WO2013148987A1 - Compositions and methods for treating intestinal bacterial infections - Google Patents

Description

COMPOSITIONS AND METHODS FOR TREATING INTESTINAL BACTERIAL

INFECTIONS

This application claims priority to U.S. Provisional Patent Application No. 61/616,707, filed March 28, 2012, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating infection by pathogenic bacteria. In particular, the present invention relates to compositions and methods for targeting virulence factors of pathogenic E. coli.

BACKGROUND OF THE INVENTION

The intestine of animals is colonized by a large number of commensal microorganisms. Millions of years of co-evolution have led this host-microbe interaction into a symbiotic relationship in which the microbiota contributes to many host physiological processes. A key role of the intestinal microbiota is protection against

Enteropathogenic E. coli (EPEC) and Enterohemorragic E. coli (EHEC) are pathogenic E. colonization and invasion by pathogens that often enter the intestinal tract, coli that are food- and waterborne pathogens that are non-invasive attaching and effacing (A/E) bacteria. As a result of the infection and ensuing intestinal inflammation, EPEC and EHEC cause severe diarrhea and other complications, leading to the death of hundreds of thousands of children worldwide each year. In addition, infection in adults can be lethal and cause significant morbidity. Unfortunately, antibiotic therapy for EPEC and EHEC infections is not effective and can result in worsening of intestinal symptoms.

New strategies to treat colitis caused by A/E pathogens are needed.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for treating infection by pathogenic bacteria. In particular, the present invention relates to compositions and methods for targeting virulence factors of pathogenic E. coli.

For example, in some embodiments, the present invention provides a method of inhibiting a biological activity of a virulence factor of a pathogenic E. coli (or other bacteria), comprising: contacting the virulence factor or a cell expressing the virulence factor with a composition that inhibits a biological activity of the virulence factor. In some embodiments, the pathogenic E. coli is EHEC or EPEC. In some embodiments, the composition is a small molecule or an antibody that specifically binds to the virulence factor. In some embodiments, the small molecule is a salicylidence acyle hydrazine compound (e.g., ME0054, ME055, ME097, MEOl 14, or ME0214) or aurodox. In some embodiments, the antibody binds specifically to a LEE factor (e.g., intimin). In some embodiments, the inhibiting treats infection by the pathogenic E. coli. In some embodiments, the inhibiting results in commensal E. coli out competing pathogenic E. coli.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

Figure 1 shows that microbiota is required for eradication of C. rodentium. A,B, SPF and

GF mice (n= 7) were infected orally with 109cfu of C. rodentium and pathogen load in feces (A) and mouse survival (B) were determined over the indicated time. C, Cells were isolated from the large intestine of SPF or GF mice on day 0, 12 and 22 after oral infection with C. rodentium, and stained for CD1 lb, F4/80, and Gr-lmAb. D, Flow cytometry analysis for inflammatory monocytes, neutrophils and CD3+ T cells in total lamina propria mononuclear cells (LPMCs). E, Histological analysis of the distal colon of SPF and GF mice infected orally with 109cfu of C. rodentium on day 12, 22 and 42 post-infection. F, Histopatho logical scores evaluating the infectious colitis phenotype induced by C. rodentium on day 12, 22 and 42 post-infection.

Figure 2 shows expression and role of ler during C. rodentium infection in SPF and GF mice.

A, ler mPvNA levels were determined by qPCR in fecal pellets of SPF and GF mice infected with C. rodentium at the indicated days post-infection. B, Expression of ler in fecal pellets of SPF and GF mice infected with the reporter ler-lux C. rodentium strain at the indicated day post-infection. C, Bioimaging of ler expression in the intestine of SPF and GF mice infected with the ler-luxC. rodentium strain. D-E, SPF and GF mice (n= 5) were infected orally with 109cfu of wild-type and Aler mutant C. rodentium, and pathogen load in feces (D) and mouse survival (E) were determined over indicated time. F, GF mice were infected with wild-type and Aler mutant C.

rodentium. At day 3 or day 21 post infection, mice were co-housed with SPF mice (1 :1).

Figure 3 shows localization of C. rodentium to intestinal niches is mediated by LEE virulence factors. A, GF mice were infected with wild-type or Aler mutant C. rodentium. B, For FISH, tissues were fixed in Carnoy's fixation. C, Transmission Electron Micrographs of C.

rodentium-mfected GF mice cecum at day 5 and day 21 post infection. D-E, GF mice were infected orally with C. rodentium. Bioimaging of ler expression of tissue-attached C. rodentiumm the cecum (top) and colon (bottom) in GF mice (D). C.rodentium burden and ler-lux luminescence in feces, cecum, and colon (E).

Figure 4 shows that similar catabolic preferences for saccharides determine the competing ability of commensal bacteria with the enteric pathogen. A, GF mice were infected with wild-type C. rodentium (Cr). At day 21 post infection, commensal E. coli (Ec) or B. thetaiotaomicron (Bt) or B. vulgatus (Bv) were inoculated. B, Total Enter obacteria (C. rodentium and E. coli) and Bacteroides culture in feces at day 0 and day 14 of competing experiment. C, Diluted fecal samples were plated onto McConkey plates with or without kanamycin. D, Carbohydrate catabolic profiles of C. rodentium and commensal bacteria strains. E, GF mice were infected with wild-type C. rodentium {Cr). F, C. rodentium mono-associated GF mice (day 21) were fed with a simple sugar diet (ssDiet) for 7 days.

Figure 5 shows antimicrobial peptide expression in intestinal epithelial cells.

Figure 6 shows tir mRNA expression in stool samples were collected on day 7 and day 12 (also on day 22 and day 42 in GF mice) after C. rodentium infection.

Figure 7 shows that bio luminescence of C. rodentiumler-lux strain represents the endogenous ler expression. A. Schematic representation of the ler-lux reporter operon fusion between the C. rodentium ler regulatory region from positions -260 to + 216 (14) and the Photorhabdus luminescens luxCDABE operon in pCS26-Pac (15). B. Expression of ler-lux in wild type C. rodentium under in vitro growth conditions. (Left panel) luminescence of ler-lux in bacteria grown in DMEM (filled circle) or LB medium (open circle). (Right panel) bacteria proliferation in DMEM (filled circle) or in LB medium (open circle). C. Endogenous ler mRNA expression in bacteria grown in DMEM or LB medium.

Figure 8 shows that down-regulation of ler- virulence is reversible. Pathogen load in feces (A) were determined over the indicated time, and histology was assessed at day 12 (B). Arrows denote marked submucosal edema and infiltration of acute inflammatory cells.

Figure 9 shows that a functional type III secretion system is not required for robust growth in

GF mice, but essential to compete with the commensal microbiota. A, B, SPF and GF mice (n= 5) were infected orally with 109cfu of wild-type and AescN (T3SS) mutant C. rodentium, and pathogen load in feces (A) and mouse survival (B) were determined over indicated time.

Figure 10 shows that ler-regulated virulence expression before conventionalization in GF mice. A, Bacterial RNAs were purified from feces, and ler expression was assessed by qPCR. B, Expression of ler in fecal pellets of GF mice infected with the ler-lux reporter C. rodentium strain at the indicated day post-infection. Figure 11 shows that intestinal inflammation does not enhance colonization of C.

rodentium lacking a functional type 3 secretion system. A, Experimental protocol of DSS- induced colitis. B, Weight (left) and disease activity index (DAI: right) of DSS-treated and untreated mice which are infected with wild-type C. rodentium or AescN mutant. C. Bacteria number in feces 2 days after C. rodentium or AescN mutant inoculation. D. SPF C57BL/6 mice were infected with C. rodentium (ampicillin resistant) or AescN mutant (kanamycin resistant), respectively, or coinfected with both strains.

Figure 12 shows that GF mice develop C. rodentium-specific immunoglobulins after infection.

Figure 13 shows that germ-free mice were able to develop Thl and Thl7 immune responses. A, Intracellular production of IFN-γ, and IL-17A was assessed in isolated colonic CD4+ T cells from SPF and GF mice on day 0, 12 and 22 after infection with C. rodentium. B, Thl and Thl7 cytokines production by isolated LP cells from SPF and GF mice on day 0, 12 after infection with C. rodentium.

Figure 14 shows Ler-dependent virulence factor-specific IgG production in C. rodentium infected mice. A. Serum IgG. B. Intestinal content IgG.

Figure 15 shows intimin specific IgG production in the intestine of C. rodentium infected mice.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the terms "detect", "detecting" or "detection" may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.

The term "derivative" of a compound, as used herein, refers to a chemically modified compound wherein the chemical modification takes place either at a functional group of the compound or backbone.

As used herein, the term "subject" refers to organisms to be treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals {e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the invention, the term "subject" generally refers to an individual who will receive or who has received treatment for a condition characterized by bacterial infection. The term "diagnosed," as used herein, refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, and the like.

As used herein the term, "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.

As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a therapeutic agent described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited intended to be limited to a particular formulation or administration route.

As used herein, the term "co-administration" refers to the administration of at least two agent(s) (e.g., a compound of the present invention) or therapies to a subject. In some

embodiments, the co-administration of two or more agents/therapies is concurrent. In some embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are coadministered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).

As used herein, the term "toxic" refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975]).

As used herein, the term "pharmaceutically acceptable salt" refers to any

pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene -2- sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and

environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

As used herein, the term "modulate" refers to the activity of a compound (e.g., a compound described herein) to affect (e.g., to promote or retard) an aspect of cellular function, including, but not limited to, bacterial growth and the like.

The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample (e.g., bacterial infection). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention. In some embodiments, "test compounds" are agents that target virulence genes.

As used herein, the term "purified" or "to purify" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for treating infection by pathogenic bacteria. In particular, the present invention relates to compositions and methods for targeting virulence factors of pathogenic E. coli and other bacteria.

I. Pathogenic E. coli

It is generally unclear, during the course of an enteric infection, how commensal bacteria and pathogens interact. This includes uncertainty regarding how virulence strategies allow pathogens to obtain an environmental niche when competing with the microbiota. Similarly, it is unclear whether commensals play a role, not only in colonization resistance, but also in the clearance of pathogens from the intestines of an infected host. Enterohemorragic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are important causes of watery diarrhea and mortality worldwide (1, 2). These Gram-negative bacteria are food- and waterborne non-invasive pathogens which attach to and colonize the intestinal tract by inducing characteristic attaching and effacing (A/E) lesions on the intestinal epithelium, leading to transient enteritis or colitis in humans (1, 2). The genome of EPEC and EPEC and related pathogens harbor the locus for enterocyte effacement (LEE) which is critical for bacterial colonization and the ability to cause pathology (3, 4). Recently, it was reported that infection with Citrobacter rodentium, a natural A/E bacterial pathogen of mice that is widely used to model human infections with EPEC and EHEC (5, 6), is associated with a significant but reversible decrease in the total number of commensal bacteria in the mouse colon ( , 8). However, the role of the microbiota in C.

rodentium colonization is unclear. Experiments conucted during the course of development of embodiments of the present invention demonstrated the role of virulence factors in growth of pathogenic E. coli.

Accordingly, embodiments of the present invention provide composisitons and methods for treating infection by pathogenic E. coli comprising inhibiting virulence factor genes and proteins.

II. Therapeutic Applications

In some embodiments, the present invention provides therapies for pathogenic E. coli (e.g., EPEC and/or EHEC). In some embodiments, therapies target virulence factors of EPEC and/or EHEC.

A. Small Molecules

In some embodiments, small molecule inhibitors of virulence factors are utilized. In some embodiments, small molecule therapeutics are salicylidence acyle hydrazine compounds.

Salicylidence acyl hydrazide compounds that have been shown to reduce the expression of T3SS effectors including those from A/E pathogens at -1-20 μΜ range (33). Examples of salicylidence acyle hydrazine compounds include, but are not limited to, ME0054, ME055, ME097, MEOl 14, and ME0214. These compounds do not inhibit bacteria growth, but can inhibit expression of several LEE virulence genes including Ler and formation of A/E lesions on bovine epithelial cells (33).

In some embodiments, small molecules are aurodox or related compounds. Aurodox is a linear polyketide compound that is a specific T3SS inhibitor of EPEC and C. rodentium (34) and targets multiple LEE virulence factors. Aurodox is commercially available (e.g., from Enzo Life Sciences, Inc. Farmingdale, NY) and has been shown to protect mice from C. rodentium infection in vivo (34).

C. Antibody Therapy

In some embodiments, the present invention provides antibodies or antigen binding partners thereof that target virulence factors of EPEC and/or EHEC. Any suitable antibody {e.g., monoclonal, polyclonal, or synthetic) may be utilized in the therapeutic methods disclosed herein. In some embodments, the antibodies used for therapy are humanized antibodies. Methods for humanizing antibodies are well known in the art {See e.g., U.S. Pat. Nos. 6,180,370,

5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference).

In some embodimetns, antibodies with defined specificity against LEE factors are used to inhibit Ler and pathogen colonization. In some embodiments, antibodies are rasied against intimin (e.g., the C-terminal 382 amino acid extracellular domain) from C. rodentium. In some embodiments, antibodies against LEE factors other than intimin are utilized. For example, in some embodiments, extracts of C. rodentium expressing LEE virulence factors and total RNA purified from the blood are utilized

In preferred embodiments, antibody based therapeutics are formulated as pharmaceutical compositions as described below. In preferred embodiments, administration of an antibody composition of the present invention results in a measurable decrease in disease {e.g., decrease or elimination of infection by pathogenic E. coli).

The present invention also includes pharmaceutical compositions and formulations that include the antibody compounds of the present invention as described below.

D. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions {e.g. , comprising pharmaceutical agents that inhibit virulence factors of pathogenic E.coli). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary {e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular

administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium

carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the acid(s) of the formulation.

Dosing 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. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

In some embodiments, the present invention provides therapeutic methods comprising one or more compositions described herein in combination with an additional agent (e.g., an antibiotic).

III. Drug Screening Applications

In some embodiments, the present invention provides drug screening assays {e.g., to screen for drugs that target virulence factors of pathogenic E. coli). In other embodiments, candidate compounds are antibodies or small molecules that specifically bind to a virulence factor and inhibit its biological function.

In one screening method, candidate compounds are evaluated for their ability to alter expression of virulence genes by contacting a compound with pathogenic E. coli and then assaying for the effect of the candidate compounds on expression of virulence genes. In some embodiments, the effect of candidate compounds on expression of a virulence gene is assayed for by detecting the level of mR A expressed by the cell. mR A expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression of virulence genes is assayed by measuring the level of polypeptide encoded by the virulence genes. The level of polypeptide expressed can be measured using any suitable method.

Specifically, the present invention provides screening methods for identifying

modulators, i.e., candidate or test compounds or agents {e.g., proteins, antibodies, peptides, peptidomimetics, peptoids, small molecules or other drugs) which have an inhibitory (or stimulatory) effect on, for example, pathogenic E. coli virulence gene expression or activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a virulence gene. Compounds thus identified can be used to modulate the activity of target gene products (e.g. , virulence genes) either directly or indirectly in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions. Compounds that inhibit the activity or expression of virulence genes in the treatment of infection by pathogenic E. coli (e.g., EPEC and/or EHEC).

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al, J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al, Proc. Nad. Acad. Sci. USA 91 : 11422 [1994]; Zuckermann et al, J. Med. Chem. 37:2678 [1994]; Cho et al, Science 261 : 1303 [1993]; Carrell et al, Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al, J. Med. Chem. 37: 1233

[1994].

Libraries of compounds may be presented in solution {e.g., Houghten, Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555- 556 [1993]), bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids (Cull et al, Proc. Nad. Acad. Sci. USA 89: 18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1 A. Materials and Methods

Animals

Specific pathogen free (SPF) C57BL/6 mice were purchased from Jackson Laboratories.

GF wild-type mice were bred and maintained at the Germ- free Animal Core Facility of the University of Michigan. GF mice were maintained in flexible film isolators and were checked weekly for germ-free status by aerobic and anaerobic culture. The absence of microbiota was verified by microscopic analysis of stained cecal contents to detect unculturable contamination. All animal studies were performed according to approved protocols by the University of Michigan Committee on the Use and Care of Animals.

C. rodentium infection

The kanamycin (Km)-resistant wild-type Citrobacter rodentium strain DBS 120

(pCRP 1 : :Tn5) was a gift of Dr. David Schauer, Massachusetts Institute of Technology. The isogenic C. rodentium Aler mutant strain has been described(^). For inoculations, bacteria were grown overnight in Luria-Bertani (LB) broth supplemented with Km (50 μg/ml) with shaking at 37°C. Mice were infected by oral gavage with 0.2 ml of PBS containing approximately 1 x 109 CFU of C. rodentium. To determine bacterial numbers in the feces, fecal pellets were collected from individual mice, homogenized in cold PBS and plated at serial dilutions onto MacConkey agar containing 50 μg/ml Km, and the number of CFU was determined after overnight incubation at 37°C. Mice were sacrificed at various time points post-infecion (p.i.), and colons were flushed with PBS and used for colonic cell isolation or fixed in 10% formalin and then processed for H&E staining.

Flow cytometric analysis

Cell surface fluorescence was assessed using a FACSCalibur analyzer and analyzed using Flow Jo software (TreeStar). Dead cells were excluded with 7-AAD staining. Non-specific Ab binding was blocked with anti-CD 16/32 Ab. FITC- PE- or APC-conjugated mAb against CDl lb (Ml/70), Gr-1 mAb (RB6-8C5), F4/80 (BM8) and CD3 (145-2C11) were from eBioscience. Isotype-matched antibodies (eBioscience) were used for control staining.

Measurement of ferexpression A plasmid containing a ler-lux transcriptional fusion was constructed by cloning a fragment spanning the C. rodentium ler regulatory region from position -260 to +216 with respect to the ler transcriptional start site (14), in plasmid pCS26-Pac containing the promoterless Photorhabdus luminescens luxCDABE operon (15). The resulting plasmid pier-lux was transformed into wild type C. rodentium by electroporation. For determination of the levels of ler expression in feces, fecal pellets were suspended in PBS at 10 mg/ml, and luminescence emitted from /er-/wx-expressing bacteria was measured using a LMax luminometer (Molecular Device). For in vivo bioluminescence imaging (BLI), the entire gastrointestinal tissues were immediately removed and placed into the light-tight chamber of the CCD camera system (IVIS200, Xenogen). Luminescence emitted from /wx-expressing bacteria in the tissue was quantified using the software program living image (Xenogen). Quantitative real time RT-PCR (qPCR) for ler and tir was performed using a SYBR green PCR master mix and the StepOne Real-time PCR system (Applied Biosystems) and normalized to the expression of the kanamycin resistance gene (Km).

Relative expression of virulence factors was determined as fold expression when compared to that of bacteria cultured in LB culture. The following primer sets were used: ler; 5'- AATATACCTGATGGTGCTCTTG-3' and 5 ' -TTCTTCC ATTC AAT AATGCTTCTT-3 ' . tir; 5'- TACACATTCGGTTATTCAGCAG-3 ' and 5 '-GACATCCAACCTTCAGCATA-3'. rrsA (16SRNA); 5 '-AGGCCTTCGGGTTGTAAAGT-3 ' and 5 '-ATTCCGATTAACGCTTGCAC-3 ' . Km (kanamycin resistant protein); 5 '-CTGAATGAACTGCAGGACGA-3 ' and 5'- ATACTTTCTCGGCAGGAGCA-3 ' .

Immunostaining for Tir

Briefly, 5 μιη paraffin sections were deparaffinized by heating at 55-65°C for 10 min, cleared with xylene, rehydrated through an ethanol gradient to water. Sections were blocked using the appropriate blocking buffer (either 2% Goat or Donkey Serum in PBS containing 1% bovine serum albumin (BSA), 0.1% Triton-XlOO (Sigma), and 0.05% Tween 20, and 0.05% sodium azide. Rat anti-sera generated against C. rodentium specific Tir (1 :5K; gift from W.

Deng) was used as the primary antibody. Epifluorescent labeling for was carried out with the appropriate secondary antibody using AlexaFluor 568-conjugated goat anti-rat IgG. Tissues were mounted using ProLong Gold Antifade reagent (Molecular Probes/Invitrogen) that contains 4 ',6'- diamidino-2-phenylindole (DAPI) for DNA staining. Sections were viewed on a Zeiss

Axiolmager microscope. Images were obtained using a Zeiss Axiolmager microscope equipped with an AxioCamHRm camera operating through Axio Vision software (Version 4.4). Fluorescence in-situ hybridization

Carnoys-fixed paraffin-embedded sections were deparaffinized and rehydrated as described above. Sections were incubated overnight at 37°C in the dark with Texas red- conjugated EUB338 general bacterial probe (5'-GCT GCC TCC CGT AGG AGT-3') and an AlexaFluor 488 conjugated GAM42a probe (5 '-GCC TTC CCA CAT CGT TT-3 ') that recognizes bacteria that belong to the γ-Proteobacter class diluted to a final concentration of 2.5 ng/μΐ each in hybridization solution (0.9 M NaCl, 0.1 M Tris pH 7.2, 30% Formamide, 0.1% SDS). Sections were then washed once in the dark with hybridization solution for 15 minutes with gentle shaking. This step was repeated once with wash buffer (0.9 M NaCL, 0.1 M TRIS pH 7.2), and sections were placed in dH20, and then mounted using Pro Long Gold Antifadereagent with DAPI (Molecular Probes) and imaged as described above. For quantification studies, the methods were carried as previously described

Transmission Electron Microscopy

GF wild-type mice were orally infected with C. rodentium. At day 5 and 21 post infection, cecums from infected mice were collected and fixed with 2.5 % glutaraldehyde in 0.1 M Sorensen's buffer (pH 7.4). Fixed tissues were then 1 %> osmium tetroxide in 0.1 M

Sorensen's buffer, sequentially dehydrated through graded alcohols and propylene oxide, and then infiltrated in Spurrs or Epon. Ultrathin sections were cut with a diamond knife, stained, and examined with Philips CM- 100 transmission electron microscope.

Carbohydrate growth array

Mouse-derived commensal bacteria used for competition experiments with C. rodentium were Escherichia coli (strain dnl5.6244.1), Bacteroides thetaiotaomicron (strain dnklv9) and B. vulgates (strain dnklv7) (22). For gnotobiotic mouse inoculations, C. rodentium and E. coli were grown aerobically overnight in LB broth with shaking. Both Bacteroides species were grown at 37°C overnight in pre -reduced chopped-meat broth (Beckton Dickinson) under an atmosphere of 5% H₂, 5%> C0₂ and 90%> N₂ in an anaerobic growth chamber (Coy Manufacturing, Grass Lake, MI). Gnotobiotic mice were fed either an autoclaved conventional PICO LAB rodent diet 5L0D (PMI Nutrition International, LLC), or an irradiated simple sugar diet, a customized version of Harlan TD.08810 in which starch and maltodextrin were replaced with sucrose (64% final w/w). For growth profiling in a custom carbohydrate growth array, each species was grown as indicated above and inoculated into 2x carbohydrate-free minimal medium as described previously (26), except that 0.5%> chopped-meat extract was added to the medium for B. vulgatus and E. coli. To quantify the amount of growth on each carbohydrate tested, the maximum absorbance (600nm) was measured for each culture after 4d of anaerobic growth, then both the starting absorbance on each substrate and the amount of growth, if any, in minimal medium lacking a carbon source were subtracted. Growth data was recorded at 10-25 min intervals for the entire 4d period and each positive growth curve was manually inspected to validate the presence of an exponential growth profile. Because each species grew to varying overall levels in the anaerobic media conditions, the growth ability on each substrate was normalized to the average growth ability of a given species on all substrates for which it demonstrated growth. By this normalization strategy, values >1 indicate above average growth; values <1 indicate below average growth.

Measurement of C. rodentium-reactiye immunoglobulin

C. rodentium-specific Ig was quantified by coating 96-well plates with heat-killed C. rodentium as described (18) using alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG, IgM or IgA Abs (Southern Biotechnology Associates, Birmingham, AL). Plates were developed using p-nitrophenyl phosphate substrate (Southern Biotechnology Associates) and OD405 values determined.

Isolation and stimulation of colonic CD4+ T cells.

Colonic CD4+ T cells were isolated from total colonic cells as described (25), using mouse CD4+ cell MACS (MiltenyiBiotec). Purified T cells were stimulated with phorbol 12- myristate 13 -acetate (50 ng/ml; Sigma- Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of GolgiStop (BD Pharmingen), and the cells producing IFN-γ and IL-17A were then stained with FITC-labeled anti-mouse IFN-ymAb (eBioscience) and PE-labeled anti-mouse IL-17A mAb (eBiosciences), and analyzed by flow cytometry. For ELISA, total LP cells were stimulated plate-bound anti-CD3 Ab (10 μg/ml) and soluble anti-CD28 Ab (2 μg/ml) for 48 h. IFN-γ, IL-17A and IL-22 in the supernatants were measured.

Measurement of anti-microbial peptide expression

At day 0 and day 12 after C. rodentium infection, colons were removed from SPF and GF mice. Colons were opened, washed with PBS and incubated in lOmM EDT A/PBS for 30 min at room temperature with gentle shaking. After EDTA treatment, colonic tissues were cut into small pieces and put into HBSS. Tissues were then shaken intensely for 10 sec, and epithelial cells were collected using a 70 μιη cell strainer. R As were isolated from colonic epithelial cells using Total R A Kit I (Omega Bio-tek), and cDNA was synthesized using High Capacity R A-to- cDNA Kit (Applied Biosystems), according to the manufacturer's instructions. Quantitative real time RT-PCR (qPCR) was performed using a SYBR green PCR master mix and StepOne Realtime PCR system (Applied Biosystems). Relative mRNA expression was calculated by the ACt method and normalized to the expression of β-actin. The following primer sets were used:

Regllly; 5 ' -TCAGGTGC AAGGTGAAGTTG-3 ' and 5 ' -GGCCACTGTTACC ACTGCTT-3 ' . PDef-l; 5 ' - AGGTGTTGGC ATTCTC AC AAG-3 ' and 5'-GCTTATCTGGTTTACAGGTTCCC- 3'.

PDef-2; 5 ' -TATGCTGCCTCCTTTTCTCA-3 ' and 5 ' -GACTTCC ATGTGCTTCCTTC-3 ' .βϋεί- 3; 5'- GTCTCC ACCTGC AGCTTTTAG-3 ' and 5 '-AGGAAAGGAACTCCACAACTGC- 3'.pDef-4; 5'- GCAGCCTTTACCCAAATTATC-3' and 5'-ACAATTGCCAATCTGTCGAA- 3'. β-actin; 5'-

AAGTGTGACGTTGACATCCG-3 ' and 5 ' -GATCC AC ATCTGCTGGAAGG-3 ' . Statistical analyses

Statistical analyses were performed using GraphPad Prism software version 5.0

(GraphPad Software Inc.). Differences between two groups were evaluated using Student's t test (parametric) or Mann- Whitney U test (non-parametric). For the multiple comparisons, statistical analysis was performed using one-way AN OVA (parametric) or Kruscal-Wallis test (non- parametric), and then the Dunnett's or Bonferroni test for parametric samples, or Dunn's test for non-parametric samples as a post-hoc test. Survival between groups of mice was compared using Log-rank (Mantel-Cox) test. Differences at <0.05 were considered significant.

B. Results

To assess the role of the microbiota in enteric infection, germ-free (GF) and specific pathogen- free (SPF) C57BL/6 mice reared in standard animal housing conditions were orally inoculated with C. rodentium. Consistent with previous results (5), the pathogen rapidly colonized the intestine of conventional SPF mice reaching maximal concentration in the feces on day 7-10 post infection (108cfu/gram), followed by a decline by day 12 and becoming undetectable by day 22 (Fig. 1A). In contrast, GF mice harbored 10-fold more C. rodentium on day 7-10 post infection, but unlike SPF mice, they were unable to clear C. rodentium even by day 42 when the experiments were terminated (Fig. 1A). Notably, all GF infected mice remained alive despite high and persistent pathogen burdens (Fig. IB).

Infection of mice with C. rodentium triggers the recruitment of neutrophils and macrophages in the intestine (9-12). To determine whether the failure of GF mice to clear the pathogen was associated with impaired inflammatory responses, the recruitment of acute inflammatory cells in the intestine of SPF and GF mice after infection by flow cytometry was monitored. In infected animals, there was an increase in the influx of neutrophils

(CD1 lb+GrlhighF4/80-) to the colon on day 12 when inflammation is known to peak, but neutrophil numbers were reduced by day 22; however, this was comparable in SPF and GF mice (Fig. 1C and ID). Likewise, the recruitment of inflammatory macrophages

CD1 lb+GrlhighF4/80+) and CD3+ T cells was similar in SPF and GF mice (Fig. 1C and ID. Consistently, histological analysis revealed comparable pathology score on day 12 post-infection which declined on day 22 in GF and SPF mice (Fig.lE and IF). Because the microbiota is known to trigger the production of several anti-microbial peptides in the colon (13), their induction in the intestine after infection with C. rodentiumm SPF and GF mice was assesed. Comparable mR A expression of the anti-microbial peptides Regllly, β-defensin-l, P-defensin-3, and P-defensin-4 after oral infection with C. rodentium in the colon of SPF and GF mice was found (Fig. 5). These results indicate that the presence of microbiota is required for the elimination of C. rodentium, and that persistence of the pathogen in the intestine of GF mice is not associated with an impaired inflammatory or anti-microbial response to the bacterium.

The expression of most LEE genes in C. rodentium and other A/E pathogens is controlled by Ler (LEEencoded regulator), a 15 kDa protein belonging to the H-NS family of nucleoid- associated proteins (3, 4, 14). Because GF mice remained healthy despite robust pathogen colonization, ler expression in SPF and GF mice was compared by real-time quantitative (qPCR) during the early (day 7) and late phase (day 12) of infection to determine whether colonizing bacteria express this critical regulatory gene. It was found that ler was expressed in the feces on day 7 post-infection in both GF and SPF mice (Fig. 2A), which correlated with robust growth of C. rodentium in the intestine (Fig. 2D). Expression of ler and tir, a Ler-regulated LEE gene and critical virulence factor that is also required for pathogen colonization, were both down-regulated on day 12 after infection in both GF and SPF mice and were not expressed at day 42 in GF mice despite the continued high level of pathogen colonization (Fig.2A, D and fig. 6). To directly monitor the expression of ler in the mouse intestine, a bioluminescent reporter C. rodentium strain in which the ler promoter is fused to the /wxCDABE operon of Photorhabdusluminescens was utilized (Fig. 7) (15). Consistent with the qPCR results, ler-lux expression was detected in the feces on day 5, but down-regulated by day 7-12 post-infection in both SPF and GF mice (Fig. 2B). ler-lux expression was visualized on day 5 post-infection in the ileum, cecum and distal colon/rectum, but was down-regulated on day 14 in both SPF and GF mice (Fig. 2C), even though the bacterial load had not declined at day 14 (Fig. 1A). The ler luminescent signal was ~ 10-fold higher in GF mice than in SPF mice which correlated with a comparably higher pathogen load in GF mice (Fig. 1A). If the C. rodentium was harvested from infected GF mice at day 21 and reinoculated into conventional SPF mice, the pathogen robustly grew in the intestines of the newly infected mice, infecting the epithelium and triggering colonic inflammation (Fig. 8), indicating that down-regulation of Ler was not due to selection of avirulent mutant bacteria in the intestine. These results indicate that Ler-dependent expression of LEE virulence genes occurs early during infection in both GF and conventional SPF mice, but is down-regulated prior to the decline of pathogen load in the intestine of conventional mice.

Since expression of virulence LEE genes is essential for pathogen colonization in the intestine of conventional SPF mice (4), it was next invenstigaed whether LEE virulence was required for colonization in GF mice. To assess this, GF and conventional SPF mice were orally infected with wild-type C. rodentium and an isogenic strain deficient in Ler {Aler), the master regulator of LEE genes (4). Wild-type, but not the Aler mutant strain, colonized SPF mice (Fig. 2D). The Aler mutant grew robustly in GF mice, reaching numbers similar to the wild-type pathogen (Fig. 2D). Both SPF and GF mice remained viable after infection with wild-type and Aler C. rodentium (Fig. 2E). These results demonstrate that in the absence of microbiota, Ler- dependent LEE virulence is not required for C. rodentium colonization and growth in the intestine. To determine whether Ler regulates the ability of the microbiota to out-compete the pathogen, GF mice were orally infected with wild-type and Aler mutant C. rodentium and the infected GF mice were naturally colonized with commensal microbes by cohousing them at day 3 or day 21 post-infection with conventional SPF mice. The burden of wild-type C. rodentium in GF mice harboring the pathogen for 21 days declined significantly by day 3 and was further reduced by 5-6-logs by day 7 after cohousing, similarly to the decline observed in mice infected with the Aler mutant for 3 and 21 days (Fig. 2F and Figure 9). In contrast, the microbiota could not out-compete the wild-type bacterium on day 3 post-infection upon co-housing with SPF mice (Fig. 2F), consistent with the observation that ler expression is active in wild-type C. rodentium at day 3 post-infection (Figure 10). The results indicate that Ler-regulated virulence is important to determine the the intestinal localization of C. rodentium and the ability of the pathogen to out- compete the microbiota.

C. rodentium induces marked inflammation in the distal colon which requires Ler- mediated expression of virulence factors including the T3SS (4). If the T3SS promotes outgrowth of C. rodentium by enabling the pathogen to colonize a niche at or near the epithelium, it would be expected that impaired colonization of the AescN mutant could not be rescued when inflammation is provided by other means. To test this, colitis was induced in mice by oral administration of 2.5 % dextran sulfate sodium (DSS), a chemical that directly damages the colonic epithelium (17) for 5 days and then infected the mice with wild-type or AescN mutant C. rodentium. Administration of DSS enhanced the colonization of the wild-type bacterium by 3 logs but not that of the AescN mutant (Fig. 11). In addition, co-infection of wild-type C.

rodentium that induces inflammation with the AescN mutant strain did not rescue colonization by the T3SS mutant strain (Fig. 11). These results indicate that inflammation promotes colonization of wild-type C. rodentium, but not in the absence of a functional T3SS.

Recent studies have indicated that the adaptive immune system and specifically IgG production and Thl7 responses against the pathogen are important for the intestinal eradication of C. rodentium (16-19). The observation that the adaptive immune system is critical for pathogen eradication prompted an examination of the role of the microbiota in the induction of specific T-cell and B-cell responses against C.rodentium. To directly test this, SPF and GF mice were orally infected with C. rodentium and the development of immunoglobulin and T-cell immune responses against the pathogen were assesed. The production of IgG, IgM, and IgA against C. rodentium in the serum was unimpaired in GF and SPF mice (Fig. 12). In line with these results, the generation of colonic IL-17 and interferon-y-producing T cells in response to C. rodentium was comparable in SPF and GF mice (Fig. 13). Thus, unimpaired induction of adaptive immunity does not explain the failure of GF mice to clear the pathogen.

During the early phase of infection, C. rodentium is known to colonize and infect the apical surface of the intestinal epithelium, a site largely devoid of commensals (20). To determine whether Ler expression controls the intestinal localization of the bacterium, GF mice were orally infected with wild-type or Aler C.rodentium and cecum and rectum were harvested on day 5 when pathogen growth and ler expression is highest and on day 21 when ler expression is down-regulated. At day 5 post-infection, Tir, a Lerdependent factor, and a marker of C.

rodentium attachment to the epithelium was seen on the intestinal surface in the cecum of GF mice infected with the wild-type C. rodentium, but not with the Aler strain (Fig. 3A). In contrast, Tir labeling was not detected on day 21 post-infection on the intestinal surface of mice infected with either wild-type or mutant C. rodentium (Fig. 3A). Consistently, the wild-type pathogen was largely associated with the epithelium whereas the Aler C. rodentium localized to the intestinal lumen on day 5 post-infection as determined by dual fluorescence in- situ hybridization (Fig. 3B). On day 21 post-infection, however, neither wild-type or Aler C. rodentium detected on the epithelium, but instead they localized to the intestinal lumen (Fig. 3B). The differential localization of the pathogen was independently verified by transmission electron microscopy that revealed C. rodentium at or near the intestinal epithelium on day 5, but not on day 21 post- infection (Fig. 3C). Aler C. rodentium did not localize at or near the epithelium on day 5 post-infection (Fig. 3C). Consistent with these results, Ler-expressing C. rodentium tightly associated to the cecum and colon was abundant in the early phase of infection in GF mice, but dramatically decreased in the later phase of the infection, despite that pathogen burdens in feces were comparable (Fig. 3D and 3E). The results indicate that the localization of the pathogen differs in the early and late phase of infection and this is controlled by LEE virulence expression.

The intestine harbors a large number of bacterial species (21). Upon infection with C. rodentium there is a significant decrease in the total number of anaerobic commensal bacteria, but an increase of aerobic γ-Proteobacteria such as E. coli (7, 8). To determine whether commensal bacteria exhibit different abilities to out-compete C. rodentium, GF mice were orally infected with C. rodentium and the infected GF mice were naturally colonized on day 21 postinfection with either E. coli or one of two different anaerobic Bacteroides species, all isolated from the intestine of SPF mice (22). The burden of C. rodentium in GF mice declined about 200- fold by day 3 and more than 500-fold by day 14 upon colonization with E. coli, but not at all, with B. thetaiotaomicron or B. vulgatus (Fig. 4A). Secondary administration of E. coli to GF mice colonized with B. thetaiotaomicron or B. vulgatus also reduced the number of C. rodentium in the feces about 500-fold (Fig. 4A). In contrast, colonization of mice harboring E. coli with B. thetaiotaomicron or B. vulgatus was not effective in further reducing the burden of C. rodentium in the feces (Fig.4A). Assessment of total Enterobacteria (E. coli and C. rodentium) and Bacteroides on day 14 post colonization with individual commensal bacteria showed comparable number of bacteria -1010 cfu/gram in the feces of GF mice (Fig. 4B). Before inoculation of commensal bacteria (dO), all Enterobacteria detected in the feces were C. rodentium (Fig. 4C). In contrast, > 99% of the total Enterobacteria in the E. coli inoculated group were E.coli after 14 days of colonization (Fig. 4C). 100% of total Enterobacteria were C. rodentium in B.

thetaiotaomicron or B. vulgatus inoculated groups, since Bacteroides strains were unable to grow under this condition (Fig. 4C). These results indicate that the differing abilities of E. coli vs B. thetaiotaomicron or B. vulgatus to out-compete C. rodentium cannot be explained simply by differences in bacterial colonization and suggest that E. coli and C. rodentium compete for similar niches in the mouse gut. To explore this concept further, the growth abilities of each of these species was analyzed on a custom carbohydrate growth array that contains most of the common mono- and polysaccharides present in plant and animal tissue (Fig. 4D and table 1). Both C. rodentium and E. coli exhibited optimal growth on monosaccharides (Fig. 4D). They were both generally deficient in the ability to grow on more complex polysaccharides; although, C. rodentium exhibited a small amount of growth on several different polysaccharides (Fig. 4D), indicating that it possesses some capacity to target more complex carbohydrate structures present in the gut. In contrast, both B. thetaiotaomicron and B. vulgatus exhibited broad abilities to catabolize both mono- and polysaccharides (Fig. 4D). These results indicate that commensal populations differ in their ability to out-compete C. rodentiun in the mouse intestine and this correlates with their ability to grow on structurally distinct carbohydrates. To further assess if competition for glycans between commensals and C. rodentium is important for pathogen eradication, GF mice on a conventional maintenance diet containing both mono- and

polysaccharides were orally infected with C. rodentium and on day 21 post-infection the infected GF mice were colonized with B. thetaiotaomicron for 7 days. On day 7 post-colonization with B. thetaiotaomicron, the mice were divided into two groups that were fed the conventional diet or a simple sugar diet containing monosaccharides but not polysaccharides. The burden of C.

rodentium in GF mice on the simple sugar diet declined ~ 200-fold by day 3, whereas no decline was observed when the mice were fed the conventional diet (Fig. 4E). The results were not explained by reduced C. rodentium burden in GF mice on the simple sugar diet because in the absence of colonization with B. thetaiotaomicron, the number of C. rodentium in feces was comparable between animals fed with the conventional and simple sugar diets (Fig. 4F).

The results indicate that during the early phase of infection, C. rodentium expresses Independent LEE genes that mediate their ability to infect the intestinal epithelium and trigger inflammatory responses (4), which in turn reduce the total numbers of colonic commensal bacteria, but increase the density of j-Proteobacteria (7, 8). These events allow the pathogen to compete with the commensal microbiota by promoting colonization of unique intestinal niches that support C. rodentium growth. This model is supported by the finding that avirulent Aler C. rodentium cannot colonize the intestines of conventional SPF mice, but grows robustly in GF mice. Thus, Ler-mediated virulence is not required for colonization and growth of the pathogen in the intestine per se, but rather is critical in colonizing the epithelial surface, allowing C.

rodentium to compete among the indigenous microbiota. Furthermore, it was shown that Ler controls the localization of the pathogen to epithelial niches that are largely devoid of commensals and may provide nutritional sources allowing the pathogen to out-compete the commensal microbiota. LEE virulence is down-regulated by day 12 of infection in the intestines of infected mice and that reduced expression correlates with C. rodentium clearance. The concomitant reduction of LEE virulence in GF mice upon ler inactivation explains the finding that microbiota-free mice can sustain robust and prolonged colonization of the pathogen without obvious adverse effects. Upon ler down-regulation, C. rodentium-mediatGd intestinal

inflammation recedes, allowing the recovery of the intestinal microbiota, which then out- competes and eradicates the pathogen not expressing LEE genes. It was found that E. coli, but not B. thetaiotaomicron and B. vulgatus exhibited broad abilities to catabolize both mono- and polysaccharides, consistent with previous reports of the broad glycan-degrading potential of various Bacteroidetes species (23, 24). The list of tested growth substrates only includes carbohydrates, and not other potential nutrients (peptides, amino acids, and lipids) that could be targeted by these species. Nevertheless, the similar catabolic preferences for monosaccharides by C. rodentium and E. coli, combined with their close phylogenetic relationship, support the idea that they would compete most directly for similar nutrient pools in vivo. In contrast, the

Bacteroides species tested have ample metabolic alternatives (i.e., plant or animal

polysaccharides) they can utilize that may limit their direct competition with C. rodentium in vivo. Because γ-Proteobacteria such as E. coli specifically accumulates after C. rodentium infection, these results indicate that this change in commensal populations benefit the host by increasing the number of commensals that can out-compete the pathogen. An important role for glycans in determining the ability of commensals to out-compete C. rodentium is supported by the finding that B. thetaiotaomicron can reduce the intestinal burden of the pathogen only when the mice are fed a simple sugar diet. Under these conditions, B. thetaiotaomicron cannot use polysaccharides and is forced to compete with C. rodentium for available monosaccharides or another common nutrient that both C. rodentium and B. thetaiotaomicron rely on in the absence of dietary polysaccharides. These findings clarify the tenuous nature of A/E pathogens, in terms of their survival/co Ionization within hosts, and therefore nutrient or probiotic shifting of the microbiota to promote commensals that directly compete with pathogens for food sources may be a useful therapeutic approach.

Table 1

Example 2

This example describes methods of screening inhibitors of s. coli virulence genes.

Use of small molecule inhibitors of LEE virulence to enhance C. rodentium eradication by the microbiota

Two classes of small molecules that have been shown to inhibit LEE virulence factors in A/E enteric pathogens are used. The first class are salicylidence acyl hydrazide compounds that have been shown to reduce the expression of T3SS effectors including those from A/E pathogens at -1-20 μΜ range. Five compounds of this class (ME0054, ME055, ME097, MEOl 14, and ME0214) are obtained from Dr. Mikael Elofsson of Umea University in Sweden. These compounds do not inhibit bacteria growth, but can inhibit expression of several LEE virulence genes including Ler and formation of A/E lesions on bovine epithelial cells.

The second class of small-molecules is represented by aurodox, a linear polyketide compound that is a specific T3SS inhibitor of EPEC and C. rodentium and therefore targets multiple LEE virulence factors. Aurodox is commercially available and has been shown to protect mice from C. rodentium infection in vivo, although its effect on bacterial loads and mechanism by which improves survival were not investigated.

First, each compound is tested at 10, 30 and 60 mg/Kg-1 or saline (mock treatment) in groups of mice (n=6). In the case of aurodox, 25 mg/Kg-1 given orally for 4 consecutive days was found to be effective in inhibiting infection-associated lethality. Thus, SPF B6 mice are given orally each compound in 5% DMSO or saline in 5% DMSO at day 0 with a gavage needle and infected 2 hrs later orally with 109 cfu of C. rodentium. Administration of the compound via the same route is given daily for 3 more days. Pathogen loads are monitored in feces at day 3, 5, 7, 12 and 14 after infection. LEE virulence gene expression (ler and two T3SS effectors tir and intimin) is quantitated on day 1, 3, 5, and 7 post-infection in fecal pellets when LEE virulence genes are induced by real-time qPCR.

Use of antibodies against LEE virulence factors to enhance C. rodentium eradication by the microbiota

The advantage of small-molecules to inhibit LEE virulence is that they are relatively inexpensive which is useful important for treatment of A E pathogen-associated colitis in underdeveloped countries. However, small-molecules may target additional pathogen or host signaling pathways. Therefore, antibodies with defined specificity against LEE factors are used to inhibit Ler and pathogen colonization. Two types of approaches and antibodies are tested. Rabbits were immunized with the purified C-terminal 382 amino acid extracellular domain of intimin from C. rodentium in CFA and after boosting, high titer antibodies against the protein were obtained. Intimin mediates the intimate interaction of the pathogen with the host epithelium and C. rodentium mutant lacking intimin cannot colonize SPF mice. Furthermore, C. rodentium- infected mice develop anti-intimin antibodies in the intestinal content and serum (Figure 15). However, mice and humans infected with enteric A/E pathogens develop antibodies against several LEE factors in addition to intimin (Figure 14). Therefore, it is possible that single or combination of antibodies against LEE factors other than intimin might be involved in the regulation of Ler expression or be more effective in reducing Ler-mediated virulence in vivo. Llamas were immunized with purified intimin or membrane extracts of C. rodentium expressing LEE virulence factors and total RNA purified from the blood was used to generate a single domain heavy-chain antibody (nanobody) phage display library. The approach to generate nanobodies using phage display library is now well established and has been successful against a wide variety of antigens including after immunization with extracts from whole bacteria. Unlike most mammals, Camelidae including llamas develop heavy-chain antibodies devoid of light chain which can be manipulated genetically and exhibit high affinity for antigen, tissue penetration and stability. Because the antigen-binding site is reduced to a single variable domain, PCR amplification of the these fragments containing antigenic diversity can be cloned into a phage display vector and electroporated into E. coli to obtain a library of 10⁶ - 10⁷ individual colonies. E. coli are infected with helper phage to generate phage particles expressing individual single variable domain antibodies. Nanobodies targeting intimin will be selected by panning the phage libraries against immobilized intimin, and by screening individual clones for intimin binding using ELISA. Intimin-binding nanobodies are screened for an inhibitory effect on TIR - intimin binding and antagonistic

nanobodies are purified to milligram scale for the use in C. rodentium infection studies in mice. To enrich for antibodies specific for LEE virulence factors, ~5 x 108 viral particles are incubated with immunotubes pre-coated with extracts from Ler-deficient C. rodentium (negative selection) and non-binding clones are panned against extracts from Ler-expressing bacteria (positive selection) to generate a library enriched for antibody clones reacting with LEE virulence factors. The enriched library for LEE virulence factors is then split into individual clones and clones reacting with extracts from wild-type C. rodentium are further screened with extracts from Ler- deficient bacteria to select antibody clones that recognize Ler-dependent factors. Nanobodies binding LEE virulence factors are purified and screened for an antagonistic effect on C.

rodentium virulence.

In order to determine which C. rodentium surface factors regulate LEE virulence, the nanobody library is screened for individual C. rodentium binding clones that display a similar regulatory function observed from the mouse polyclonal antibodies. For this, the ler-lux C.

rodentium reporter strain is incubated with nanobody extracts from positive C. rodentium binding clones (initially pooled per 10-20) and screen for clones that influence Ler expression as read out by luminescence assay. Positive pools are split into individual clones to identify the unique clones that demonstrate Ler regulation. Upon identification of Ler-active nanobodies, their molecular target on the C. rodentium surface is pulled down by co-immunoprecipitation and/or tandem affinity purification, and identified using mass spectrometry. The same strategy is used to test the ability of polyclonal rabbit and llama monoclonal antibody clones to inhibit LER expression and to promote the eradication of C. rodentium in vivo. It is first tested whether administration of rabbit anti-intimin antibodies inhibit Ler expression and pathogen colonization in the intestine of pathogen-infected mice. The rabbit sera with high titer anti-intimin antibodies is used to purify specific IgG using an intimin-affinity column. Because oral administration of antibodies is known to be effective in inducing regulatory activity in the gut, it is tested whether oral administration of purified rabbit IgG against intimin or control IgG can affect the virulence and colonization of C. rodentium in vivo. In these experiments, conventional SPF mice (n=6) are infected orally with the ler-lux C. rodentium reporter strain and the mice are given orally anti-intimin IgG (40 mg/kg) or control IgG on the day 0, 1 and 2 post infection. Pathogen burden in feces and Ler expression in the feces and the intestine of individual mice is measured by bio luminescence on day 1, 3, 7, 12, 15 post-infection and compared with that of mock-treated mice. It is contemplated that inhibition of LEE virulence by specific antibodies will promote pathogen eradication in a microbiota-dependent manner given that the loads of Ler-deficient C. rodentium are not reduced at all in GF mice. To test this, anti-intimin antibody or control IgG is given orally to groups of SPF and GF mice infected with the ler-lux C. rodentium reporter strain and pathogen load and Ler expression is measured in the feces as outlined above. References

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All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method of inhibiting a biological activity of a virulence factor of a pathogenic E. coli, comprising: contacting said virulence factor or a cell expressing said virulence factor with a composition that inhibits a biological activity of the virulence factor.

2. The method of claim 1, wherein said pathogenic E. coli is selected from the group consisting of enterohemorragic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC).

3. The method of claim 1, wherein said composition is selected from the group consisting of a small molecule and an antibody that specifically binds to said virulence factor.

4. The method of claim 3, wherein said small molecule is a salicylidence acyle hydrazine compound.

5. The method of claim 4, wherein said salicylidence acyle hydrazine compound is selected from the group consisting of ME0054, ME055, ME097, MEOl 14, and ME0214.

6. The method of claim 3, wherein said small molecule is aurodox.

7. The method of claim 3, wherein said antibody binds specifically to a LEE factor.

8. The method of claim 7, wherein said LEE factor is intimin.

9. The method of claim 1 , wherein said inhibiting treats infection by said pathogenic

E. coli.

10. The method of claim 9, wherein said inhibiting results in commensal E. coli out competing said pathogenic E. coli.