "therapeutically effective amount" means an amount of a compound or composition which is administered to a subject in need thereof, necessary to effect a beneficial change in the severity of a disease or disorder, or prevent such disease, in said subject. This amount should also be within specific pharmacological ranges, to avoid toxic effects by over-dosing. For example, in the present invention, a therapeutically effective amount of at least one of NIeE, NIeC, NIeD or NIeB, for the treatment of colitis would be the amount of these proteins administered to a subject which would induce a beneficial change in the subject, alleviating, ameliorating, or preventing the recurrence of said colitis, without causing detrimental side-effects, or causing only mild side-effects. It is understood that the therapeutically effective amount is not an absolute term and depends on subjective circumstances, such as the subject's age, health, weight, and various other statistics, as described in the and specifically determined by the attendant physician or other person skilled in the art after an evaluation of the subject's conditions and requirements. It should be further noted that for the method of treatment and prevention provided in the present invention, said therapeutic effective amount, or dosage, is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the immunomodulatory agents used by the invention or any composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the combined composition of the invention is administered in maintenance doses, once or more daily.

It should not be overlooked that as a further aspect, the invention contemplates the use of at least one of the bacterial NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, for the preparation of a pharmaceutical composition for the prevention, treatment, or amelioration of an immune-related disorder.

According to certain embodiments, the invention provides the use of at least one of NIeE, NIeC, NIeD and NIeB proteins, optionally with an additional therapeutic agent, in the preparation of a pharmaceutical composition for the prevention, treatment, or amelioration of an immune-related disorder.

In another embodiment, the invention involves the use a therapeutically effective amount of NIeE, NIeC and NIeB proteins. It yet another embodiment, the invention contemplates the use of the NIeE, NIeC, NIeB and NIeD proteins.

It should be noted that the invention relates to the use of any possible combinations of the NIeE, NIeC, NIeB and NIeD proteins, or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof or any optional combinations thereof with an additional therapeutic agent. It should be further appreciated that any of the possible combinations described herein above for any of the compositions of the invention are also applicable in any of the uses according to the invention.

An important aspect of the disclosed invention is a tissue-targeted delivery system of an immunomodulatory protein. The delivery systems of the invention comprise a non- virulent/attenuated Type-Three Secretion System (TTSS)-expressing microorganism. According to certain embodiments, such attenuated microorganism comprises nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. It should be appreciated that the nucleic acid sequences are operably linked to TTSS secretion signal sequences.

Type three secretion systems, often written Type III secretion system and abbreviated TTSS or T3 S S, as used herein, is a protein appendage found in several Gram-negative bacteria. In pathogenic bacteria, the structure is used to secrete proteins that help the bacteria infect multicellular, eukaryotic organisms. The proteins are secreted directly from the bacterial cell into the host cells using a needle-like structure which is the hallmark of the TTSS. Examples of bacteria expressing TTSS are Shigella Spp., Salmonella, Escherichia coli, Burkholderia, Yersinia, Chlamydia, Pseudomonas and the plant pathogens Erwinia, Ralstonia, Rhizobium, Vibrio, and Xanthomonas.

The TTSS is composed of approximately 30 different proteins. TTSSs are essential for the pathogenicity of many pathogenic bacteria. Defects in the TTSS may render a bacterium non-pathogenic.

Most TTSS genes are laid out in operons. These operons are located on the bacterial chromosome in some species and on a dedicated plasmid in other species. Salmonella, for instance, has a chromosomal region in which most TTSS genes are gathered, the so-called Salmonella pathogenicity island (SPI). Shigella, on the other hand, has a large virulence plasmid on which all TTSS genes reside. It is important to note that many pathogenicity islands and plasmids contain elements that allow for frequent horizontal gene transfer of the island/plasmid to a new species. Effector proteins that are to be secreted through the needle need to be recognized by the system, since they float in the cytoplasm together with thousands of other proteins. Recognition is done through a secretion signal, a short sequence of amino acids located at the beginning (the N-terminus) of the protein (usually within the first 20 amino acids), that the needle complex is able to recognize. As opposed to other secretion systems, the secretion signal of TTSS proteins is never cleaved off the protein. However, the nature of the secretion signal and the mechanism of its recognition are poorly understood, though methods for predicting which bacterial proteins can be transported by the Type III secretion system have been developed.

As indicated above, the invention uses the a TTSS-based system for specific delivery and translocation of the immuno-modulatory effector proteins of the invention, namelly, at least one of the NIeE, NIeC, NIeB and NIeD proteins, or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof and any combinations thereof. The attenuated microorganism, preferably, attenuated bacteria used by the delivery system of the invention comprise nucleic acid sequences encoding said immuno-modulatory effector proteins of the invention. For efficient recognition and delivery of the desired proteins by the TTSS system, the encoding nucleic acid sequences are operably linked to nucleic acid sequences encoding a "TTSS secretion signal". Proteins of interest that need to be delivered into cells using the TTSS system need to have specific signal sequences (tags) preferably at their N-terminals. The injected proteins have two signals in their N-terminus. The first located at residues -1-30 and is recognized by the TTSS and the second in residues -30-90 and is recognized by a chaperon that increases the injection efficiency. It should be appreciated that the nucleic acid sequences encoding the secretion TTSS signal may be also taken from other known injected effectors such as Tir, EspH, EspZ, EspF and others. According to a specific embodiment, the TTSS secretion signal may be taken from any one of the NIeE, NIeC, NIeD and NIeB proteins.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. Non-limiting examples for nucleic acid sequences encoding the effector proteins of the invention, the NIeE, NIeC, NIeB and NIeD proteins of SEQ ID NO. 2 or 90, 4, 8 and 6, or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, are nucleic acid sequences comprising any one of SEQ ID NO. 1, 3, 7 or 5, respectively.

It should be also noted that the nucleic acid sequences encoding the immuno-modulatory proteins of the invention may be endogenous or native to the attenuated microorganism used by the delivery system of the invention. Alternativelly, such sequences may be heterologous. In order to be delivered and secreted by the delivery system of the invention the encoding nucleic acid sequenes should be operably linked to sequences encoding the TTSS secretion signal as indicated above.

The term "operably linked" is used herein for indicating that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

In some embodiments, the microorganism comprised within the tissue-targeted delivery system according to the invention optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein. It should be noted that examples for additional therapeutic agent may also include other bacterial effectors, for example, EPEC effectors such as the NIeHl, that exhibit anti-inflammatory effect.

Specific embodiments of the invention relate to the delivery system, wherein the immunomodulatory protein inhibits at least one of an NF-κB, JNK and p38 mediated signal transduction pathways, thereby leading to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a cell of a target tissue. In more specific embodiments, the immunomodulatory protein of the delivery system of the invention is an anti-inflammatory protein. More specifically, as shown by the Examples, NIeE and NIeB inhibit IKB phosphorylation and thereby increase its stability, via inhibition of IKKβ activation, NIeC was shown as a p65 -specific Zn metalloprotease, and NIeD is a JNK-specific Zn metalloprotease. NIeD was further shown by the invention as leading to specific cleavage of different isoforms of p38.

It should be understood that the modular different combinations described herein before for the compositions of the invention also apply for any of the delivery systems of the invention.

In some embodiments, the delivery system of the invention comprises non- virulent/attenuated TTSS expressing microorganisms comprising nucleic acid sequences encoding NIeE. Such nucleic acid sequences may comprise for example the nucleic acid sequence as denoted by SEQ ID NO. 1. In another embodiment, the delivery system of the invention optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein. According to another embodiment, the delivery system of the invention comprises nucleic acid sequences encoding NIeE and at least one of NIeC, NIeD and NIeB proteins.

In particular embodiments, the delivery system of the invention comprises non- virulent/attenuated TTSS expressing microorganisms comprising nucleic acid sequences encoding NIeC. In certain embodiments such nucleic acid sequences may comprise for example the nucleic acid sequence as denoted by SEQ ID NO. 3. In yet another embodiment the delivery system optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein. According to another embodiment, the delivery system of the invention comprises nucleic acid sequences encoding NIeC and at least one of NIeE, NIeD and NIeB proteins.

In other embodiments, the delivery system of the invention comprises non- virulent/attenuated TTSS expressing microorganisms comprising nucleic acid sequences encoding the NIeD protein. In certain embodiments such nucleic acid sequences may comprise for example the nucleic acid sequence as denoted by SEQ ID NO. 5. In yet another embodiment the delivery system optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences. According to another embodiment, the delivery system of the invention comprises nucleic acid sequences encoding NIeD and at least one of NIeE, NIeC and NIeB proteins.

In particular embodiments the delivery system of the invention comprises non- virulent/attenuated TTSS expressing microorganisms comprising nucleic acid sequences encoding NIeB protein. In certain embodiments such nucleic acid sequences may comprise for example the nucleic acid sequence as denoted by SEQ ID NO. 7. hi yet another embodiment the delivery system optionally further comprises at least one nucleic acid sequence encoding a therapeutic protein, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences. According to another embodiment, the delivery system of the invention comprises nucleic acid sequences encoding NIeB and at least one of NIeE, NIeC and NIeD proteins.

It should be appreciated that according to certain embodiments the delivery system of the invention comprises nucleic acid sequences encoding the NIeE, NIeC and NIeB proteins. It yet another embodiment, delivery system of the invention comprises nucleic acid sequences encoding the NIeE, NIeC, NIeB and NIeD proteins.

It should be noted that according to certain embodiments the delivery system of the invention comprises nucleic acid sequences encoding any combinations of the NIeE, NIeC, NIeB and NIeD proteins or any optional combinations thereof with an additional therapeutic protein. It should be further appreciated that any of the possible combinations described herein before for any of the compositions and methods of the invention are also applicable in any of the delivery systems of the invention. It should be further noted that as such, the delivery systems of the invention provide modular and versatile system enabling creation and design of specific compositions suitable for treating different particular disorders.

In specific embodiments, the delivery system of the invention may comprise non- virulent/attenuated TTSS expressing microorganisms that may be any one of Enteropathogenic E. coli (EPEC), Enterohemorrhagic E. coli (EHEC), Yersinia enterocolitica, Yersinia pseudotuberculosis, Salmonella typhi, Salmonella enterica, Pseudomonas aeruginosa, Vibrio cholerae, Shigella sp. Bordetella Pertussis, Chlamydia trachomatis and Citrobacter rodentium, or any combinations thereof.

In one specific embodiment, the TTSS-expressing microorganism used by the delivery system of the invention may be any one of EPEC or EHEC. EPEC and EHEC are members of the Enter obacteriaceae. Like other Proteobacteria they have Gram-negative stains, and they are facultative anaerobes. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals. Most members of Enterobacteriaceae have peritrichous Type I fimbriae involved in the adhesion of the bacterial cells to their hosts.

In some specific embodiments, the non-virulent/attenuated Type-Three Secretion System (TTSS)-expressing microorganism is EPEC.

Enteropathogenic E. coli (EPEC) induce a profuse watery, sometimes bloody, diarrhea. They are a leading cause of infantile diarrhea in developing countries. Pathogenesis of EPEC involves a plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables localized adherence of bacteria to intestinal cells and a non fimbrial adhesin designated intimin, which is an outer membrane protein that mediates the final stages of adherence. They do not produce heat-stable (ST) or heat-labile (LT) toxins.

Adherence of EPEC strains to the intestinal mucosa is a very complicated process and produces dramatic effects in the ultrastructure of the cells resulting in rearrangements of actin in the vicinity of adherent bacteria. The phenomenon is sometimes called "attachment and effacing" of cells. The infectious dose of EPEC in healthy adults has been estimated to be 10⁶ organisms.

In other embodiments, the non- virulent/attenuated Type-Three Secretion System (TTSS)- expressing microorganism is EHEC. Enterohemorrhagic E. coli (EHEC) are recognized as the primary cause of hemorrhagic colitis (HC) or bloody diarrhea. EHEC are characterized by the production of verotoxin or Shiga toxins (Stx). Although Stxl and Stx2 are most often implicated in human illness, several variants of Stx2 exist. There are many serotypes of Stx-producing E. coli, but only those that have been clinically associated with HC are designated as EHEC. Of these, O157:H7 is the prototypic EHEC and most often implicated in illness worldwide. The infectious dose for O157:H7 is estimated to be 10 - 100 cells; but no information is available for other EHEC serotypes. Nothing is known about the colonization antigens of EHEC but fimbriae are presumed to be involved. The bacteria do not invade mucosal cells as readily as Shigella, but EHEC strains produce a toxin that is virtually identical to the Shiga toxin. The toxin plays a role in the intense inflammatory response produced by EHEC strains. The toxin is phage encoded and its production is enhanced by iron deficiency.

The terms "attenuated" or "non- virulent bacteria" in connection with any of the delivery systems of the invention refer to a bacteria that has the capacity to colonize a particular target tissue, but attenuated or reduced capacity to cause disease for example due to lack of several effector genes, or deletion of the genes coding for the type IV, or I pili or other bacterial toxins. These terms are described in more details herein after in connection with attenuated probiotic bacteria provided by the invention.

In another aspect of the invention, the inventors contemplated a composition comprising at least one delivery system of an immunomodulatory protein comprising a non- virulent/attenuated TTSS-expressing microorganism. Such attenuated microorganisms may comprise nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and combinations thereof. As mentioned herein before, the nucleic acid sequences are operably linked to TTSS secretion signal sequences. The composition of the invention may also further comprise a pharmaceutically acceptable carrier or excipient. Non- limiting examples for nucleic acid sequences encoding the different NIeE, NIeC, NIeD and NIeB proteins of the invention and any combinations thereof are described herein before for the delivery system of the invention and are also applicable for any of the compositions described herein.

It should be appreciated that examples for nucleic acid sequences encoding the immunomodulatory proteins of the invention are mentioned above in connection with the delivery system of the invention and are also applicable for any of the compositions and methods using the delivery system.

More specifically, some embodiments relate to the composition of the invention, wherein the delivery system optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein. It should be understood that such nucleic acid sequences also comprise TTSS secretion signal.

In yet further particular embodiments, any of the compositions of the invention may further comprise at least one additional therapeutic agent. It should be understood that the therapeutic agent may be a protein, a small molecule or any other drug. Examples for additional therapeutic agents are discussed herein before in connection with other compositions of the invention. It should be appreciated that any example of said agents is also applicable for compositions comprising the delivery systems of the invention and for any methods and uses thereof.

As indicated above, the compositions or the pharmaceutical compositions of the invention described herein are modular and may comprise different TTSS bacteria encoding different combinations of the immuno-modulatory proteins of the invention. It should be appreciated that any of the combinations mentioned herein above in connection with other compositions or methods of the invention are also applicable herein.

In one particular embodiment, the composition comprises TTSS bacteria encoding NIeE, NIeC and NIeB proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. It yet another embodiment, the compositions of the invention comprise TTSS bacteria encoding the NIeE, NIeC, NIeB and NIeD proteins, proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof.

In some embodiments, the composition according to the invention leads to the inhibition of at least one of an NF-κB, JNK and p38 mediated signal transduction pathways.

More specifically, as shown by the Examples, NIeE and NIeB inhibit IKB phosphorylation, via inhibition of IKKβ activation, and therefore increase IKB stabilization and inhibit nuclear translocation of NF-κB. NIeC was shown as a p65- specific Zn metalloprotease, and NIeD is a JNK-specific Zn metalloprotease, and thus inhibits JNK mediated signal transduction. NIeD was further demonstrated by the invention as cleaving different isoforms of p38 and therefore may inhibit p38 signaling. Therefore, the composition of the invention leads to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a cell of a target tissue. Such composition may be applicable in the treatment of immune-related disorders where modulation of the inflammatory response may be desired.

Thus, the invention further provides a pharmaceutical composition comprising at least one delivery system of an immunomodulatory protein according to the invention. Such composition is specifically suitable for preventing, treating, or ameliorating an immune- related disorder. As mentioned above, an immune-related disorder according to the invention may be any one of an inflammatory disease, an autoimmune disease and a malignant or non-malignant proliferative disorder.

In other embodiments, the compositions of the invention are specifically suitable for oral administration, however it should be noted that the administration may include intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, transdermal, intranasal, mucosal, topical or subcutaneous administration, or any combination thereof.

In a further aspect, the invention is directed to a method for preventing, treating, or ameliorating an immune-related disorder in a subject in need thereof. The method of the invention comprises the step of administering to the subject a therapeutically effective amount of a tissue-targeted delivery system of an immunomodulatory protein or any composition comprising the same. The delivery system used by the method of the invention comprises a non- virulent/attenuated TTS S -expressing microorganism comprising nucleic acid sequences encoding at least one of the immuno-modulatory NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof. It should be noted that the nucleic acid sequences are operably linked to TTSS secretion signal sequences.

In specific embodiments, the delivery system used by the method according to the invention, optionally further comprises nucleic acid sequences encoding at least one additional therapeutic protein. It should be appreciated that both versions of the methods of the invention may involve an additional step of administering at least one additional therapeutic agent that may be an additional protein or a small molecule or drug. Examples for other therapeutic agents are mentioned herein above in connection with the compositions of the invention and are applicable in these methods as well.

It should be understood that the methods of the invention comprise the use of the delivery system of the invention include all possible combinations of the therapeutic proteins NIeE, NIeC, NIeB and NIeD, as indicated herein before.

It is important to note that the inventors also envisioned the modularity of the therapeutic compositions and delivery systems of the invention. As discussed earlier, the inventors have established that NIeE and NIeB inhibit IKB phosphorylation, via inhibition of IKKβ activation, leading to increased stability of IKB. NIeC was shown as a p65-specific Zn metalloprotease, and NIeD is a JNK and p38-specific Zn metalloprotease. Thus, NIeE, NIeC and NIeB inhibit NF-κB signaling and NIeD inhibits JNK signaling. NF-κB, p38 and JNK signaling induce transcription of pro-inflammatory target genes, however while NF-κB activation inhibits pro-apoptotic gene transcription, JNK and p38 signaling is pro- apoptotic. By customizing the combinations of the therapeutic proteins of the invention, optionally, with combined additional agents delivered to target cells, one may therefore modulate apoptosis and pro- or anti-inflammatory gene expression. In this manner, the compositions, methods and delivery systems of the invention may be customized to specific requirements. As a non-limiting example, Inflammatory bowel disease (IBD), for example, Crohn's disease and Colitis patients will benefit from different combinations and doses of NIeE, NIeC and NleB,for inducing an anti-inflammatory response. Addition of NIeD may reduce the anti-apoptotic effect of this composition by blocking c-Jun and p38 pathways. It should be understood that the action of the immuno-modulatory proteins of the invention may be optionally augmented with other therapeutic proteins or agents. In the other hand, patients suffering from proliferative disorders will benefit from treatment with pro-apoptotic NIeC, NIeE and NIeB, that block NF-κB, also optionally augmented with other therapeutic proteins or agents. Thus, in one specific embodiment the method of the invention using a TTSS tissue- targeted delivery system may be particularly applicable for the treatment of an inflammatory bowel disease (IBD), such as Crohn's disease and Colitis.

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine. The major types of IBD are Crohn's disease and ulcerative colitis (UC). Other forms of IBD include collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's Syndrome and indeterminate colitis.

As indicated above, Ulcerative colitis is a form of colitis, a disease of the intestine, specifically the large intestine or colon, that includes characteristic ulcers, or open sores, in the colon. The main symptom of active disease is usually constant diarrhea mixed with blood, of gradual onset. Crohn's disease, also known as granulomatous, and colitis, is an inflammatory disease of the intestines that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea (which may be bloody), vomiting, or weight loss. Crohn's disease is thought to be an autoimmune disease.

The main difference between Crohn's disease and UC is the location and nature of the inflammatory changes. Crohn's can affect any part of the gastrointestinal tract, from mouth to anus (skip lesions). Ulcerative colitis, in contrast, is restricted to the colon and the rectum. It should be noted that by using EPEC attenuated bacteria in the targeting delivery system of the invention the proteins of the invention may be targeted to the GI (gastro intestinal), for treating Crohn's disease. Specific targeting of the immunomodulatory proteins of the invention to the colon, using EHEC, may be applicable in treating colitis.

It should be noted that the methods and compositions of the invention may be applicable also for any other immune-related disorders. Examples for such disorders include: rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, Type I diabetes (IDDM), Sjogren's syndrome, autoimmune thyroid disease, sarcoidosis, autoimmune uveitis, autoimmune hepatitis, hypersensitivity lung diseases, hypersensitivity pneumonitis, delayed-type hypersensitivity, interstitial lung disease (ILD), scleroderma, dermatitis, iritis, conjunctivitis, keratoconjunctivitis, cutaneous lupus erythematosus, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Graves ophthalmopathy, amyotrophic lateral sclerosis (ALS), primary biliary cirrhosis, ileitis, chronic inflammatory intestinal disease, celiac disease, irritable bowel syndrome, neurodegenerative diseases, ataxiatelangiectasia, asthma, psoriasis, atherosclerosis, and combination of any of the above, said inflammatory bowel diseases comprising Crohn's and ulcerative colitis, said interstitial lung disease (ILD) comprising idiopathic pulmonary fibrosis and ILD associated with rheumatoid arthritis, said Dermatitis comprising atopic dermatitis and eczematous dermatitis and said neurodegenerative disease comprising MS (multiple sclerisis).

An important facet of the invention is the consideration of toxic effects caused by administration of high doses of the therapeutic proteins of the invention. The introduction of high concentrations of any compound, particularly the bacterial irnrnuno-modulatory effector proteins of the invention, into cells may be cytotoxic, and particularly so in the case of effector proteins which may fatally block signaling pathways when present in high intracellular levels. The invention herein overcomes this obstacle by providing several effector proteins which act in different points along signal transduction pathways. Thus, the combined effect elicited by the activity of the effector proteins may be an additive effect, where the total effect is the sum of all inhibitory effects afforded by the effector proteins, or a synergistic effect, where the total effect exceeds the sum of all inhibitory effects afforded by the effector proteins. These effects are accomplished without producing significant toxic effects since each effector is present in sub-toxic levels within the target cells, and while exerting the desired inhibitory effect, possibly in an additive or synergistic manner with other effector proteins, it does not reach cytotoxicity threshold. A non-limiting example of such an effect is the combination of NIeE, NIeC and NIeB, which inhibit IKB phosphorylation via inhibition of IKKβ activation, and p65 activity by its degradation. Combinations of at least one of these proteins with NIeD also target the JNK and p38 signaling pathways.

In some embodiments, the method for the prevention, treatment, or amelioration an immune-related disorder of the invention concerns improvement of at least one undesired manifestation of the disease, comprising an increase in disease free periods, decrease in acute disease periods (in time and severely), decrease in severity of the disease, improvement in life quality, decreased mortality, decrease in the rate of disease progression as well as prophylactic treatment before disease occurs.

Since the method of the invention involve the use of attenuated live microorganism, specifically, bacteria, it will be understood that such methods may optionally comprise an additional step of treatment termination by addition of antibiotics for elimination of the microorganism comprising the delivery system of the invention, or the withdrawal of an ingredient that is necessary to the microorganism.

As contemplated by the invention, the administration step of the above method comprises oral, intravenous, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

The recognition and targeting of a certain tissue by the delivery system of the invention is based on the specific recognition and colonization of a target tissue by a specific attenuated microorganism, specifically, bacteria used by the invention.

The invention therefore provides in a further aspect, a method for tissue targeted delivery of an immunomodulatory agent. The targeting method of the invention comprises the step of administering to a subject in need thereof an immuno-modulatory effective amount of a tissue-targeted delivery system of an immunomodulatory protein or any composition comprising the same. The delivery system used by the targeting method is according to the invention and described herein.

As mentioned above, tissue targeting of the delivery system of the invention is based on the specific recognition and colonization of a target tissue by a specific microorganism used, specifically, the differential bacterial tropism. Examples of TTSS bacteria and the tissues they recognize include for example Yesinia enterocolitica, which is targeted to the lymph nodes and Salmonella typhi that is targeted to the liver and spleen. EPEC is targeted preferably to the small intestine while EHEC is targeted preferably to the colon. Therefore, an important aspect of the invention is the use of a tissue-targeted delivery system of the invention for specific delivery of different combinations of the immunomodulatory proteins of the invention for treating different immune-related disorders. Thus, in one specific embodiment, the targeting method of the invention may use a delivery system comprising attenuated EPEC bacteria expressing any combination of the NIeE, NIeC, NIeB and NIeD proteins of the invention or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof for treating GI (gastro intestinal) associated inflammatory disorders, such as IBD.

It should be noted that any of the methods of the invention may also involve use of at least one additional therapeutic agent for enhancement and completion of the therapeutic effect.

The various compositions and methods of the invention use the terms "target cells" and "target tissues" as cells or cells in a specific tissue which are susceptible to the introduction of the immunomodulatory proteins of the invention through the TTSS- expressing microorganisms of the invention, or through any other means. It should be appreciated that such cells may be any one of cell culture, tissues or organs, in vivo or in vitro. In certain specific embodiment such target cells may be intestinal epithelial cells. The target cells are the cells where modulation of intracllular signaling by the proteins of the invention is desirable. ,

The present invention contemplates a delivery system for inroducing the effector proteins of the invention, and optionally, additional other therapeutic proteins into target cells. However, it should be appreciated that the therapeutic compositions of the invention may also be introduced into target cells by alternative means. For example, liposomes may be used to deliver therapeutic agents to target cells. The term "liposome" as referred to herein, means closed vesicles composed of a lipid assembly in a membrane form and an aqueous phase within the membrane. The liposome used in the present invention is not particularly limited. Details of the preparation of the liposome is described in JP Patent Publication (Kokai) No. 9-208599A (1997) and so on. Furthermore, the liposome according to the present invention may comprise a substance that improves particular cell (target cell) tropism for the purpose of specifically injecting a substance to the particular cell. Examples of the substance that improves target cell tropism can include, but not particularly limited to: antibodies; antibody fragments; sugar chains as ligands against sugar chain receptors on cell surfaces, such as glucose, galactose, mannose, and fucose; sialic acid and derivatives thereof; transferrin and derivatives thereof as ligands against peptide receptors on cell surfaces; and folic acid derivatives against folic acid receptors. Most prepferably, liposomes of the invention will comprise a sustance that improves targeting of the liposomes to epithelia of either the small intestine, the colon, or both.

The liposome according to the present invention comprises any combination of the therapeutic proteins of the invention, and optionally additional therapeutic proteins and therapeutic agents therewithin. Most preferably, the intended compounds carried by the liposomes and introduced into the target cells are the compositions of the invention, most preferably, said inteneded compounds comprising at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory bacterial proteins and any functional homologies, variants, fragments, derivatives, mixtures and any combinations thereof, and optionally further comprises at least one additional therapeutic protein or agent.

An important advantage of the use of liposomes is the ease with which one may customize their payload. Liposomes may be easily loaded with any one or a combination of the effector proteins of the invention, as well as other therapeutic agents or proteins. Thus, modularity of this therapeutic system is conveniently achieved using liposomes.

As described above, the immunomodulatory proteins of the present invention are generally administered in the form of a pharmaceutical composition comprising the therapeutic proteins of this invention that may be supplemented with a pharmaceutically acceptable carrier or diluent, and optionally an additional therapeutic protein and/or a therapeutic reagent. Alternatively, the immunomodulatory proteins of the present invention may be also comprised within a delivery system, targeting the immuno- modulatory proteins to specific cells, tissues or organs. Since the invention contemplates several therapeutic proteins and many combinations thereof, with or without other therapeutic proteins or agents, possibly delivered by various delivery systems, such as EPEC, EHEC, liposomes, or any other delivery system, it is clear that it would be advantageous to provide means to dispense each component of the system individually, or in specific pre-determined combinations. Thus, the immuno-modulatory proteins used by this invention can be administered either individually in a kit or together in any conventional oral or mucosal dosage form.

More particularly, since the present invention relates to the treatment of diseases and conditions with a combination of active ingredients which may be administered separately, the invention also relates as a further aspect, to combining separate pharmaceutical compositions in kit form. The kit includes at least two separate dosage forms: (a) at least one of the therapeutic proteins of the invention, which may be supplemented with other therapeutic protein/s of the invention or otherwise, and a pharmaceutically acceptable carrier or diluent in a dosage form. The dosage form may also comprise an appropriate delivery system; (b) optionally, at least one other therapeutic protein or agent, and a pharmaceutically acceptable carrier or diluent in another dosage form. This dosage form may also comprise an appropriate delivery system; and (c) container means for containing the various dosage forms. The kit may include two or more dosage forms, and optionally at least one additional therapeutic protein or agent, as some disorders may benefit from treatment with more complex combinations of the therapeutic proteins and agents of the invention. Thus, multiple dosage forms are contemplated, each containing at least one of the therapeutic proteins of the invention, which may be supplemented with other therapeutic protein/s or additional therapeutic agent/s and a pharmaceutically acceptable carrier or diluent.

Thus, according to certain embodiments, a kit provided by the invention may comprise: a. at least one of:

(i) NIeE protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeE protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form; (ii) NIeC protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeC protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form;

(iii) NIeD protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeD protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a third unit dosage form; (iv) NIeB protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeB protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a fourth unit dosage form; and optionally

b. at least one additional therapeutic protein, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said therapeutic protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a unit dosage form; and optionally

c. at least one additional therapeutic agent or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a unit dosage form; and d. container means for containing said unit dosage forms.

More specifically, the kit includes container means for containing separate compositions; such as a divided bottle or a divided foil packet however, the separate compositions may also be contained within a single, undivided container. Typically the kit includes directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

According to one embodiment, the kit of the invention is intended for achieving a therapeutic effect in a subject suffering from an immune-related disorder. Achieving a therapeutic effect is meant for example, where the kit is intended for the treatment of a specific disorder, the therapeutic effect may be for example slowing the progression of the treated condition.

The invention further provides a method of treating, ameliorating, preventing or delaying the onset of an immune-related disorder in a subject in need thereof comprising the step of administering to said subject a therapeutically effective amount of the unit dosage forms comprised in a kit according to the invention.

It should be appreciated that the multiple components of the kit may be administered simultaneously.

Alternatively, said multiple dosage forms are admim^'stered sequentially in either order.

More specifically, the kits described herein can include a composition as described, or in separate multiple dosage unit forms, as an already prepared liquid oral dosage form ready for administration or, alternatively, can include the composition as described as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral dosage form. When the kit includes a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral administration), the kit may optionally include a reconstituting solvent. In this case, the constituting or reconstituting solvent is combined with the active ingredient to provide liquid oral dosage forms of each of the active ingredients or of a combination thereof. Typically, the active ingredients are soluble in so the solvent and forms a solution. The solvent can be, e.g., water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to oils, alcohols, such as ethanol, glycerin, and glycols, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

Another aspect of the invention relates to an attenuated EPEC or EHEC pro-biotic bacteria expressing intact Tir (translocated intimin receptor) and Intimin encoding genes, wherein the attenuation is caused by deletion or inactivation of at least one of: a. at least one gene encoding type IV pilli and type I pilli;

b. a gene encoding the effector Map (Mitochondrial-associated protein). It should be noted that in some embodiments any other bacterial effector may be deleted. Examples for such effectors include EspF, EspG, EspG2, EspH, EspZ, EspJ, EspL2, NIeA, NIeF, NIeG; and

c. genes encoding exotoxins selected from the group shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins, hemolysin, EspE, EspP and LifA;

In particular embodiments, the attenuated bacteria express an intact TTSS and at least one effector protein, specifically, effectors selected from NIeE, NIeC, NIeD and NIeB (also denoted by SEQ ID NOs. :2 OR 90, 4, 6 and 8, respectively), and,optionally, any one of NIeA, NIeBl, NleB2, NIeEl, NleE2, NIeF, NIeG, NIeHl and NleH2.

Finally, the last aspect of the invention relates to a nutraceutical composition comprising as an active ingredient an effective amount of attenuated EPEC or EHEC pro-biotic bacteria expressing intact Tir (translocated intimin receptor) and Intimin encoding genes, wherein the attenuation is caused by deletion or inactivation of at least one of:

a. at least one gene encoding at least one of type IV pilli and type I pilli;

b. a gene encoding the effector Map (Mitochondrial-associated protein), and optionally any further effector; and

c. genes encoding exotoxins selected from the group shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins and hemolysin; said composition optionally further comprises a pharmaceutically acceptable carrier.

According to one specific embodiment, the attenuated EPEC or EHEC comprised within the nutraceutical composition of the invention express at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory effector proteins of the invention.

The present invention provides a nutraceutical composition comprising as an active ingredient an effective amount of attenuated EPEC or EHEC probiotic bacteria. The term "probiotic" microorganisms or bacteria (probiotics) relates to living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond basic nutrition. The beneficial effects that probiotics may induce are numerous. Few examples are: the modulation of host immune functions, the reduction of diarrhea, the reduction of lactose intolerance, the prevention of colon cancer, the improvement or prevention of constipation, the in situ production of vitamins, and the modulation of blood lipids. In domesticated and aquatic animals they also can improve growth, survival and stress resistance associated with diseases and unfavorable culture conditions. Therefore, there is considerable interest in including probiotics into human foodstuffs and into animal feed. The term "nutraceutical" is intended to encompass any consumable matter intended for the consumption by humans or by animals, such as domesticated animals, for example cattle, horses, pigs, sheep, goats, and the like, that provides health and medical benefits, including the prevention and treatment of disease. In some embodiments, the nutraceutical composition disclosed herein includes standard food products, pelleted feeds and pet food (for example a snack bar, crunchy treat, cereal bar, snack, biscuit, pet chew, pet food, and pelleted or flaked feed for aquatic animals). In preferred embodiments, the nutraceutical composition disclosed herein includes probiotic beverages, such as yoghurts, fruit juice, fermented beverages such as kefirs, coconut juice, wheat juice and others. Different known methods can achieve a long shelf-life while maintaining high levels of bacterial viability. The probiotic bacteria may be added to the beverage in, for example, freeze- dried or frozen form.

The probiotic micro-organisms are preferably mixed in a concentrated wet paste form or frozen paste form (for example a probiotic paste of >10% solids) with the other protective substances. Microorganisms may also be mixed, directly after fermentation, with the protective components described herein followed by hydrogel formation and a drying process thereafter. For example, probiotic micro-organisms are mixed with the protective materials such as a saccharide, for example trehalose, sucrose, lactose or maltodextrin, a protein, for example egg albumen, soy protein isolate or hydrolysate either alone or in combination and a polysaccharide, for example, agarose, alginate or chitosan either alone or in combination. A hydrogel is then formed in a desired shape and size or sliced after hardening the gel according to established procedures known to persons skilled in the art. If micro-matrix particles are required, then the hydrogel can be sliced or extruded and then dried using a variety of drying techniques, for example fluidized bed drying, freeze drying, air drying, convention oven drying or another adequate drying process. The dry probiotic substance is then ground and sieved to preferred sizes. If flakes or treats are required, then the molded or otherwise pre-shaped or sliced hydrogel is preferably dried in a vacuum drier or freeze drier at a temperature above the freezing point of the hydrogel. The pre-shaped dried flake or treat is then ready for packaging alone or in combination with other food products.

The bacteria that are used to make the compositions and delivery systems of the invention are preferably those that infect via the oral route. The bacteria may be those that colonize mucosal surfaces, preferably GI tract mucosal surfaces. The bacteria are generally Gram- negative. Non-limiting examples of the bacteria used in the invention are EPEC or EHEC from the genera Escherichia.

The bacteria used in the nutraceutical compositions and the delivery systems of the invention are "non-virulent" and/or "attenuated". Such bacteria preferably contain a mutation in one or more genes that encode virulence factors, preferably more. This is so that the risk of the bacterium reverting to the virulent state is minimized which is clearly important for the use of the bacterium as a human or animal pharmaceutical composition. It will generally be desirable to introduce more than one mutation so as to reduce the risk of attenuation yet further

A number of genes that are candidates for second and further mutations are known. Some non-limiting examples include, genes encoding type IV pilli and type I pilli, a gene encoding the effector Map (Mitochondrial-associated protein) and genes encoding exotoxins such as shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins, hemolysin, EspC and EspP. It should be noted that these attenuated bacteria may carry mutations in other effectors, for example, any one or at least one of EspF, EspG, EspG2, EspH, EspZ, EspJ, EspL2, NIeA, NIeBl, NleB2, NIeC, NIeD, NIeEl, NleE2, NIeF, NIeG, NIeHl and NleH2.

The mutations are non-reverting mutations. These are mutations that show essentially no reversion back to the wild-type when the bacterium is used as pro-biotic bacteria. Such mutations include insertions and deletions. Insertions and deletions are preferably large, typically at least 10 nucleotides in length, for example from 10 to 600 nucleotides. The bacterium used in the invention preferably contains only defined mutations, i.e. mutations which are characterized. The attenuating mutations may be constructed by methods well known to those skilled in the art. For example, cloning the DNA sequence of the wild- type gene into a vector, e.g. a plasmid or cosmid, and inserting a selectable marker into the cloned DNA sequence or deleting a part of the DNA sequence, resulting in its inactivation. A deletion may be introduced by, for example, cutting the DNA sequence using restriction enzymes that cut at two points in the coding sequence and ligating together the two ends in the remaining sequence. A plasmid carrying the inactivated DNA sequence can be transformed into the bacterium by known techniques. It is then possible by suitable selection to identify a mutant wherein the inactivated DNA sequence has recombined into the chromosome of the bacterium and the wild-type DNA sequence has been rendered non-functional in a process known as homologous recombination.

The pro-biotic bacteria of the invention are non-virulent. The term "virulence" or "virulent", as used herein, refers to the degree of pathogenicity of a microorganism as indicated by case fatality rates and/or its ability to invade the tissues of the host; the competence of any infectious agent to produce pathological effects. The ability of bacteria to cause disease is described in terms of the number of infecting bacteria, the route of entry into the body, the effects of host defense mechanisms and bacterial virulence factors. Host-mediated pathogenesis is often important because the host can respond aggressively to infection with the result that host defense mechanisms do damage to host tissues while the infection is being countered.

The virulence factors of bacteria are typically proteins or other molecules that are synthesized by protein enzymes. These proteins are coded for by genes in chromosomal DNA, bacteriophage DNA or plasmids. Bacteria use quorum sensing to synchronise release of the molecules. These are all proximate causes of morbidity in the host.

In specific embodiments, the nutraceutical composition of the invention further comprises additional pro-biotic bacteria.

In other embodiments, the pro bacteria further comprised in the nutraceutical composition of the invention is selected from the group consisting of: Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bifidus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus delbruekii, Lactobacillus thermophilus, Lactobacillus fermentii, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus gasseri, Pediococcus cerevisiae, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, Bifidobacterium lactis, Bifidobacterium bulgaricus, Bifidobacterium breve, Bifidobacterium subtilis, strains of the genera Bacillus, Bacteroides, Enterococcus and Leuconostoc, and combinations thereof

It should be further noted that the nutraceutical composition of the invention may further comprise an additional component selected from the group consisting of prebiotics; fiber; vitamins; minerals; metals; elements; plant-derived components; fungal-derived components, carotenoids; anti-oxidants and combinations thereof.

The invention further concerns a transgenic non- virulent/attenuated TTSS-expressing bacteria comprising heterologous nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, said transgenic bacteria optionally further comprises at least one heterologous nucleic acid sequence encoding an additional therapeutic protein, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include site-directed mutagenesis, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology ,by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3^rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, NY.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention. EXAMPLES

Experimental Procedures

Bacterial strains, plasmids, and primers

Bacterial strains, plasmids, and primers used in this study are listed in Table 1, 2, and 3A- 3B, respectively. Deletions in the EPEC chromosome were constructed using the primers listed in Tables 3A-3B, as described [Datsenko, K.A. and Wanner, B.L., Proc Natl Acad Sci U S A 97(12):6640-6645 (2000)]. For bacterial expression, the genes were cloned in pSAlO, pCX341, pET28a or pET52 as described [Nadler, C, et al., Infect Immun 74(2): 839-849 (2006)]. For this cloning procedure, genes were amplified by PCR, using the primers listed in Tables 3A and 3B. Formation of plasmid-borne nleE-blaM fusions were carried out as described [Mills, E., et al., Cell Host Microbe 3(2): 104-113 (2008)] using the plasmid pCX341 and primers as listed in Table 3 A. The

DNA was amplified from EPEC ΔIE6 mutant and that of nleE_m from EPEC ΔIE2 mutant.

For formation of mCherry fusions, the EGFP gene of pEGFP-Nl (Clonetech) was excised from the plasmid using the Notl and BamHI and replaced by mCherry taken from pREST- mCherry, resulting in pMS2841. This plasmid was transformed intopSC4141 by introduction of a unique seal site at the mCherry 3 ', eliminating its stop codon in the process. This was done using QuikChange site-directed mutagenesis kit (Stratagene #200518-5) and primers listed at Table S3. This plasmid was further used to create the transcriptional fusion: mCherry-nleEIE6-6his (pSC4144) and mCherry-nleEIE2-6his (pSC4350), using primers listed at Table 3A.

References [1], [2] and [3] relate to [Jarvis, K.G., et al., Proc Natl Acad Sci U S A 92(17):7996-8000 (1995)], [Nadler, C, et al., Infect Immun 74(2):839-849 (2006)] and the present invention, respectively.

Table IB: Bacterial strains used in the study ofnleC and nleD

References [2], [3], [4] and [5] relate to [Nadler, C, et al., Infect Immun 74(2):839-849 (2006)], the present invention, [Nadler, C, et al., PLoS Pathogens 6(l):el000743 (2010)] and [Wiles, S., et al., Cell Microbiol 6(10):963-972 (2004)], respectively.

Table 2A: Plasmids used in the study ofnleE and nleB

References [3], [6] and [7] relate to the present invention, [Schlosser-Silverman, E., et al., J Bacteriol 182(18):5225-5230 (2000)] and [Mills, E., et al., Cell Host Microbe 3(2):104- 113 (2008)], respectively. Table 2B: Plasmids used in the study ofnleC and nleD

References [3], [6], [7] and [8] relate to the present invention, [Schlosser-Silverman, E., et al., J Bacteriol 182(18):5225-5230 (2000)], [Mills, E., et al., Cell Host Microbe 3(2): 104- 113 (2008)] and [Kallunki et al., Genes Dev 8: 2996-3007 (1994)], respectively.

F - forward; R - reverse. Table 3B: Primers used in the analysis ofnleC and nleD

F - forward; R - reverse.

Protein extraction. Western blot analysis and immunoprecipitation

Protein extraction and Western blot analysis were performed as described [Nadler, et al., Infect Immun 74(2):839-849 (2006)]. Blots were developed using one of the following antibodies: anti-p65-C-terminus domain (SC-372, Santa Cruz, 1 : 1000 in TBS), anti-p65- N-terminus domain (SC-109, Santa Cruz, 1 :1000 in TBS), anti-JNK (554285, BD Pharmingen, 1 : 1000 in TBS), anti-HA, anti-6His (27471001, GE Healthcare, 1 :3000 in TBS), anti-ERK (m5670, Sigma, 1 :2500 in TBS), anti-c-Jun (H79, SCBT), anti-phospho- c-Jun (Cell Signaling), Agarose-protein-A (Sigma) beads coated with anti-HA or anti- FLAG antibody were used for immunoprecipitation. Analysis of IKB stability and IKB and IKKβ phosphorylation

HeLa cells (9X10⁵) in 4 cm plates were infected with a 1 :100 dilution in DMEM of bacteria grown overnight statically at 37°C (multiplicity of infection, MOI -1:100). Following a 3 hours infection in 5% CO₂, at 37°C, the medium was replaced with fresh DMEM with or without 10 ng/ml TNFα for 40 min. When indicated, the infecting bacteria were supplemented with 0.01 mM IPTG at 1.5 hours post inoculation. To terminate the infection, cells were washed with 3 ml of cold TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), scraped with 1 ml of cold TBS, collected and centrifuged, (800xg, 2 minutes at 4°C). The pellet was resuspended in 40 μl lysis buffer (0.5% Triton-XlOO, 2OmM Tris-HCl pH 7.2, 0.2 mM VO₄, 10 mM NaF, 30 μl of complete inhibitor Roche) and centrifuged (20,000xg, 3 minutes at 4°C). Supernatant was either transferred for protein quantification assay (BCA assay) or to a tube with loading dye (LDS sample buffer, NuPAGE), boiled for 10 min, and then centrifuged (20,000xg, 3 min). Quantification of samples was performed using bicinchoninic acid (BCA) and copper sulfate. Equal protein concentration for each sample was then loaded on SDS-PAGE gel, transferred to PVDF membrane, and reacted with antibodies against IKB (1:1000), Tubulin (1:2500), or phospho-IκB (1:1000, Cell Signaling). When indicated, 20 mM MG132 (1:1000) was used. Protein band density was quantified using TINA software (version 2.09) and the percentage of the unphosphorylated IKB was determined by calculating the relative phosphorylated IKB out of the total IKB shown for each lane. IKKβ analysis was done as described for IKB, except that induction time with TNFα was reduced to 10 minutes and with IL lβ it remained 20 minutes. IKK detection was preformed by western blot analysis using anti-IKKβ antibody (1:1000, Cell Signaling Technology, #2684) and Phospho-IKKα (Serl80)/IKKβ (Serl81) antibody (1:1000, Cell Signaling Technology, #268 IS).

Nuclear cytoplasmic fractionation

HeLa cells (2.8><10⁶) were seeded in 10 cm plates. The next day, the cells were infected with EPEC for 3.5 hours as described. Cells were then washed, treated with 20 ng/ml TNFα in DMEM for 30 min., washed with cold PBS, scrapped, transferred to Eppendorff tubes and centrifuged (5 minutes, 660 g, 4°C). Then, the pellet was resuspended in 7 times the volume of Hypotonic Lysis Buffer (HLB, 1OmM HEPES pH 7.6, 0.ImM EDTA, 0.1 mM EGTA, 2mM DTT, 1OmM KCl, ImM PMSF, 0.75mM Spermidine, 0.15mM Sperimide, 2OmM PNPP, lμM okadaic acid and 5μg/ml protease inhibitor), incubated on ice for 15 minutes and then 0.2% NP40 was added gently following gentle mixing for several minutes. The lysate was then centrifuged (5 minutes, 2600 g, 4°C), the supernatant (cytoplasmic fraction) was recovered and the pellet (nuclear fraction) was washed with HLB once and then resuspended in 100 μl Nuclear Extraction Buffer (NEB, 210 mM HEPES pH 7.6, 0.2 mM EDTA, 2 mM EGTA, 0.5 mM DTT, 25% Glycerol, 0.42 M NaCl, 20 mM glycerophosphate, 29 mM PNPP, 1 μM okadiac acid, 1 mM NaVO4, 5 μg/ml protease inhibitor, 0.75 mM Sperimidine, 0.15 mM Sperimide). The nuclear lysets were then vortexed, mixed vigorously (1400 rpm, 30 min., 4°C) and clarified (20,000 g, 10 min, 4°C). Protein concentrations were determined (BCA kit, Sigma), adjusted and the extracts were used for western analysis using anti-NF-κB p65 antibodies (Santa Cruz, SC372). The quality of the fractionation was confirmed using tubulin as a cytoplasmic marker and fibrillarin as a nuclear marker.

Expression and translocation of NIeE-BIaM fusions

To determine translocation levels, overnight cultures of wild-type EPEC containing plasmids expressing NIeE-BIaM were diluted 1:50 in DMEM and used to infect HeLa cells for 3 h. Cells were then washed and stained with CCF2 for 2.5 hours as described [Charpentier, X. and Oswald, E., J Bacteriol 186(16):5486-5495 (2004)], washed in cDMEM, excited at 405 nm, and then emission at 465 nm and 535 nm was recorded (SPECTRAFluor, TECAN). The amount of translocation was determined as described [Charpentier, X. and Oswald, E., J Bacteriol 186(16):5486-5495 (2004)]. As a negative control, the inventors used EPEC expressing unfused BIaM (Vector). To determine expression levels, the unattached bacteria were harvested, washed, and lysed by repeated freezing and thawing in PBS containing 1 mM EDTA, 1 mg/ml lysozyme, and 0.1% Triton-X100. The BIaM activity in the lysate was determined using nitrocefm as substrate and the rate of product accumulation per number of bacteria (OD 600) was determined as described [Mills, E., et al., Cell Host Microbe 3(2): 104-113 (2008)].

IL-8 secretion assay

For the NIeE and B experiments disclosed by Examples 4 and 5, HeLa cells (7*10^s) in 6 wells plates were inoculated with a 1:100 dilution in DMEM of bacteria grown overnight statically at 37°C (multiplicity of infection, MOI-1: 100) and incubated for 3 h (5% CO₂, 37°C). To terminate the infection and induce IL8 expression, the medium was replaced with fresh DMEM supplemented with 2% FCS, lOOug/ul gentamicin and with or without 10 ng/ml TNFα and incubated for additional 3 h. Cells were than washed with 2 ml of cold TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), scraped with 1 ml of cold TBS, collected and centrifuged, (800 g, 2 minutes, 4°C). RNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (EPICENTRE Biotechnologies) and used to synthesize cDNA with the Verso cDNA kit (Thermo scientific). hHPRT transcript levels were used to normalize total RNA levels in samples. Real time analysis was than conducted using Absolute Blue QPCR SYBR Green (Thermo scientific) in a real-time cycler (Rotor-Gene 6000, Corbett).

For the NIeC and D experiments disclosed by Examples 15, 16 and 19, HeLa cells (8X10⁴) were seeded in 24-well plate. The next day, the cells were infected with EPEC culture as described above. After 3.5 h, supernatants were replaced with 300 μl DMEM, 2% FCS, and 50 μg/ml gentamycin with or withoutlO ng/ml TNFα. After 16 h, 100 μl of cleared supernatant taken from each well was used for IL-8 measurements using Human CXCL8/IL-8 Quantikine immunoassay assay (R & D), according to the manufacturer's instructions.

Transfection of HeLa cells and p65 staining

HeLa cells were transfected with 1 μg of plasmid DNA using ExGen500 (Fermentas), as recommended by the manufacturer, or were not transfected. After 24 h, the medium was replaced with fresh DMEM containing, or not containing, 10 ng/ml TNFα. After 1 h, cells were fixed (3.7% PFA in PBS for 10 minutes and washed with PBS), perforated (with 0.25% Triton-XIOO in PBS for 10 minutes and washed twice with PBS) and blocked (2% BSA in TBS) at 4°C for 16 hours. Cells were then stained using anti-p65 (SC 109, Cell Signaling) antibodies (1 :300 in TBS) overnight and further stained with CY-488 goat anti- rabbit (Cell Signaling) (1:1000 in TBS) for 1 h. Slides were analyzed by fluorescent microscopy.

Purification of NIeD. NIeC and NIeC-El 84A

E. coli BL21 (DE3) expressing His-tagged proteins were grown to OD ~ 0.3. Expression was induced by IPTG (1 mM) and cultures were grown at 20°C for 18 hours. Alternatively, expression was induced by the autoinduction method at the same physical settings [Studier, F. W., Protein Expr Purif 41(l):207-234 (2005)]. Cells were collected (6000 rpm, 7 min., 4°C) and resuspended in binding buffer (20 mM imidazole, 300 mM NaCl, 20 mM Tris, pH 8, 0.02% Triton X-100 and 6 mM or 3 mM or no β- mercaptoethanol). Suspensions were lysed with French Press (Thermo Scientific), cleared (45,000 rpm, 45 min., 4°C) and protein were bound to Ni-NTA beads (Novagen), followed by 3 washing steps on a dripping column (step 1 buffer: 300 mM NaCl, 20 mM imidazole, 20 mM Tris, pH 8, step 2 buffer: 600 mM NaCl, 30 mM imidazole, 20 mM Tris, pH 8 and step 3 buffer: 300 mM NaCl, 40 mM imidazole, 20 mM Tris, pH 8). Elution was performed with elution buffer (50 mM NaCl, 300 mM imidazole, 20 mM Tris, pH 8) and eluted samples were equilibrated overnight against dialysis buffer (50 mM NaCl, 20 mM Tris, pH 8) at 4° C with 5000 MW cutoff SnakeSkin^® dialysis tubes (Thermo Scientific). Equilibrated samples were purified with Mono-Q anion exchange column (GE healthcare) utilizing linear gradient with elution buffer (500 mM NaCl, 20 mM Tris, pH 8). Selected fractions were further purified by size exclusion column (GE healthcare) equilibrated against 50 mM NaCl, 20 mM Tris, pH 8. The collected fractions were pooled and concentrated to 44 mg-ml^-1, 34.5 mg-ml^'1, and lβmg-mT¹ (NIeC, NIeC- E 184 A and NIeD, respectively).

In vitro assay for NIeD and NIeC activity.

Purified recombinant JNK2 (15 μg, a gift from D. Engeleberg the Hebrew University) was mixed with purified NIeD (0.375 μg) in 40 μl of reaction buffer (50 mM Tris-HCl pH 7.5, 2 mM CaCl₂, 50 mM NaCl). In the case of NIeC, purified NIeC (0.4 μg) was mixed with cleared HeLa cells extracts in 20 μl of reaction buffer (5OmM Tris pH 7.5, 2 mM CaCl₂, 5mM NaCl). Where specified, 1, 10-phenanthroline was added (5 mM, Sigma). Reactions were carried out for the indicated period of time at room temperature and stopped by the addition of SDS loading buffer. Digestion products were visualized using Western blot analysis or SDS-PAGE followed by coomassie staining. Example 1

EPEC inhibit IKB degradation andNF-κB activation by a TTSS-dependent mechanism

The ability of EPEC to either inhibit or induce NF-κB activation is controversial. Therefore, this point was re-examined using HeLa cells as host cells and IKB stability as a read-out for NF-κB activation. Importantly, TNFα treatment strongly stimulates NF-κB activation in these cells, but they exhibit minimal NF-κB activation upon stimulation with bacterial PAMPs, thus allowing the uncoupling of infection and NF-κB activation. HeLa cells were infected with EPEC culture for 3 h, during which the bacteria injected the TTSS effectors into host cells. The infected cells were then treated with 10 ng/ml TNFα to activate the NF-κB and at different time points post TNFα-induction, cellular lysates were subjected to western analysis. As shown by Figure IA, TNFα treatment induced rapid degradation of IKB in uninfected cells (N/I) or cells infected with EPEC TTSS-deficient mutant (escN::kaή). In contrast, IKB in cells infected with wild-type EPEC remained stable.

Next, the inventors assessed whether the stabilization of IKB by EPEC was associated with inhibition of NF-κB translocation to the nucleus. To monitor NF-κB activation, a reporter cell line (AGS SIB02) stably expressing the NF-κB subunit p65 fused to GFP was used. Cells were infected with wild-type EPEC or, as a negative control, with EPEC TTSS mutant (escV::kan). After 3 hours of infection, cells were washed, induced with TNFα, and at 15, 45, 60, and 75 minutes post TNFα-induction, the cells were fractionated into cytoplasmic and nucleus fractions and the amount of p65 in the different fractions was determined by immunoblot using anti-p65 antibody. As shown by Figure IB, wild type EPEC, but not the esc V mutant, blocked translocation of p65 to the nucleus, thus supporting the notion that EPEC inhibit NF-κB activation by a TTSS-dependent mechanism [Hauf, N. and Chakraborty, T., J Immunol 170(4):2074-2082 (2003); Maresca, M., et al., Cell Microbiol 7(12):1749-1762 (2005)], and suggesting that EPEC deliver into infected cells one or more effectors that inhibits NF-κB activation.

NIeH has been proposed as such an effector since it is similar to OspG, a Shigella effector that inhibits NF-κB activation [Kim, D.W., et al., Proc Natl Acad Sci U S A 102(39):14046-14051 (2005)]. However, Figure 1C demonstrates that an EPEC strain, in which both nleH alleles were deleted, still inhibited IKB degradation, similarly to wild- type EPEC, suggesting that NIeH is not required for blocking IKB degradation under the experimental conditions used by the present invention.

Example 2

NleE and NleB are required for IKB stabilization

To identify putative effector(s) that block IKB degradation, a comparison of the genome of EPEC to that of non-pathogenic E. coli Kl 2 was undertaken, identifying large EPEC- specific regions that contain, or possibly contain, effector genes. Based on this comparison, a set of 15 EPEC strains, each deleted of one EPEC-specific large chromosomal region (Table 4) was constructed. Altogether, 770 EPEC-specific ORFs were deleted. The inventors then tested the capacity of each of the deleted strains to inhibit IKB degradation upon TNFα treatment. One of the strains, deleted of the IE6 region [Dean, P. and Kenny, B., Curr Opin Microbiol 12(l):101-109 (2009)], could not inhibit IKB degradation (data not shown). Further systematic deletion analysis, presented by Figures 2A and 2B, defined two effector-encoding genes, nleB and nleE, required for stabilizing IKB. Deletion of nleE strongly reduced the bacteria's capacity to stabilize IKB, but a complete deficiency in IKB stabilization was observed only in the strain deleted of both nleB and nleE (Fig. 2B). To corroborate the notion that NleE is required for IKB stabilization, the inventors complemented a strain deleted of the nleBΕ region with plasmids containing nleB, nleE, or nleBE, and found that expression of NleΕ, but not of NleB, partially restored ΕPΕC's capacity to stabilize IKB (Fig. 2C). Importantly, full IKB protection was achieved in strains expressing both NleB and NleΕ (Fig. 2C). A mutant expressing only NleB showed only low level of IKB protection (Fig. 2C). Taken together, these results suggest that NleB and NleΕ, located at the IΕ6 region, are necessary for stabilizing IKB and that this activity is contributed mainly by NleE (Fig. 2B). The inventors therefore focused their attention on NleE.

(a) the island nomenclature used by [Iguchi, A., et al., J Bacterid 191(l):347-354 (2009)] is in brackets, (b) based on [Iguchi, A., et al., J Bacteriol 191(l):347-354 (2009)].

Example 3

NleEIE2 is not required for IKB stabilization

EPEC encode two very similar nleE alleles. One allele, identified in the screen of the invention, is located in the IE6 region and the other is in the IE2 region [Iguchi, A., et al., J Bacteriol 191(l):347-354 (2009)]. It was initially found by the inventors that deletion of the IE6 region, but not of the IE2 region, caused deficiency in inhibition of IKB degradation (data not shown). However, the two proteins, NIeE_1E2 and NIeE_IE6, are identical, apart from an internal deletion of 56 residues in NIeEi_E2 (Fig. 3, and SEQ ID NOs.: 89 and 90, respectively), and this similarity between the two proteins urged the inventors to determine the activity of each of the two proteins. The inventors first tested their ability to complement IKB destabilization in a strain deleted of nleEIE6. To this end, each of them were expressed on a plasmid carrying an identical promoter and ribosomal binding site. The results of Figure 5A clearly showed that only NIeE_IE6, but not NIeEi_E2, was able to attenuate IKB degradation. These results indicate that NIeE_IE6 is either not active in the host cell or is not translocated into the host cell. To differentiate between these two possibilities, the abovementioned plasmid where both proteins were fused to the β-lactamase translocation reporter protein (BIaM) was used. The plasmids were introduced into EPEC and the ability to translocate them into infected cells was tested. It was found that both NIeEi_E2-BIaM and NIeE_IE6-BIaM were expressed at similar levels in the bacteria (Fig. 5B). Importantly, however, only NIeEiE₆ was translocated into the host cell (Fig. 5C), suggesting that NIeEi_E2 is a cryptic effector. Cumulatively, these results define NIeEi_E6, but not NIeE_IE6, as the effector needed for inhibition of IKB degradation. Example 4

NIeE is required for full inhibition of ^' TNFa-induced IL-8 expression

To further substantiate their results, the inventors used IL-8 expression as an additional read-out for NF-κB activation. Briefly, HeLa cells were infected with different EPEC strains or remained uninfected. Then, cells were washed and treated for 3 hours with TNFα and gentamycin, to kill the remaining bacteria. RNA was then extracted from the cells and the amount of produced IL-8 mRNA was measured by real time PCR. As shown by Figure 6 A, in comparison to non infected cells or cells infected with EPEC escV mutant, both wild type and the ΔIE2 mutant exhibit a -100 fold repression of IL8 expression (Fig. 6A). In contrast, the nleE mutant exhibited a partial, less then 10 fold, repression of IL8 expression and this was moderately complemented by plasmid expressing native NIeE. A more severe deficiency in repression of IL8 expression was exhibited by a double mutant nleBE (Fig. 7). Furthermore, a plasmid expressing nleBE restored IL8 repression to that seen in wild type EPEC (Fig. 7). Upon testing the amount of secreted IL8 protein instead of production of IL8 mRNA, similar results were obtained (Fig. 8). Taken together these results show that (i) NIeE is required for full inhibition of IL-8 expression, (ii) NIeB also contributes to this repression and iii) a putative TTSS effector(s), other then NIeB and NIeE might function in parallel to inhibit IL-8 expression.

Example 5

NIeE is required to inhibit the EPEC-induced IL8 expression

The IL8 expression assay was found to be much more sensitive then testing translocation to the nucleus or the IkB degradation assay. This is probably since the latter are very transient events while the mRNA tends to accumulate, increasing the signal/noise ratio. Interestingly, using the IL8 expression assay the inventors found that infection with the esc V mutant was sufficient to induce IL8 expression in HeLa cells, albeit not as strong as that induced by TNFα (data not shown). This activation is possibly via the activity of flagellin, LPS or other PAMPs. Thus, the inventors next asked whether NIeE also inhibits the EPEC-induced IL8 expression. To this end the experiment described in Fig. 6A was repeated, but TNFα was omitted. The experiment revealed that even the non infected cells produce certain levels of IL8 mRNA, but upon infection with EPEC esc V mutant, a -10 fold increase in IL8 expression was observed (Fig. 6B). In contrast, the EPEC wild type (or the ΔIE2 mutant) exhibited strong repression of the EPEC-induced IL8 expression. Importantly, the nleE mutant exhibited only a partial capacity to repress the self-induced IL8 expression. Similar results where observed when the double mutant nleBE was used instead of nleE mutant (Fig. 7). However, both the nleE or the nleBE, mutants were not as deficient in IL8 repression as the esc V mutant (Fig. 6B and 7). Thus, the inventors predict that additional putative effector might function in parallel to NIeB and NleE to repress IL8 expression. In conclusion, the results clearly show that i) EPEC mediate a TTSS- dependent repression of self-induced IL8 expression; and ii) NleE is required for full repression of the EPEC-induced IL8 expression.

Example 6

NleE, expressed by HeLa cells, inhibits NF-κB translocation to the nucleus

The inventors next examined whether NleE is sufficient for inhibition of NF-κB activation in the absence of the infecting bacteria and other putative effectors. To this end, the inventors constructed a vector expressing mCherry fused to NleE (mCherry-NleE) and used it for transient transfection of HeLa cells. As shown in Figure 9, untransfected cells (Fig. 9A) or cells transfected with either the mCherry-NleE vector or a vector expressing mCherry alone (Fig. 9B) were stimulated with TNFα for 1 hour, or remained untreated. Next, these cells were fixed, stained with anti-p65 antibody, and analyzed by fluorescent microscopy to determine both the ability of the transiently expressed NleE to inhibit TNFα-induced migration of p65 to the nucleus and to determine its localization in the expressing cells. The TNF treatment induced strong migration of p65 to the nucleus in the untransfected cells (Fig. 9A), and in cells transfected with the negative control vector (Fig. 9B two upper panels and 9C). As shown by Figures 9B (two lower panels) and Figure 9C, the transiently expressed mCherry-NleE induced a strong inhibition of p65 translocation to the nucleus. The expressed mCherry and mCherry-NleE were similarly distributed in the cells, predominantly in the cytoplasm (Fig. 9B). These results indicate that NIeEi_E6 is sufficient for inhibition of NF-κB migration to the nucleus presumably by IKB stabilization. The capacity of NleE to block NF-κB was next compared to that of NIeB. Therefore, HeLa cells were transfected with plasmids expressing only mCherry (indicated as mCherry), or mCherry fused to NleE (NleE) or NIeB (NIeB). The transfected cells were treated with TNFα and analyzed by microscopy as described in Figures 9B and 9C. As shown by Figure 9D, inhibition of nuclear translocation of NF-κB by NIeB is comparable with the inhibition caused by NIeE.

Similar analysis using NIeE_1E2 instead of NIeE₁E₆ show that while NIeEi_E6 inhibited p65 translocation to the nucleus, NIeEi_E2 lost this ability (Fig. 10), highlighting the importance for NIeE activity of the region between residues 49-115, which is deleted in NIeEi_E2 as indicated in Figure 3 and also denoted as SEQ ID NO.: 89.

Example 7

NIeE inhibits phosphorylation of IKB

Different NF-κB activating pathways converge at the level of IKK phosphorylation, which subsequently leads to IKB phosphorylation, targeting it to ubiquitination and proteasome-mediated degradation [Karin, M. and Ben-Neriah, Y., Annu Rev Immunol 18(621-663 (2000)]. The inventors thus tested whether NIeE inhibits the TNFα-induced IKB phosphorylation. Cells were infected with different EPEC strains followed by TNFα treatment. The levels of IKB and phospho-IκB were then determined by Immunoblot analysis with the appropriate antibodies and the relative accumulation of unphosphorylated IKB was determined. For a negative control, cells infected with the escN mutant, which cannot stabilize IKB, were used (Fig. HA). Indeed, in cells infected with this mutant IKB phosphorylation increased, followed by its degradation. However, the addition of proteasome inhibitor (MGl 32) resulted in accumulation of phosphorylated IKB (Fig. 11). As a parental strain, the ΔIE2 strain (WTΔIE2) was used. Like wild-type EPEC, ΔIE2 efficiently protected IKB from degradation (Fig. 1 IA and 1 IA). Importantly, the accumulated IKB in these cells was mostly unphosphorylated. In contrast, the corresponding nleE mutant failed to induce accumulation of unphosphorylated IKB, exhibiting a phenotype similar to that of the escN mutant (Fig. HA). Taken together, these results indicate that wild-type EPEC stabilizes IKB by preventing its phosphorylation and that NIeE is required for this activity. Indeed, complementing the AnIeE mutant with a plasmid expressing NIeE restored the bacteria's capacity to induce the accumulation of unphosphorylated IKB (Fig. 1 IA). These results suggest that one can restore the inability of the AnIeE mutant to prevent IKB degradation by two alternative approaches: (i) by treatment with proteosome inhibition, to inhibit phospho-IκB degradation, or (ii) by complementation with a plasmid expressing nleE, to block IKB phosphorylation. To compare the efficiency of these two treatments, the inventors infected HeLa cells with the ΔIE2 strain (WTΔIE2), AnIeE mutant, or with the AnIeE mutant complemented either by proteosome inhibitor (MGl 32) treatment upon TNFα induction, or by a plasmid expressing nleE. The results presented by Figure HB show that both treatments similarly stabilized the IKB. However, the first treatment led to a strong phosphorylation of the accumulated IKB whereas when NleE was added, the accumulated IKB remained unphosphorylated (Fig. 1 IB). These results further support the notion that NleE stabilizes IKB by inhibiting its phosphorylation.

Example 8

NleE inhibits IKKβ activation induced by TNFa or ILlβ

The signaling pathways induced by the TNF receptor (TNFR) is different from that induced by the ILl or TLR receptors, but both converge at the level of IKK activation by TAKl as demonstrated by Figure HC [Hayden, M.S. and Ghosh, S., Cell 132(3):344-362 (2008)]. The inhibition of the self-induced IL8 expression by NleE (Fig. 6B), suggests that NleE functions downstream to the pathways converging point. To directly test this prediction, the inventors tested whether EPEC is capable of inhibiting ILlβ-induced degradation of IKB. AS clearly shown by Figure HD, it was discovered that wild type EPEC, but not the nleE mutant, inhibited the ILlβ-induced IKB degradation. These results confirmed that NleE functions downstream to the signaling converging point. The inventors next tested whether NleE can block the phosphorylation and thus activation of IKKβ. To this end, the inventors extracted proteins from cells, which were infected with different strains and then treated with TNFα or ILlβ as indicated by Figure HE. The extracted proteins were subjected to Western analysis using anti-IKKβ, anti-phospho- IKK, anti-IκB and anti-phospho-IκB antibodies. The results presented by Figure HE show that treatment with either TNFα or ILlβ induced IKB and IKK phosphorylation in non infected cells or cells infected with the esc V mutant. It was also found that wild type EPEC, but not the nleE mutant, inhibited this IKK phosphorylation. The same inhibition is noted for the IkB phosphorylation, in the wildtype strain, However, due to IkB degradation, less protein is noted and thus its phosphorylation cannot be seen (Fig. 1 IE). Complementation with plasmid expressing wild type nleE allele only partially, but consistently restored the inhibition of IKKβ phosphorylation (Fig. HE). These results suggest that NIeE blocks activation of IKKβ. Taken together the results indicate that NIeE blocks the NF-κB signaling cascade downstream to the converging point of the TNFα and IL lβ signaling pathways, but upstream to IKB phosphorylation, possibly by direct blocking TAKl or IKKβ activation as illustrated by the scheme of Figure 12.

Example 9

EPEC induce JNK cleavage

TNFα induces activation of the MAP3K TAKl that in turn activates both IKKβ and JNK phosphorylation. The inventors showed in the previous examples that NIeE and NIeB block IKKβ activation. Therefore, the inventors tested whether these effectors inhibit also JNK activation. To this end, HeLa cells were infected with wild type or various mutant EPEC and tested for JNK activation. As clearly shown in Figure 13 A and 14A) EPEC induced cleavage of JNK in a TTSS-dependent, but NleBE-independent manner. In contrast, Figure 14B demonstrates that the closely related MAP kinase, ERK, remained unaffected upon EPEC infection. Thus, the inventors concluded that EPEC inject into the infected cells effector protein(s) other then NIeE and NIeB that specifically destabilize JNK. This finding corroborates similar previous observations [Ruchaud-Sparagano et ah, Cell Microbiol 9:1909-1921 (2007)].

Example 10

NleD is required for JNK clipping

To identify the bacterial gene encoding for the effector that clips JNK, the inventors screened a collection of mutant EPEC with large chromosomal deletions presented by Table IB, and found that deletion of the PP4 prophage of EPEC [Iguchi, A., et al., J Bacteriol 191(l):347-354 (2009)], renders EPEC deficient in inducing JNK cleavage, as seen in Figure 14 A. In contrast, mutants with deletion of other chromosomal regions induced JNK degradation as effectively as wild type EPEC as also shown by Fig. 14 A. More detailed deletion analysis depicted in Figure 13A identified the nleD gene as required for JNK clipping. As shown by Figure 15, NIeD contains a conserved motif; HEXXH, also denoted by SEQ ID NO.:9, typical for Zn metalloproteinase. This motif is conserved in other homo logs of NIeD shown by Figure 15. To test whether this motif is required for JNK clipping, the inventors complemented the nleD deletion mutant with plasmids expressing either wild type NleD, or a mutated NleD, where the glutamic acid of the HEXXH motif (SEQ ID NO.: 9), was replaced by alanine (NIeD-E 143A). As shown by Figure 13 A, the wild type NleD, but not NIeD-E 143 A, restored the capacity of EPEC to induce JNK clipping. Analysis of the clipping kinetics, presented in Figures 13B and 14C, showed that the JNK clipping initiated 30 minutes after infection, and after 150 minutes intact JNK could no longer be detected in the infected cells. Furthermore, Figure 14D shows that the NleD homologues in other AE pathogens are also involved in JNK clipping. Taken together, the results show that i) NleD is required for the EPEC-induced cleavage of JNK and ii) it is likely that NleD is Zn metalloprotase that cleaves JNK.

Example 11

Ectopically-expressed NIeD cleaves native JNK

To examine whether NIeD is sufficient for JNK clipping, a mammalian expression vector expressing mCherry fused to NIeD (mCherry-NleD) was constructed and used for transient transfection of HeLa cells, thus testing the ability of these fusion proteins to cleave endogenous JNK. As controls, cells transfected with plasmids expressing mCherry or mCherry fused to NIeD-E 143E mutant were used. Proteins were next extracted from the transfected cells and analyzed by Western immunoblot using anti-JNK antibodies. As shown by Figure 13 C, the ectopically-expressed NIeD induced partial clipping of JNK. The relatively low JNK clipping found in this experiment probably reflected the fact that only -10-20% of the cells were transfected, as evaluated by microscopic examination of the percentage of mCherry expressing cells. In contrast, expression of mCherry-NleD- E 143 A was not associated with JNK clipping and generation of JNK fragments (Fig. 13C). Notably, the mCherry, mCherry-NleD and mCherry-NleD-E143A, were similarly distributed in the HeLa cells, suggesting that NIeD is localized to the host cell cytoplasm. These results indicate that NIeD is sufficient for JNK clipping and additional EPEC factors are not required for this activity.

To determine whether the cleavage inactivates JNK, the inventors next examined if NIeD expression affects the phosphorylation state of JNK targets. The experiment focused on a key JNK target, c-Jun, which is phosphorylated by JNK at serine 63/73 and thronine 91/93 following exposure to UV radiation [Derijard et al., Cell 76:1025-1037 (1994); Hibi et al., Genes Dev 7:2135-2148 (1993); Yogev et al., Cancer Res 68: 1398-1406 (2008)]. HEK293 cells transfected with control vector, or plasmids expressing either NIeD or NIeD-E 143 A, were UV irradiated and then c-Jun phosphorylation levels evaluated. As shown by Figure 13D, expression of NIeD reduced significantly the levels of c-Jun phosphorylation, whereas expression of the NIeD-E 143 A mutant had little effect. These results indicate that NIeD both cleaves and inactivates JNK. Example 12

NIeD activity is independent of mammalian host cell factor

Next, the inventors examined whether or not NIeD requires a co-factor, or an activation step in the mammalian host cells. To this end, two plasmids were co-transformed into E. coli laboratory strain (BL21); one expressing 6xHis-JNK2, and the other 6xHis-NleD. Proteins were then extracted from the bacteria and used for Western blot analysis with anti-JNK antibody. As shown by Figure 13E, JNK was efficiently clipped in the E. coli cytoplasm in an NleD-dependent manner. JNK clipping was not evident in the absence of NIeD (Fig. 13E). These data confirm that JNK clipping is mediated by NIeD and does not require a host-specific cell factor. Given that NIeD activity is independent of host factors, the inventors tested whether purified 6xHis tagged NIeD could cleave purified JNK2 in vitro (Fig. 13F). As shown by Figure 13 F, when wild type NIeD and JNK2 were mixed at a molar ratio of 1 :40 JNK was cleaved readily. Notably, this cleavage was inhibited by the Zn metalloprotease specific inhibitor phenanthroline (Fig. 13F). For unknown reasons, JNK were only partially digested by NIeD in vitro. Nevertheless, taken together, the results show that NIeD is a Zn-metalloproteinase that specifically clips JNK.

Example 13

NIeD cleaves within the activation loop ofJNK2

To define the NIeD digestion site the inventors took advantage of a JNK2 construct with an N-terminal HA tag. The activation loop of this JNK protein is flanked by two epitopes, the N-terminal HA epitope and an epitope localized in the C-terminal domain that is recognized by the anti-JNK monoclonal antibody and depicted in Figure 16A. HEK293 cells were transfected with HA-JNK expressing plasmid and then infected with wild type EPEC or nleD mutant. After 2.5 h, immunoprecipitation was performed using anti-HA antibodies. Western blot analysis using either anti-HA (Fig. 16B) or anti-JNK (Fig. 16C) antibodies was employed to examine the precipitated proteins, which comprised full length HA-tagged JNK and N-terminal JNK fragments. Figure 16B shows that an NleD- dependent, N-terminal JNK fragment of -23 kDa was recognized by anti-HA antibody. In parallel, distinct C-terminal JNK fragments of -33 kDa were recognized by anti-JNK antibody, as shown in Figure 16C. These data indicate that the C-terminal portion of the clipped HA-JNK must remain in complex with the HA-tagged N-terminus fragment. Moreover, based on the predicted sizes of these C- and N- terminal fragments generated by NIeD it appears that NIeD cuts JNK2 somewhere within the activation loop illustrated by Figure 16A. Notably, in the Western blots the C-terminal fragment of JNK frequently appeared as a doublet (Fig. 13B, 13C and 16C) suggesting that NIeD clips JNK at two proximate sites. Figures 17A-16C illustrate experiments performed using JNKl instead of JNK2, which generated similar results. Taken together, these results show that NIeD cleaves JNK at its activation loop and the two generated fragments remain in complex.

To define more rigorously the cleavage point, purified N-terminally tagged 6xHis-JNK2 was incubated with purified NIeD. Initially, the reaction products were characterized using Western blot analysis with anti-6xHis antibody, shown by Figure 16D, and in parallel, by SDS-PAGE followed by Coomassie Blue staining, shown by Figure 16E; the former to detect specifically N-terminal products and the latter all generated products. Notably, similarly sized C- and N- terminal JNK fragments were observed in this in vitro study as in the in vivo analysis described above (compare Fig. 16B and C to 16D and E). Next, a band corresponding to the C-terminal product of JNK was excised from the gel depicted in Figure 16E, and subjected to mass spectrometry analysis. The inventors discovered that JNK is clipped within the activation loop between Alal73 and Argl74. The inventors further predicted that an additional NIeD cleaving site might be located in close proximity downstream to Rl 74, but this could not be detected by the analysis. A model of the JNK structure (taken from PDB 3E7O; Fig. 16F) highlights that extensive surface interactions between the C and N terminal JNK domains are not interrupted by such cleavage of the activation loop (Fig. 16F), which accords with the presented immunoprecipitation data.

Example 14

NleD cleaves different isoforms ofp38

In the course of investigating NIeD, the inventors determined whether other major stress signal transducers were affected by NIeD. For this purpose HEK 293T cells were transfected with vectors expressing either JNK2 (positive control), p38α, p38β, p38γ or p38δ with N-terminal HA tag. The transfected cells were then infected with either wild type or ΔnleD strains and total protein was extracted and subjected to a western blot analysis using a monoclonal anti-HA antibody, as shown by Figure 18. As can be seen, WT EPEC induced the degradation of JNK2 and all p38 isoforms. In contrast, ΔnleD mutants did not degrade neither JNK2, as expected, nor any p38 isoform. Thus, NIeD is responsible for the cleavage of p38 isoforms, and therefore may be involved in inhibition of p38 signaling.

Example 15

NIeC, but not NIeD, inhibits TNFa-induced IL-8 expression

Induction of IL-8 expression by TNFα sometimes involves not only NF-κB but also AP-I and JNK (Kang et al., 2007; Roger et al, 1998). As described earlier herein, EPEC infection interferes with this induction of IL-8. To delineate whether NIeD plays a role in repressing IL-8 expression during EPEC infection, the inventors compared the capacity of two strains to repress TNFα-induced IL-8 expression: a mutant with the IE6 region deleted (IE6 contains the nleBE genes, previously reported to inhibit IL-8 expression); and a mutant with both IE6 and PP4 regions deleted, the latter region containing nleD. HeLa cells were infected with the relevant strain, exposed to TNFα and then harvested to assay IL-8 mRNA levels by Real Time PCR. Figure 19A shows that the ΔIE6 mutant was partially deficient in repressing IL-8 expression, whereas the double mutant (ΔIE6, ΔPP4) was completely deficient. These results appeared to corroborate the expectation of the inventors that nleD, a gene within the PP4 region that was shown to inactivate JNK, contributes to IL-8 repression. However, as Figure 19A demonstrates, complementation analysis using expression plasmids revealed that nleD was not the gene within the PP4 region mediating IL-8 repression. The inventors therefore tested other genes that are located in PP4 including nleC and nleG. Importantly, nleC, and not nleD or nleG, was found to influence IL-8 repression, seen in Figure 19 A. Generation of an EPEC mutant bearing a precise nleC deletion confirmed that this indeed is the gene within the PP4 region that mediates IL-8 repression during EPEC infection (Fig. 19B). Notably, nleC is located in the EPEC genome immediately upstream to nleD, and like NIeD, also contains a conserved Zn metalloprotease signature motif, HEXXH (also denoted by SEQ ID NO.: 9). As shown by Figure 20, his motif is conserved in all homologes of NIeC. Importantly, Figure 19B shows that only a plasmid expressing NIeC but not one expressing NIeC- E 184 A (a mutant in the HEXXH motif) restored the ability of the nleC mutant EPEC to block TNFα-induced IL-8 expression. In conclusion, these results show that NleC is a mediator of EPEC -dependent IL-8 repression and possibly exerts this effect via proteolysis of a key component in the NF-κB pathway.

Example 16

Ectopically expressed NleC is associated with reduced levels ofp65

To investigate how NleC represses IL-8 induction, the inventors next examined if NleC influences TNFα-induced translocation of NF-κB to the nucleus. To this end, HeLa cells were transfected with vectors expressing mCherry, mCherry fused to NleC (mCherry- NIeC), or mCherry fused to NIeC-El 84A (mCherry-NleC-E184A), exposed the cells to TNFα and evaluated p65 localization and levels by fluorescent microscopy using anti-p65 antibody. Interestingly, Figure 19C shows that both nuclear and cytoplasmic p65 staining was considerably diminished in cells expressing mCherry-NleC, whereas strong p65 staining was evident in cells expressing mCherry or mCherry-NleC-E184A. Indeed, quantification revealed that only 6% (n = 64) of the mCherry-NleC expressing cells, exhibited some p65 staining, whereas 96% (n = 53) and 97% (n = 67) of the cells expressing mCherry or mCherry-NleC-E184A, respectively, exhibited clear p65 staining. These results indicate that NleC destabilizes p65, possibly via its putative protease activity. Example 17

NIeC clips p65 and reduces nuclear p65 levels

To test the hypothesis that NIeC mediates p65 proteolysis, the inventors infected HeLa cells with various EPEC strains, then separated cytoplasmic from nuclear proteins and subjected these two fractions to Western analysis using anti-p65 antibodies. The ΔIE2 strain was used as wild type in these experiments, as this strain exhibits wild type phenotype with respect to NF-κB inhibition, as shown earlier. Figure 21 A clearly shows that ΔIE2 EPEC, but not a TTSS deficient mutant (AescV), induced marked reduction in nuclear p65 levels and partial clipping of both nuclear and cytoplasmic p65. To isolate the role of NIeC from effects induced by NIeB and NIeE, the inventors took advantage of a triple mutant ΛnIeBE, ΛnIeC. As expected, this mutant was deficient in causing reduction of nuclear p65 and in p65 clipping (Fig. 21A). Figure 21A also shows that the capacity of this triple mutant to reduce nuclear levels of p65 and stimulate p65 clipping was restored by a plasmid expressing NIeC, but not by a plasmid expressing NIeC-El 84A. When considering this experiment (Fig. 21) and the previous one (Fig. 19C), it should be taken into account that EPEC injects relatively low levels of natively expressed NIeC compared to the levels attained when NIeC is ectopically expressed in the host cell (data not shown). This data indicate that (i) injected NIeC clips p65 and (ii) that p65 clipping leads to reduced nuclear p65 levels.

To determine which end of p65 is cleaved by NIeC, HeLa cells were infected with nleC mutant (AnIeC) complemented with either plasmid only (vector) or vector expressing wild type NIeC (pnleC). After 3 hours cells were extracted and the lysates were subjected to a western blot analysis using two different anti-p65 antibodies; one raised against the p65 N-terminus region and the other against the p65 C-terminus region. Importantly, both antibodies reacted with the full lengthen p65, but only the anti-p65-C-ternimus antibody reacted with the cleaved form of p65, as illustrated by Figure 2 IB. These finding indicate that NIeC mediate the clipping of the p65 N-terminal domain. Example 18

NIeC clips p65 in vitro

To confirm the premise that NIeC mediates p65 clipping, the inventors tested whether NIeC cleaves p65 in vitro. Ideally, it would be desirable to use purified components, but the inventors were unable to generate pure p65 due to its poor stability and solubility in E. coli (data not shown). Therefore, the inventors employed extracts of HeLa cells, which were mixed with purified NIeC, or NIeC-El 84A. Western blot analysis of the mixtures was performed using anti-p65 antibodies to detect the reaction products, as shown by Figure 21C. In this assay NIeC, but not NIeC-El 84A, effectively clipped p65 (Fig. 21C). Importantly, phenanthroline, a Zn metalloprotease inhibitor, blocked NleC-induced p65 clipping (Fig. 21C). Taken together, these results indicate that i) NleC-mediated p65 clipping is not dependent on any other EPEC factor, ii) p65 clipping requires the Zn metalloprotease activity of NIeC and iii) it is likely that NIeC directly cuts p65.

Example 19

NIeB, NIeE and NIeC cooperate to achieve full NF-κB inhibition

Figure 19B shows that the nleC mutant is only partially deficient in repressing TNFα- induced IL8 expression. Similar partial deficiency was shown for nleBE mutant. These findings indicate that concerted activity of NIeB, NIeE and NIeC is required to achieve full NF-κB repression. The inventors tested this hypothesis using IL8 secretion as readout for NF-κB activity. Cells were infected for 3 hours with different EPEC strain, washed and treated with TNFα for 16 h. Figure 22 presents medium IL8 levels using ELISA. As positive controls (full repression of IL8 secretion), cells infected with EPEC ΔIE2 (used here as wild type EPEC) or cells which were neither infected nor treated with TNFα were used. As a negative control (no repression of IL8 secretion), cells which were not infected, but treated with TNFα were used. As shown by Figure 22, the EPEC AnIeBE mutant was only partially deficient in repressing IL8 secretion, but the AnIeBE, AnIeC triple mutant was almost completely deficient in repressing IL8 secretion. Importantly, plasmids overexpressing either NIeC or NleBE were sufficient to restore almost full repression of IL8 secretion. Taken together, these results suggest that under native expression condition, both NIeC and NleBE are required for full NF-κB repression. Yet, upon overexpression, which is associated with increase injection of these proteins by EPEC (data not shown), each, NIeC, or NIeBE, are sufficient to achieve almost full repression of NF-κB signaling.

Example 20

Proposed mechanism of action for TTSS-dependent EPEC effectors

Figure 23 depicts the proposed mechanism of EPEC effectors action. EPEC attach to host cells, activating IL-I receptor/TLR-mediated pro-inflammatory signaling cascades, which may be augmented by local IL-I β and TNFα release. EPEC use the TTSS to inject into the host cells NIeB, NIeC, NIeD, NIeE and NIeHl. NIeB inhibits signaling from TNFR and IL-I receptor/TLR, upstream of TAKl. NIeE inhibits IKB phosphorylation and degradation. NIeC cleaves cytoplasmic and nuclear p65, destabilizing it. NIeD cleaves JNK, and NIeHl inhibits p65/p50-mediated transcription. Thus, NIeB, NIeC, NIeE and NIeHl inhibit various stages along the NF-κB signaling cascade, while NIeD inhibits the JNK signaling cascade. Both NF-kB and JNK signaling cascades activate expression of pro-inflammatory target genes, therefore the concerted activities of the effectors prevent local inflammation.

Table 5 indication of Sequence listing numbers and description

Claims

CLAIMS:

1. A composition comprising as an active ingredient at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins or any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, said composition optionally further comprises a pharmaceutically acceptable carrier or excipient.

2. The composition according to claim 1 optionally further comprising at least one additional therapeutic agent.

3. The composition according to any one of claims 1 and 2, wherein said composition leads to the inhibition of at least one of an NF-κB, JNK and p38 mediated signal transduction pathways, thereby leading to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a target cell.

4. A pharmaceutical composition according to claim 3, for preventing, treating, or ameliorating an immune-related disorder.

5. The composition according to any one of claims 1 and 2, comprising as an active ingredient a therapeutically effective amount of NIeE, NIeC, NIeB and optionally, NIeD immuno-modulatory proteins, said composition optionally further comprises a pharmaceutically acceptable carrier or excipient.

6. A method for preventing, treating, or ameliorating an immune-related disorder in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeD and NIeB immuno- modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof and any compositions comprising the same.

7. The method according to claim 6, comprising the step of administering to said subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and optionally at least one additional therapeutic agent or any compositions or combined compositions comprising the same.

8. The method according to any one of claims 6 and 7, wherein said method leads to inhibition of at least one of an NF-κB, JNK and p38 mediated signal transduction pathway, thereby leading to at least one of an anti-inflammatory response, an anti- apoptotic effect or a pro-apoptotic effect in a cell of said treated subject

9. The method according to claim 8, wherein said immune-related disorder is any one of an inflammatory disease, an autoimmune disease and a malignant or non-malignant proliferative disorder.

10. The method according to any one of claims 6 and 7, comprising the step of administering to said subject a therapeutically effective amount of at least one of NIeE, NIeC, NIeB and optionally, NIeD immuno-modulatory proteins and any functional homologues, variants, fragments, derivatives and mixtures thereof and any compositions comprising the same.

11. Use of at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, for the preparation of a pharmaceutical composition for the prevention, treatment, or amelioration of an immune-related disorder.

12. The use according to claim 11, of at least one of NIeE, NIeC, NIeD and NIeB proteins and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, and optionally, at least one additional therapeutic agent, for the preparation of a pharmaceutical composition for the prevention, treatment, or amelioration of an immune-related disorder.

13. A tissue-targeted delivery system of an immunomodulatory protein comprising a non-virulent/attenuated Type-Three Secretion System (TTSS)-expressing microorganism comprising nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

14. The tissue-targeted delivery system according to claim 13, wherein said microorganism optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein, said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

15. The delivery system according to claim 1, wherein said immunomodulatory protein inhibits at least one of an NF-κB, JNK and p38 mediated signal transduction pathway, thereby leading to at least one of an anti-inflammatory response, an anti- apoptotic effect or a pro-apoptotic effect in a cell of a target tissue.

16. The delivery system according to any of claims 13 and 14, wherein said system comprises a non-virulent/attenuated TTSS-expressing microorganism comprising nucleic acid sequences encoding NIeE, NIeC, NIeB and optionally, NIeD immuno-modulatory proteins.

17. A composition comprising at least one delivery system of an immunomodulatory protein comprising a non-virulent/attenuated TTSS-expressing microorganism comprising nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB immuno- modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures and combinations thereof, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences, said composition further comprises a pharmaceutically acceptable carrier or excipient.

18. The composition according to claim 17, wherein said delivery system optionally further comprises at least one nucleic acid sequence encoding an additional therapeutic protein, said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

19. The composition according to any one of claims 17 and 18, wherein said composition further comprises an additional therapeutic agent.

20. The composition according to claim 18, for the inhibition of at least one of an NF- KB, JNK and p38 mediated signal transduction pathway, thereby leading to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a cell of a target tissue.

21. A pharmaceutical composition according to claim 20, for preventing, treating, or ameliorating an immune-related disorder.

22. A method for preventing, treating, or ameliorating an immune-related disorder in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a tissue-targeted delivery system of an immunomodulatory protein or any composition comprising the same, said delivery system comprises a non-virulent/attenuated TTSS-expressing microorganism comprising nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologies, variants, fragments, derivatives, mixtures and any combinations thereof, wherein said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

23. The method according to claim 22, wherein said delivery system optionally further comprises nucleic acid sequences encoding at least one additional therapeutic protein, said nucleic acid sequences are operably linked to TTSS secretion signal sequences.

24. The method according to any one of claims 22 and 23, wherein said immunomodulatory protein inhibits at least one of an NF-κB, JNK and p38 mediated signal transduction pathway, thereby leading to at least one of an anti-inflammatory response, an anti-apoptotic effect or a pro-apoptotic effect in a cell of a target tissue in said subject

25. The method according to claim 24, wherein said immune-related disorder is any one of an inflammatory disease, an autoimmune disease and a malignant or non-malignant proliferative disorder.

26. The method according to any one of claims 22 and 23, wherein said delivery system comprises a non- virulent/attenuated TTSS-expressing microorganism comprising nucleic acid sequences encoding NIeE, NIeC, NIeB and optionally, NIeD immunomodulatory proteins and any functional homologues, variants, fragments, derivatives and mixtures thereof.

27. Use of a tissue-targeted delivery system of an immunomodulatory protein comprising a non-virulent/attenuated TTSS-expressing microorganism comprising nucleic acid sequences encoding at least one of NIeE, NIeC, NIeD and NIeB immuno-modulatory proteins and any functional homologues, variants, fragments, derivatives, mixtures and any combinations thereof in the preparation of a composition for the prevention, treatment, or amelioration of an immune-related disorder.

28. A kit for achieving a therapeutic effect in a subject in need thereof comprising: a. at least one of:

i. NIeE protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non- virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeE protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form;

ii. NIeC protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeC protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form;

iii. NIeD protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeD protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a third unit dosage form; iv. NIeB protein and any functional homologues, variants, fragments, derivatives, mixtures, any combinations thereof, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said NIeB protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a fourth unit dosage form; and optionally b. at least one additional therapeutic protein, or a tissue-targeted delivery system comprising non-virulent/attenuated TTSS-expressing microorganisms comprising nucleic acid sequences encoding said therapeutic protein or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a unit dosage form; and optionally

c. at least one additional therapeutic agent or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a unit dosage form; and

d. container means for containing said unit dosage forms.

29. An attenuated EPEC or EHEC pro-biotic bacteria expressing intact Tir (translocated intimin receptor) and intimin encoding genes, wherein the attenuation is caused by deletion or inactivation of at least one of:

a. at least one gene encoding type FV pilli and type I pilli;

b. a gene encoding the effector Map (Mitochondrial-associated protein); and

c. genes encoding exotoxins selected from the group shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins, hemolysin; EspC, EspP and LifA.

30. A nutraceutical composition comprising as an active ingredient an effective amount of attenuated EPEC or EHEC pro-biotic bacteria expressing intact Tir (translocated intimin receptor) and intimin encoding genes, wherein the attenuation is caused by deletion or inactivation of at least one of:

a. at least one gene encoding at least one of type IV pilli and type I pilli;

b. a gene encoding the effector Map (Mitochondrial-associated protein), and optionally any further effector; and c. genes encoding exotoxins selected from the group consisting of shiga toxins, verotoxins, heat labile toxins, heat stable enterotoxins and hemolysin; said composition optionally further comprises a pharmaceutically acceptable carrier.

31. The nutraceutical composition according to claim 30, wherein said composition further comprises additional pro-biotic bacteria.