WO2009121848A2 - Use of pseudomonas aeruginosa alkaline protease (apra) and inhibitors thereof in medicine and agriculture - Google Patents
Use of pseudomonas aeruginosa alkaline protease (apra) and inhibitors thereof in medicine and agriculture Download PDFInfo
- Publication number
- WO2009121848A2 WO2009121848A2 PCT/EP2009/053758 EP2009053758W WO2009121848A2 WO 2009121848 A2 WO2009121848 A2 WO 2009121848A2 EP 2009053758 W EP2009053758 W EP 2009053758W WO 2009121848 A2 WO2009121848 A2 WO 2009121848A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- apra
- flagellin
- alkaline protease
- pseudomonas aeruginosa
- inhibitor
- Prior art date
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
The invention relates to an inhibitor of Pseudomonas aeruginosa alkaline protease for use in the treatment or prevention of infectious diseases in humans, animals and plants. The treatment suitably comprises inhibiting the interference of Pseudomonas aeruginosa alkaline protease with a microbial molecule that is recognized by receptors involved in innate immunity, in particular flagellin. The receptors involved in innate immunity are Toll-like receptors, in particular the Toll-like receptor 5 (TLR5), or the flagellin locus 2 (FLS2) receptor.
Description
USE OF PSEUDOMONAS AERUGINOSA ALKALINE PROTEASE (AprA) AND INHIBITORS THEREOF IN MEDICINE AND AGRICULTURE
FIELD OF THE INVENTION The present invention relates to the new use of the
Pseudomonas aeruginosa alkaline protease (AprA) and inhibitors thereof in medicine and agriculture, in particular for the treatment of infections with Pseudomonas aeruginosa, other Pseudomonas bacteria and even other Gram- negative bacteria in humans, animals and plants.
BACKGROUND OF THE INVENTION
Pseudomonas is a common environmental Gram-negative bacterium, which acts as an opportunistic pathogen. Normally, human hosts counteract this microorganism effectively via the innate immune system (Luczak et al (2000) Microbes Infect 9:1051-60). However, immunocompromised patients like severe burn victims and cystic fibrosis patients are extremely sensitive to Pseudomonas aeruginosa infections. Pseudomonas aeruginosa is known for its resistance to antibiotics.
The bacterium is naturally resistant to many antibiotics due to its tendency to colonize surfaces in a biofilm which makes the cells impervious to therapeutic concentrations of antibiotics. Since its natural habitat is the soil, living in association with the bacilli, actinomycetes and molds, it has developed resistance to a variety of their naturally-occuring antibiotics. Moreover, Pseudomonas maintains antibiotic resistance plasmids, both R-factors and RTFs, and it is able to transfer these genes by means of the bacterial mechanisms of horizontal gene transfer (HGT), mainly transduction and conjugation.
Two extracellular proteases have been associated with virulence that exert their activity at the invasive
stage: elastase and alkaline protease. Elastase has several activities that relate to virulence. The enzyme cleaves collagen, IgG, IgA, and complement. It also lyses fibronectin to expose receptors for bacterial attachment on the mucosa of the lung. Elastase disrupts the respiratory epithelium and interferes with ciliary function. So far, alkaline protease was reported to interfere with fibrin formation. Together, elastase and alkaline protease destroy the ground substance of the cornea and other supporting structures composed of fibrin and elastin. Elastase and alkaline protease together are also reported to cause the inactivation of gamma interferon (IFN) and tumor necrosis factor (TNF) .
The innate immune system detects and rapidly responds to invasion of micro-organisms. Pattern recognition receptors like the Toll-like receptors (also identified herein as "TLR") recognize various conserved patterns of micro-organisms and play a crucial role in the innate immune system. Stimulation of these receptors triggers activation of phagocytes and production of pro-inflammatory cytokines. Each TLR recognizes specific microbial molecules. For example, TLR4 detects LPS, while TLR2 recognizes bacterial lipoproteins and lipoteichoic acid.
Toll-like receptor 5 is of crucial importance in gram negative recognition. It recognizes flagellin, the basic and conserved part of a bacterial flagellum. Flagella consist of a basal body, the flagellar hook and a filament which serves as a propeller. The filament is formed by polymerization of flagellin. These structures are essential for bacterial motility and colonization. Flagellin consists of four domains, the conserved DO-Dl domains and the variable D2-D3 domains. The TLR5 recognition site of flagellin lies within the Dl domain, which is buried in the flagellar filament and only accessible in the monomeric form
of flagellin. During infection and growth some flagellin is released, which is capable of stimulating TLR5. In this way one receptor can recognize all motile bacteria. Only monomeric flagellin is recognized by TLR5, not the flagellum itself.
Plants have evolved a similar sensing system for flagellin as mammals. In Arabidopsis thaliana, stimulation of the plasma-membrane-located receptor FLS2 results in the activation of defence responses, such as callose deposition and the production of pathogenesis-related proteins.
Flagellin recognition contributes to the resistance of Arabidopsis to the bacterial pathogen Pseudomonas syringae . Although TLR5 and FLS2 serve a similar function in pathogen recognition, the composition of the receptor, as well as the downstream signaling pathways differ considerably. Furthermore, FLS2 recognizes a different epitope of flagellin than does TLR5. A peptide, called flg22, consisting of 22 amino acids derived from the highly conserved N-terminal region of P. syringae flagellin activates FLS2 even better than purified flagellin.
Some bacteria evolved strategies in which they manipulate flagellin to preserve motility while TLR5 activation is impaired. Immune evasion is a generally accepted important strategy for bacteria to survive in the human host. Immune evasion molecules are crucial for this survival and bacteria particularly evade the human innate immune system.
SUMMARY OF THE INVENTION According to the invention it was found that Toll- like receptor 5 (TLR5) and plant receptor flagellin sensitive locus 2 (FLS 2) recognition can be evaded by alkaline protease of P . aeruginosa . Toll-like receptors (TLRs) recognize various conserved molecules of micro-
organisms. Activation of these receptors is a crucial step in antimicrobial defence. The ligand of TLR5 and FLS2 is flagellin, which is the major component of the bacterial flagellum. Flagellin polymerizes to form long filaments, which are essential for the motility and virulence of flagellated bacteria. TLR5 recognizes a conserved and functionally essential part of monomeric flagellin.
Bacteria secrete proteins that interfere with different pathways of the innate immune system, thereby they evade recognition and killing. According to the invention, a protein was identified and characterized that is secreted by P . aeruginosa as an inhibitor of TLR5 activation.
Human embryonic kidney cells transfected with TLR5 were incubated with bacterial supernatant and stimulated with flagellin. IL-8 production of HEK-TLR5 cells was measured as readout for TLR5 activation. Recombinant AprA was expressed and isolated from E.coli. Flagellin and flagellar filaments were isolated from P . aeruginosa .
Supernatants of several bacteria were screened for TLR inhibitors and it was identified that P . aeruginosa inhibits TLR5 activation. After fractionation of the supernatant by ion-exchange chromatography and gel filtration the protein of interest was identified by mass spectrometry as alkaline protease (AprA) of P . aeruginosa. AprA is a zinc-metalloprotease that forms a gene cluster together with its inhibitor Aprl and AprD, AprE and AprF, which are necessary for the secretion of AprA.
Recombinant AprA isolated from E.coli also inhibits TLR5 activation, while Aprl blocked this effect. To identify the mechanism of action, the effect of AprA was tested on TLR5 and flagellin. HEK-TLR5 cells were still functional after incubation with AprA, while flagellin was cleaved by alkaline protease. Monomeric flagellin of P . aeruginosa and S . typhimurium were both cleaved in the same way according to
SDS-PAGE. Interestingly, flagellar filaments were not susceptible to degradation by AprA.
It was thus found that AprA of P . aeruginosa is an inhibitor of TLR5 activation and thereby the first bacterial inhibitor described to date with such a function. AprA cleaves flagellin, however flagella are not affected. In this way, bacteria preserve their motility, while they evade recognition via TLR5.
It was furthermore found that ectopic expression of the AprA inhibitor Aprl in crop plants augments the perception of and response to AprA-producing pathogens by the plants, resulting in enhanced disease resistance.
Moreover, it lowers the threshold of detection of crop plants to disease suppressive biocontrol bacteria, such as Pseudomonas spp . , and thereby boosts the level of induced systemic resistance (ISR) to future pathogen attack.
DETAILED DESCRIPTION OF THE INVENTION
Based on their knowledge of immune evasion, the inventors hypothesized that bacteria secrete proteins that interfere with recognition of Toll-like receptors. In their search for Toll-like receptor antagonists they found an activity in the supernatant of Pseudomonas that inhibited TLR5 signalling. After purification using Ion exchange chromatography and gel filtration the metalloproteinase AprA was identified as the active substance. The mechanism of action was revealed as the protease action of AprA on flagellin (the TLR5 ligand) , not on TLR5 itself. AprA cleaves monomeric flagellin, not the flagellum. Knock out strains confirmed this finding.
Here a novel mechanism of P. aeruginosa was indentified to prevent recognition by TLR5 and FLS2, while motility is not affected. The unique features of this
bacterial strategy implement use in antibacterial as well as anti-inflammatory therapy.
The invention thus relates to inhibitors of AprA or homologues or derivates thereof for use in medicine and more in particular for use in the treatment of infectious diseases. The treatment suitably comprises inhibiting the interference of Pseudomonas aeruginosa alkaline protease with a microbial molecule that is recognized by receptors involved in innate immunity in humans, animals or plants. Such receptor is suitably the Toll-like receptor 5 (TLR5)
TLR5) or the plant receptor flagellin sensitive locus 2 (FLS 2) .
Furthermore, in vitro studies demonstrated that Aprl relieves the AprA-mediated immuno-suppressive effects in mammalian and plant hosts (Fig. 18) . Therefore, it is beneficial for host cells to produce Aprl and subsequently secrete this inhibitor to the site where pathogenic Pseudomonas bacteria release AprA. In that case, Aprl prevents bacterially-produced AprA from degrading flagellin monomers and thereby favours activation of host defence signaling. In addition, this Aprl-mediated inhibition of AprA also prevents flagellin-degradation in the rhizosphere and results in increased perception of and response to beneficial ISR-inducing bacteria. According to a further aspect thereof, which is particularly related to the treatment or prevention of infections in plants, the invention relates to the use of the Aprl-gene to make transgenic plants that express Aprl to prevent Gram negative bacterial infections therein. More in particular, the invention relates to the use of the Aprl- gene to make transgenic plants that express Aprl to prevent infections in plants. The invention also relates to a transgenic plant that has no or a lowered susceptibility to being infected by Gram negative bacteria, which transgenic
plant comprises the Aprl-gene in its genome. The Gram negative bacteria are suitably Pseudomonas spp .
An inhibitor of AprA is defined as any compound that interferes with the ability of AprA to break down proteins, in particular the breakdown of bacterial flagellin. Examples of such inhibitors are the Pseudomonas protein Aprl (SEQ ID NO : 2 ) , antibodies against AprA that neutralize the function of AprA, analogues of antibodies such as, but not restricted to, camel bodies, binding bodies, Fab fragments, F(ab')2 fragments, single chain antibodies, etc., blocking peptides, peptidomimetics, small molecules that block the specific function of AprA which is the breakdown of bacterial flagellin in such way that flagellin loses it ability to activate TLR5. The gene for alkaline protease has been identified before. The whole genome of P. aeruginosa PAOl was published (Stover et al . 2000, Nature 406:959-964) and alkaline protease was identified therein under the name AprA (PA1249) . The amino acid sequence of AprA from P. aeruginosa strain PAOl is:
[SEQ ID NO:1]
MSSNSLALKGRSDAYTQVDNFLHAYARGGDELVNGHPSYTVDQAAEQILREQASWQKAPG DSVLTLSYSFLTKPNDFFNTPWKYVSDIYSLGKFSAFSAQQQAQAKLSLQSWSDVTNIHF VDAGQGDQGDLTFGNFSSSVGGAAFAFLPDVPDALKGQSWYLINSSYSANVNPANGNYGR
QTLTHEIGHTLGLSHPGDYNAGEGDPTYADATYAEDTRAYSVMSYWEEQNTGQDFKGAYS SAPLLDDIAAIQKLYGANLTTRTGDTVYGFNSNTERDFYSATSSSSKLVFSVWDAGGNDT LDFSGFSQNQKINLNEKALSDVGGLKGNVSIAAGVTVENAIGGSGSDLLIGNDVANVLKG GAGNDILYGGLGADQLWGGAGADTFVYGDIAESSAAAPDTLRDFVSGQDKIDLSGLDAFV NGGLVLQYVDAFAGKAGQAILSYDAASKAGSLAIDFSGDAHADFAINLIGQATQADIVV
The invention thus relates to the use of alkaline protease inhibitor (Aprl) and other inhibitors to inhibit this TLR5 inhibiting function of AprA. Aprl is also produced
by P. aeruginosa PAOl (Stover et al . 2000, Nature 406:959- 964) identified therein under the name Aprl (PA1250) . The amino acid sequence of Aprl from P. aeruginosa strain PAOl is :
[SEQ ID NO: 2]
MSASAKLSRMVCLLCGFFSTGISMASSLILLSASDLAGQWTLQQDEAPAICHLELRDSEV AEASGYDLGGDTACLTRWLPSEPRAWRPTPAGIALLERGGLTLMLLGRQGEGDYRVQKGD GGQLVLRRATP
According to a further aspect thereof the invention relates to Pseudomonas aeruginosa alkaline protease or a derivative thereof for use in medicine, in particular for use in anti-inflammatory therapy. The anti- inflammatory therapy is suitably effected by interfering with molecules that are recognized by receptors involved in innate immunity.
According to a first embodiment of this aspect of the invention, AprA and also homologues of AprA and derivatives thereof can be used as vaccines in the strategy to inhibit the function of AprA and are thus of therapeutical advantage in infections with Pseudomonas or other bacteria containing AprA homologues. In a further embodiment AprA and homologues and derivatives thereof can be used as molecules to combat inflammatory diseases where flagellin is involved.
Such homologues or derivatives must be functional. Derivatives may for example be fragments, such as peptides, truncated proteins, chimeric proteins comprising at least a functional part of AprA and another part, or peptidomimetic versions of the protein.
Homologues are intended to encompass allelic variants of the P. aeruginosa as well as homologues from other bacteria. So far AprA homologues have been described
in many bacteria. The following bacteria contain an AprA- homologue and are potential pathogens for humans, animals or plants: Pseudomonas aeruginosa, Pseudomonas entomophila, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas mendocina, Pseudomonas syringae, Serratia proteamaculans, Erwinia carotovora, Yersinia intermedia, Yersinia pseudotuberculosis, Yersinia pestis, Yersinia bercovieri, Yersinia mollaretii, Yersinia frederiksenii, Yersinia enterocolitica, Yersinia intermedia. According to the invention, the application of inhibitors of AprA enhances the recognition by the human innate immune system considerably, thereby enhancing clearance of bacteria by the subject's own immune system, thereby reducing morbidity and mortality due to infections. Furthermore, AprA itself or derivatives thereof can be used as anti-inflammatory compounds to combat inflammatory diseases .
The invention will be further illustrated in the Examples that follows and that are given for illustration purposes only and are not intended to limit the invention in any way. Reference is made to the following figures.
FIGURES Figure 1 Schematic representation of the gene cluster for
AprA and Aprl on the genome of Pseudomonas aeruginosa strain PaOl. (PaOl genes Pal245-Pal251, Tigr (Stover et al . 2000, Nature 406: 959-964) )
Figure 2
AprA prevents flagellin-induced interleukin-8 production by HEK/TLR5 cells. HEK/TLR5 cells were incubated with 10 μg/ml AprA for 15 minutes and subsequently challenged with increasing concentrations recombinant
flagellin (from Salmonella typhimurium) . After 6 hours the IL-8 concentration in the cell culture supernatant was measured by ELISA. IL-8 levels are expressed as OD at 450 nm after subtraction of background values and a representative experiment is shown.
Figure 3
AprA concentration dependent inhibition of flagellin-induced HEK/TLR5 cell activation. HEK/TLR5 cells were treated with increasing concentrations AprA and challenged with 1 ng/ml flagellin. After 6 hours IL-8 was measured in the supernatant by ELISA.
Figure 4 AprA also inhibits homologous flagellin-induced
HEK/TLR5 activation. Cells were preincubated with 10 μg/ml AprA and challenged with increasing concentrations native flagellin isolated from Pseudomonas aeruginosa strain PaO25. After 6 hours IL-8 was measured in the supernatant by ELISA.
Figure 5
AprA does not affect TLR5. HEK/TLR5 cells were treated with AprA purified from P. aeruginosa for 15 minutes and directly stimulated or washed before stimulation with increasing concentrations flagellin from S. typhimurium.
After 6 hours IL-8 was measured in the supernatant by ELISA.
Figure 6
Degradation of flagellin by AprA. Flagellin was mixed with 0, 3, 1, 0.3, 0.1, 0.03 and 0.01 μg/ml AprA for 60 minutes at 370C in PBS and protein degradation analyzed on a 12% SDS-PAGE by Coomassie staining. Native flagellin isolated from Pseudomonas PaOl with flagella Type A (gel A) and Pa025 with flagella Type B (gel B) were compared with
recombinant flagellin from type B Pseudomonas (gel C) and Salmonella (gel D) . Molecular weight markers in kDa are indicated.
Figure 7
Flagellin degradation by AprA in time. Flagellin of S. typhimurium was incubated for 0, 1, 3, 10, 30 and 60 min at 370C with AprA purified from P. aeruginosa . In the right panel flagellin degradation by AprA (30 min) in the presence of EDTA is shown.
Figure 8
Intact flagella are not degraded by AprA. Native intact and denaturated (20 min at 7O0C) flagella from Pseudomonas PaOl were mixed with increasing concentrations AprA for 60 min at 370C and analyzed on a 12% SDS-PAGE for flagellin degradation.
Figure 9 Aprl prevents the AprA-mediated flagellin degradation. Recombinant flagellin from P. aeruginosa (A) and S. typhimurium (B) was mixed with 1 μg/ml AprA in the absence or presence of 0, 0.01, 0.03, 0.1, 0.3, 1 and 3 μg/ml Aprl for 60 min and analyzed on a 12% SDS-PAGE for degradation.
Figure 10
Aprl prevents the AprA-mediated inhibition of TLR5 activation by flagellin. HEK/TLR5 cells were challenged with 1 ng/ml flagellin of S. typhimurium in the presence of an increasing concentration AprA premixed with or without Aprl at 0, 0.1, 0.3, 1 or 3 μg/ml. After 6 hours IL-8 production was measured by ELISA.
Figure 11
Lack of AprA gene preserves Pseudomonas flagellin activity for TLR5 activation. Dilutions of bacterial culture supernatants, collected from wild-type (Wt) and isogenic AprA or Aprl knock-out (KO) Pseudomonas strains, were used as flagellin source and added to HEK/TLR5 cells. After 6 hours IL-8 production was measured by ELISA.
Figure 12 Recombinant AprA inactivates endogenous flagellin.
Bacterial supernatants were incubated for 15 min at 370C with exogenous recombinant AprA (3 μg/ml) and dilutions added to HEK/TLR5 cells. After 6 hours IL-8 production was measured by ELISA.
Figure 13
Recombinant Aprl prevents the AprA-mediated flagellin inactivation in bacterial supernatant. Wild-type and knock-out Pseudomonas strains were grown in the presence of 3 μg/ml exogenous recombinant Aprl and dilutions of the culture supernatants were added to HEK/TLR5 cells. After 6 hours IL-8 production was measured by ELISA.
Figure 14 Endogenously produced AprA still inactivates exogenously recombinant flagellin. Bacterial culture supernatant (33%) of wild-type (Wt) and AprA knock-out (KO) strains was mixed with an increasing concentration recombinant flagellin and added to HEK/TLR5 cells. Flagellin mixed with buffer served as control stimulus. After 6 hours IL-8 production was measured by ELISA.
Figure 15
AprA knock-out strains do not produce AprA. Bacterial culture supernatant of wild-type (Wt) , AprA and Aprl knock-outs (KO) was analyzed for the presence of AprA protein by western blotting with a rabbit-anti-AprA antiserum. Recombinant AprA at 0.3 and 3 μg/ml served as reference controls.
Figure 16 AprA prevents recognition of flagellin in
Arabidopsis . A. thaliana La-er seedlings were incubated or not with 500 nM flg22 or P. aeruginosa flagellin that was preincubated with 3 μg/ml AprA when indicated. After treatment for 24 h, seedlings were stained for callose deposition by aniline blue and fluorescence was photographed under UV light. lOμg/ml Aprl was added after AprA treatment or prior to AprA treatment (bottom panel) .
Figure 17 AprA prevents flagellin-induced growth inhibition.
Seedlings were grown axenically for 10 days in MS medium in the presence of 50OnM flg22 or P. aeruginosa flagellin that was preincubated with 3μg/ml AprA when indicated and subsequently photographed. lOμg/ml Aprl was added after AprA treatment or prior to AprA treatment (bottom panel) .
Figure 18
Proposed mechanism for AprA. P. aeruginosa excretes AprA, which degrades free flagellin in the surrounding of the bacterium, whereas polymeric flagellin present in flagella is not affected. In this way flagellin is not recognized by TLR5 and FLS2 and thereby P. aeruginosa escapes activation of the innate immune system in both mammals and plants. AprA is secreted in one step over both
membranes and is not present in its active form in the cytoplasm of the bacterium
EXAMPLES EXAMPLE 1
Materials and Methods
Reagents
Dulbecco's modified Eagle's medium (DMEM) and Iscoves modified Dulbecco's medium (IMDM) (Invitrogen) , fetal bovine serum (Gibco) , Normocin and Blasticidin (both Invivogen) were used for cell and bacterial culture.
Pseudmonas aeruginosa strain PaO25, alkaline protease rabbit antiserum and plasmids pJFl and pAG302 were a gift from Prof. J. Tommassen (University of Utrecht, The Netherlands) .
IL-8 ELISA kit and high performance ELISA buffer (HPE) were purchased from Sanquin (Amsterdam, The Netherlands) .
Clinical isolates of P. aeruginosa and S. typhimurium were obtained within the UMC Utrecht. Competent E.coli TOPlOF' and BL21 (DE3) pLys were purchased from Invitrogen .
HEK-TLR5 cells assay Human embryonic kidney cells transfected with human TLR5 (Invivogen) were maintained in DMEM supplemented with 10% FCS, lOug/ml Blasticidin and lOOug/ml Normocin. Monolayers of HEK/TLR5 cells were preincubated with bacterial supernatant or purified proteins and subsequently stimulated with flagellin for 6 hours at 370C. Supernatant of cells was harvested and stored at -2O0C for analysis. Samples were diluted 100 times in HPE buffer and IL-8 levels were measured by ELISA following manufacturer's protocol. IL-8 levels are expressed as OD at 450 nm.
To measure NF-kB activation HEK/TLR5 cells were transiently transfected 2-3 days before stimulation with a NF-kB reporter plasmid pHIV-CAT (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH; (Nabel et al . Nature (1987) 326 ( 6114) : 711-3) . Transfected cells were stimulated with LPS or flagellin for five hours at 370C. Cells were lysed with lysis buffer/substrate (Promega) according to manufacturer's protocol. Chemiluminescence was measured using a Centro LB 960 microplate luminometer (Berthold, Germany) . NF-kB activation is expressed as stimulation index, which represents the ratio between stimulated and control cells.
Isolation and purification of alkaline protease and alkaline protease inhibitor
Pseudomonas aeruginosa was cultured overnight in IMDM and supernatant was collected by centrifugation and filtration. Supernatant was applied on a Q sepharose XL column and eluted with PBS + 2M NaCl pH 7.4 using an Akta FPLC system (GE Healthcare) . Active fractions were pooled and concentrated by freeze-drying and resupended in PBS before gel filtration with a Superdex 75 column (GE Healthcare) . Subsequently active fractions were TCA precipitated and separated on SDS-PAGE and stained with silver. Proteins of interest were identified by mass- spectometry (Alphalyse, Danmark)
Isolation of recombinant alkaline protease from E.coli was performed using two plasmids, pAG302 resistant for kanamycin and pJFl resistant for chloramphenicol containing the PaOl genes AprA/Aprl and AprD/AprE/AprF, respectively. The two plasmids were transformed into E.coli BL21 and positive clones resistant for chlormaphenicol and kanamycin were selected. Protein expression was induced by addition of ImM Isopropyl β-D-1-thiogalactopyranoside (IPTG)
for 3-4 hours at 370C. Supernatant was collected and diafiltrated using a proflux M-12 system (Millipore) . AprA was applied on a Q sepharose XL column and eluted with phosphate buffered 2M NaCl pH 7. Fractions containing AprA were concentrated using a 3kD filter (Millipore) and applied on a superdex 75 column. Purity was checked with SDS-PAGE and Coomassie blue staining.
The gene Aprl of Pseudomonas aeruginosa strain PaOl with an 5' Xba restriction site, 6x His-tag, enterokinase cleavage site and a 3' EcoRl restriction site was synthesized by Baseclear (Leiden, The Netherlands) . The Aprl construct was ligated in a pRSET B vector and transformed in E.coli ToplOF' . Protein expression was performed in E.coli BL21 and alkaline protease inhibitor was purified under denaturing conditions according to manufacturer' s instructions using a His trap FF column (GE healthcare) . Purified denatured protein was diluted ten times in PBS and concentrated on a His trap column. Purity of Aprl was checked on SDS-PAGE and the protein was dialyzed against PBS.
Flagellin Isolation
Pseudomonas aeruginosa PaO25 (flagellin type B) and clinical isolate (flagellin type A) were grown overnight in Lucia Broth medium and bacteria were pelleted by centrifugation . Pellets were resuspended in PBS and flagella were sheared from bacteria, followed by centrifugation at 8,000 x g for 15 min to pellet bacteria. To isolate flagella the supernatant was centrifuged at 100,000 x g for 60 min. Ultracentrifugation pellets were resuspended in PBS and purity was checked by SDS-PAGE. Isolated filaments were heated at 7O0C for 20 min to depolymerize filaments.
Constructs for recombinant S. typhimurium and P. aeruginosa flagellin were generated by an overhang extension
polymerase chain reaction (PCR) as described previously (Bestebroer; Blood (2007) 109: 2936-2943) . Briefly, genes flic of S. typhimurium (clinical isolate) and flagellin type B of P. aeruginosa strain PaO25 were cloned directly downstream of the 6x His-tag and enterokinase cleavage site of the pRSET B vector. PCRs were performed using VentR DNA polymerase (New England Bio Labs) .
After verification of the sequence the vector was transformed into E.coli BL21 and protein expression was performed as described for AprA. His-tagged flagellin was isolated by lysing bacteria with cellytic B (Sigma) supplemented with DNAse/RNAse and protease inhibitor cocktail (Roche) followed by purification with an His trap FF column. The protein was eluted with 0.5M Imidazole in PBS and dialyzed against PBS. Purity was checked with SDS-PAGE and Coomassie Blue staining.
P. aeruginosa knockout
P. aeruginosa knockout mutants AprAl, AprA2 and Aprl were obtained from the Pseudomonas aeruginosa PaOl transposon mutant library (University of Washington, Jacobs et al. (2003) PNAS 100:14339-44). Bacteria were grown overnight in IMDM and supernatant was harvested by centrifugation and filtration. 3μg/ml recombinant alkaline protease or lOμg/ml Aprl was added to the culture medium before inoculation with the mutants. HEK/TLR5 cells were stimulated with overnight cultured supernatant of the wild- type and knockout strains and IL-8 levels were measured by ELISA.
SDS-PAGE
Recombinant alkaline protease and flagellin of P. aeruginosa or S. typhimurium in PBS were incubated for 1 h (unless otherwise specified) at 370C. Cleavage products
were analyzed with SDS-PAGE and stained with Coomassie blue. Flagellar filaments isolated from P. aeruginosa were heated to generate monomeric flagellin or untreated for polymeric flagellin before cleavage with alkaline protease. To block protease activity assay was performed in the presence of 60 mM EDTA.
The presence of AprA in the supernatant of P. aeruginosa mutants was checked by Western Blot using rabbit alkaline protease antisera. Recombinant AprA or bacterial supernatant was separated with SDS-PAGE. Proteins were transferred to a Immobilon-P membrane (Millipore) and blocked with 4% ELK in PBS + 0.05% Tween. Subsequently blots were incubated with 1/500 diluted rabbit AprA antiserum followed by HRP conjugated goat-anti-rabbit IgG.
Results
Cloning and expression of Pseudomonas Alkaline Protease
Alkaline protease (AprA) of Pseudomonas aeruginosa is a secreted 5OkD zinc metalloprotease, which is crystallized together with its inhibitor Aprl . Both proteins form a genetic cluster together with the genes AprD, AprE and AprF (Fig. 1) which are crucial for the secretion of AprA. A construct was used in E.coli that contains the complete set of genes required for proper excretion of AprA into the medium. The protein was purified with ion-exchange and size-exclusion chromatography.
AprA inhibits TLR5 mediated cell activation
Recombinant AprA blocked the flagellin-induced IL- 8 production by HEK/TLR5 cells completely, even at relatively high flagellin concentrations of 10 and 100 ng/ml (Fig. 2) . To determine the potency of AprA, different concentrations were used to inhibit the flagellin-induced IL-8 production by HEK/TLR5 cells. Complete inhibition of
cell activation was observed with 1 μg/ml AprA with an IC50 of ± 200 ng/ml (Fig. 3) . Initial experiments used recombinant flagellin from Salmonella typhimurium, a commonly used ligand for TLR5 in literature. Flagellin was isolated from Pseudomonas aeruginosa and also served as a potent agonist for HEK/TLR5 stimulated IL-8 production. In addition, 10 μg/ml AprA also completely inhibited the Pseudomonas flagellin response (Fig. 4) . These data demonstrate that Pseudomonas aeruginosa AprA is an effective inhibitor of TLR5 mediated cell activation.
AprA Interacts not with TLR5
To determine the mechanism of TLR5 inhibition, the effect of AprA on TLR5 and flagellin was investigated separately. Initial experiments were performed by adding flagellin and AprA together to the HEK/TLR5 cells resulting in an impaired IL-8 production. To establish an effect on TLR5, cells were incubated with AprA for 30 minutes, washed and subsequently stimulated with flagellin. Washing between AprA incubation and addition of flagellin returned TLR5 activation to control levels (Fig. 5) . This indicates that TLR5 is not affected by AprA and still responds to its ligand flagellin.
AprA cleaves flagellin
Another mechanism to block TLR5 recognition is neutralization or proteolysis of flagellin. To test this hypothesis, isolated flagellin was incubated with AprA and analyzed by SDS-PAGE for degradation. Two different Pseudomonas flagellins, type A and B with slightly different molecular mass were used and showed a comparable degradation pattern when mixed with increasing concentrations AprA (Fig. 6A and 6B) . Identical cleavage patterns were observed with recombinant Pseudomonas flagellin type B of strain PaOl
(Fig. 6C) . Moreover, AprA also cleaves recombinant flagellin from Salmonella typhimurium (Fig. 6D) , although this preparation was not so homogenous after purification. At higher concentrations of AprA, flagellin type B and Salmonella flagellin is completely degraded and no Coomassie stained products could be observed. Cleavage of flagellin by AprA is time dependent and the addition of EDTA allows only the first cleavage steps (Fig. 7) . Because the recombinant flagellin was cloned and expressed with an N-terminal His- tag, an antibody against the His-tag was used in Western blotting and indicated cleavage of the flagellin N-terminus (data not shown) . Furthermore, sequencing of cleavage products suggested that both the N- and C-terminal parts of Pseudomonas aeruginosa flagellin were removed by AprA (data not shown) .
Flagellar filaments are not susceptible to AprA cleavage
Flagella are essential for bacterial motility and virulence and flagellin is the most abundant protein of the flagellum. Secretion of a protease that cleaves off flagella from Pseudomonas would be a disadvantage for the bacterium. Therefore, we tested whether AprA also degrades complete flagellar filaments isolated from Pseudomonas . Incubation of AprA together with flagellin polymers did not result in any degradation in contrast to the monomeric flagellin obtained via depolymerization of the flagellar filaments (Fig. 8) . Even at high AprA concentration no cleavage of flagellar filaments is observed. AprA inactivates only monomeric flagellin that is released in bacterial supernatant by degradation of flagella, whereas complete flagella themselves resist cleavage.
Alkaline protease inhibitor blocks AprA-mediated cleavage
Immediately downstream of the AprA gene there is a gene encoding an alkaline protease inhibitor (Aprl; Fig. 1) . Kinetic studies with this 11.5kD periplasmic protein revealed a very high affinity for AprA (Kd of 4pM; Feltzer et al. J Biol Chem. (2000) 275:21002-9). This natural occurring inhibitor was also cloned, expressed and isolated from E.coli as a His-tagged protein. At equal molar ratio Aprl effectively prevents cleavage of flagellin, while increasing Aprl concentration blocks cleavage completely. Both Pseudomonas and Salmonella flagellin are protected by
Aprl against AprA-induced proteolysis (Fig. 9) . Importantly, incubation of AprA with Aprl before addition of flagellin restored activation of HEK/TLR5 cells dose-dependently (Fig. 10) . Equal molar ratios AprAiAprI inhibits activation of HEK/TLR5 cells for at least fifty percent.
Pseudomonas AprA knockout activates TLR5
Purified recombinant alkaline protease effectively blocks flagellin-induced TLR5 activation. Screening experiments with rough Pseudomonas culture supernatant showed consistent but weak inhibition. The presence of other proteins in rough supernatant may affect flagellin cleavage as well as low AprA concentrations. To address the importance of this protease, we used an AprA knockout (ΔaprA) Pseudomonas strain. HEK/TLR5 cells were stimulated with overnight culture supernatant of wildtype, ΔaprA (two different KO strains) or ΔaprI Pseudomonas strains.
In the absence of AprA, Pseudomonas supernatant triggers TLR5 activation due to the release of flagellin. Surprisingly, the supernatant of wildtype or ΔaprI Pseudomonas did not initiate IL-8 production at all (Fig. 11) . These data indicate that AprA degrades flagellin released in the supernatant during overnight growth of the wildtype and Aprl knockout strain. Deletion of the gene for
AprA preserves the released flagellin, resulting in activation of TLR5. Dilution of the supernatant results in less activation of TLR5 due to a decreasing flagellin concentration. Addition of recombinant AprA to the culture supernatant of the ΔaprA strain prevented TLR5 activation as observed for the wild-type strain.
To investigate, whether wild-type and ΔaprA produce flagellin in the supernatant, the culture medium was supplemented with Aprl . As expected, Aprl abolished AprA mediated cleavage of flagellin resulting in activation of TLR5 (Fig. 13) . These results indicate that Pseudomonas aeruginosa secretes enough AprA to cleave flagellin that is released by degradation of flagella during bacterial growth. Addition of exogenous flagellin to HEK/TLR5 cells in the presence of the supernatants shows a shift in the flagellin- induced IL-8 response with wild-type supernatant. The endogenous flagellin was already degraded, but the AprA present in the supernatant is still able to inactivate part of the freshly added flagellin. The AprA knock-out supernatant already contains sufficient amounts of intact flagellin for a full blown IL-8 response (Fig. 14) .
To verify that the AprA knockout strains indeed did not produce AprA in the supernatant, the culture supernatant was TCA precipitated and probed with a specific antibody against AprA by western blotting. As expected, while wildtype and Aprl knockout strain secreted AprA while the two AprA knock-out strains did not (Fig. 15) . Growth of the knockout strains was comparable with wildtype.
EXAMPLE 2
Materials and Methods
Arabidopsis callose deposition assay
Seeds of A. thaliana ecotype Landsberg erecta (La- er) were vapor face sterilized and sown on Murashige-Skoog
(MS; Sigma) medium containing 0.6% Plant Agar (Duchefa) and 1% (w/v) sucrose (Sigma) . After a two-day vernalization period at 40C, plates were transferred to growth chambers with an 8-h day (200 μEm^.sec"1 at 240C) and 16-h night (2O0C) cycle for seven days.
Three seedlings were transferred to a single well containing 1 ml MS containing 1% (w/v) sucrose and the components required for the treatments as indicated in the text. After 24 h the medium was replaced by 1 ml 96% EtOH followed by incubation overnight for removal of chlorophyll. The next day, decolorized seedlings were washed in 0.07 M phosphate buffer (pH 9) and subsequently incubated with the same buffer containing 0.01% aniline blue (water blue; Merck) . Samples were placed in the dark for a period of 20 h at RT. Microscopic slides were prepared in a matrix of fresh aniline blue. Observations were performed with a fluorescence microscope (Olympus Ax70 with Olympus U-RFL-T) with UV filter (bandpass 340 to 380 nm, long-path 425 nm) (Pozo et al. (2008) New Phytol. 180:511-523.
Arabidopsis growth assay
Ten vapor-face sterilized seeds of A. thaliana (La-er) were transferred to a well of 24-well-plates containing 1 ml MS medium with 1% (w/v) sucrose and treatment-specific components. Seeds were vernalized by putting the 24-well plates at 40C for two days. Subsequently, plates were transferred to growth chambers with an 8-h day (200 μEm^.sec"1 at 240C) and 16-h night (2O0C) cycle. Differences in growth rate were monitored after 7-10 days by photography.
Results
AprA interferes with flagellin recognition by Arabidopsis
Like mammals, plants also possess an innate immune system in which the detection of flagellin monomers also results in the activation of effective immune responses (Felix et al . (1999) Plant J. 18:265-276). In Arabidopsis, the FLS2 receptor has been demonstrated to specifically interact with a conserved 22-amino acid motif (flg22) of flagellin monomers (Gomez-Gomez et al . (2001) Plant Cell 13:1155-1163). Upon interaction of FLS2 with flagellin monomers or flg22, several downstream defense mechanisms are activated, amongst which the deposition of callose polymers (Gomez-Gomez et al . (1999) Plant J. 18:277-284).
Furthermore, as a result of the activation of energy-costly defense mechanisms, treatment with flagellin or flg22 has a negative effect on Arabidopsis growth. To assess whether AprA activity can also prevent flagellin- induced defense activation in plants, we studied the effect of AprA on flagellin- or fIg22-induced callose deposition and growth inhibition of Arabidopsis seedlings. P. aeruginosa flagellin monomers triggered callose deposition (Fig. 16) and affected growth (Fig. 17) to a slightly less extent than did flg22, which is in line with earlier observations (Felix et al . (1999) Plant J. 18:265-276). Preincubation of flagellin or flg22 with AprA abolished callose deposition (Fig. 16) and restored plant growth to control levels (Fig. 17) . This result indicates that AprA disrupts the active epitope of flagellin that is normally recognized by FLS2. Moreover, as addition of AprA completely neutralizes the effects of the peptide flg22, these results suggest that AprA cleaves flagellin at least within this 22-amino acids long conserved motif. Addition of Aprl prior to AprA treatment of flagellin neutralized the AprA-mediated effects in Arabidopsis, while Aprl had no effect when added after AprA treatment of flagellin (Fig. 16 and 17) .
Claims
1. Inhibitor of Pseudomonas aeruginosa alkaline protease for use in the treatment or prevention of infectious diseases in humans, animals and plants.
2. Inhibitor as claimed in claim 1, wherein the treatment comprises inhibiting the interference of Pseudomonas aeruginosa alkaline protease with a microbial molecule that is recognized by receptors involved in innate immunity.
3. Inhibitor as claimed in claim 2, wherein the receptors involved in innate immunity are Toll-like receptors or the flagellin locus 2 (FLS2) receptor.
4. Inhibitor as claimed in claim 3, wherein the Toll-like receptor is the Toll-like 5 receptor.
5. Inhibitor as claimed in claim 2, 3 or 4, wherein the microbial molecule is flagellin.
6. Inhibitor as claimed in any one of the claims 1-5, wherein the inhibitor is selected from: a) the Pseudomonas aeruginosa protein Aprl (SEQ ID
NO : 2 ) or a fragment, homologue or derivative thereof; b) an antibody against Pseudomonas aeruginosa alkaline protease; c) an analogue or fragment of an antibody against Pseudomonas aeruginosa alkaline protease; and d) a molecule that blocks the breakdown of bacterial flagellin by Pseudomonas aeruginosa alkaline protease .
7. Inhibitor as claimed in claim 6, wherein the analogues or fragments of antibodies against Pseudomonas aeruginosa alkaline protease are selected from camel antibodies, binding bodies, Fab fragments, F(ab')2 fragments, single chain antibodies, etc.
8. Inhibitor as claimed in claim 6, wherein the molecule that blocks the breakdown of bacterial flagellin by Pseudomonas aeruginosa alkaline protease is selected from blocking peptides, peptidomimetics, small molecules, etc. 9. Inhibitor as claimed in any one of the claims 1 tot 4, wherein the infectious disease is caused by Pseudomonas species, Salmonella species, Serratia species, Erwinia, Yersinia species, and other bacterial species containing AprA homologues. 11. Pseudomonas aeruginosa alkaline protease or a derivative thereof for use in medicine and agriculture.
12. Pseudomonas aeruginosa alkaline protease or a derivative thereof for use in anti-inflammatory therapy.
13. Pseudomonas aeruginosa alkaline protease or a derivative thereof as claimed in claim 12, wherein the anti¬ inflammatory therapy is effected by interfering with molecules that are recognized by receptors involved in innate immunity.
14. Pseudomonas aeruginosa alkaline protease or a derivative thereof as claimed in claim 13, wherein the molecules that are recognized by receptors involved in innate immunity is monomeric flagellin.
15. Pseudomonas aeruginosa alkaline protease or a derivative thereof as claimed in claim 13 or 14, wherein the receptors involved in innate immunity are Toll-like receptors or the flagellin locus 2 (FLS2) receptor.
16. Pseudomonas aeruginosa alkaline protease or a derivative thereof as claimed in claim 15, wherein the Toll- like receptor is Toll-like receptor 5. 17. Vaccine for inhibit the function of AprA, comprising AprA or a homologue or derivative thereof.
18. Vaccine as claimed in claim 17, for use in the prophylaxis of infections with Pseudomonas or other bacteria containing AprA homologues.
19. Transgenic plant that has no or a lowered susceptibility for infection with Gram negative bacteria, which transgenic plant comprises the Aprl-gene in its genome . 20. Transgenic plant as claimed in claim 19, wherein the Gram negative bacteria are Pseudomonas spp .
21. Method for producing a transgenic plant as claimed in claim 19 or 20, comprising the introduction of the Aprl gene into the genome of a plant.
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| CN110746485A (en) * | 2019-10-09 | 2020-02-04 | 天津科技大学 | Screening of novel alkaline protease inhibitory peptides |
| CN114591936A (en) * | 2022-03-29 | 2022-06-07 | 山东大学 | Mutant type algin lyase and application thereof |
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| JPH03277278A (en) * | 1990-02-21 | 1991-12-09 | Suntory Ltd | Alkaline protease gene derived from pseudomonas-aeruginosa |
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| JPH03277278A (en) * | 1990-02-21 | 1991-12-09 | Suntory Ltd | Alkaline protease gene derived from pseudomonas-aeruginosa |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110746485A (en) * | 2019-10-09 | 2020-02-04 | 天津科技大学 | Screening of novel alkaline protease inhibitory peptides |
| CN114591936A (en) * | 2022-03-29 | 2022-06-07 | 山东大学 | Mutant type algin lyase and application thereof |
| CN114591936B (en) * | 2022-03-29 | 2023-11-28 | 山东大学 | Mutant algin lyase and application thereof |
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