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Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds*

  • Dai Wang
    Footnotes
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Caroline E. Zetterström
    Footnotes
    Affiliations
    Department of Chemistry, Linnaeus v, University of Umeå, SE-90187 Umeå, Sweden
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  • Mads Gabrielsen
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Katherine S.H. Beckham
    Footnotes
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Jai J. Tree
    Footnotes
    Affiliations
    Zoonotic and Animal Pathogens Research Laboratory, Immunity and Infection Division, The Roslin Institute and R (D)SVS, Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom
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  • Sarah E. Macdonald
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Olwyn Byron
    Affiliations
    School of Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Tim J. Mitchell
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • David L. Gally
    Footnotes
    Affiliations
    Zoonotic and Animal Pathogens Research Laboratory, Immunity and Infection Division, The Roslin Institute and R (D)SVS, Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom
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  • Pawel Herzyk
    Affiliations
    Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Arvind Mahajan
    Affiliations
    Zoonotic and Animal Pathogens Research Laboratory, Immunity and Infection Division, The Roslin Institute and R (D)SVS, Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom
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  • Hanna Uvell
    Footnotes
    Affiliations
    Umeå Centre for Microbial Research and Laboratories for Molecular Infection Medicine Sweden, Linnaeus v, University of Umeå, SE-90187 Umeå, Sweden
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  • Richard Burchmore
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Brian O. Smith
    Affiliations
    School of Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

    Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Mikael Elofsson
    Correspondence
    To whom correspondence may be addressed. Tel.: 46-90-786-9328; Fax: 46-90-86-9995
    Footnotes
    Affiliations
    Department of Chemistry, Linnaeus v, University of Umeå, SE-90187 Umeå, Sweden

    Umeå Centre for Microbial Research and Laboratories for Molecular Infection Medicine Sweden, Linnaeus v, University of Umeå, SE-90187 Umeå, Sweden

    Laboratories for Chemical Biology Umeå, Department of Chemistry, Linnaeus v, University of Umeå, SE-90187 Umeå, Sweden
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  • Andrew J. Roe
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-141-3302980; Fax: 44-141-3304600
    Affiliations
    Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Author Footnotes
    * This work was generously supported by Medical Research Scotland Grant 223 ORG (to D. W., A. J. R. and R. B.) and Biotechnology and Biological Sciences Research Council (Swindon, United Kingdom) Grant BB/G011389/1 (to A. J. R. and M. G.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2, Tables S1–S5, and Protocol S1.
    1 Both authors contributed equally to this work.
    2 Supported by the Swedish Research Council.
    3 Supported by the Wellcome Trust.
    4 Supported by the Knut and Alice Wallenberg Foundation and Vinnova.
Open AccessPublished:July 01, 2011DOI:https://doi.org/10.1074/jbc.M111.233858
      A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

      Introduction

      As the prevalence of antibiotic-resistant strains increases, targeting virulence determinants of pathogenic bacteria has become an attractive alternative to the use of traditional bactericidal antibiotics (
      • Cegelski L.
      • Marshall G.R.
      • Eldridge G.R.
      • Hultgren S.J.
      ,
      • Clatworthy A.E.
      • Pierson E.
      • Hung D.T.
      ,
      • Escaich S.
      ,
      • Lynch S.V.
      • Wiener-Kronish J.P.
      ,
      • Rasko D.A.
      • Sperandio V.
      ). A key feature of this strategy is that the virulence-blocking compounds spare the endogenous microflora and thereby exert less selective pressure, which in turn should reduce development of resistance. One potential target is the bacterial type three secretion system (T3SS),
      The abbreviations used are: T3SS
      type three secretion system
      AUC
      analytical ultracentrifugation
      HSQC
      heteronuclear single quantum coherence
      ecTpx
      E. coli O157 Tpx
      QSAR
      quantitative structure-activity relationship
      ypTpx
      Y. pseudotuberculosis Tpx
      MEM
      minimum Eagle's medium
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      SNP
      single-nucleotide polymorphism.
      a conserved protein injection organelle that is central to the virulence of many human, animal, and plant pathogens including Chlamydia sp., enteropathogenic and enterohemorrhagic Escherichia coli, Pseudomonas aeruginosa, Salmonella sp., Shigella sp., and Yersinia sp. (
      • Hueck C.J.
      ,
      • Coburn B.
      • Sekirov I.
      • Finlay B.B.
      ). With the T3SS, the pathogen translocates effector proteins into the cytosol of the host cell and thereby creates a niche that allows bacterial growth. A class of virulence-blocking compounds, the salicylidene acylhydrazides, was originally identified as putative T3SS inhibitors in Yersinia pseudotuberculosis (
      • Kauppi A.M.
      • Nordfelth R.
      • Uvell H.
      • Wolf-Watz H.
      • Elofsson M.
      ). In a number of publications the compounds have been shown to be broadly effective in negatively affecting the function of the T3SS in a number of pathogenic bacteria including Chlamydia sp. (
      • Muschiol S.
      • Bailey L.
      • Gylfe A.
      • Sundin C.
      • Hultenby K.
      • Bergström S.
      • Elofsson M.
      • Wolf-Watz H.
      • Normark S.
      • Henriques-Normark B.
      ,
      • Bailey L.
      • Gylfe A.
      • Sundin C.
      • Muschiol S.
      • Elofsson M.
      • Nordström P.
      • Henriques-Normark B.
      • Lugert R.
      • Waldenström A.
      • Wolf-Watz H.
      • Bergström S.
      ,
      • Slepenkin A.
      • Enquist P.A.
      • Hägglund U.
      • de la Maza L.M.
      • Elofsson M.
      • Peterson E.M.
      ,
      • Wolf K.
      • Betts H.J.
      • Chellas-Géry B.
      • Hower S.
      • Linton C.N.
      • Fields K.A.
      ), Salmonella typhimurium (
      • Hudson D.L.
      • Layton A.N.
      • Field T.R.
      • Bowen A.J.
      • Wolf-Watz H.
      • Elofsson M.
      • Stevens M.P.
      • Galyov E.E.
      ,
      • Negrea A.
      • Bjur E.
      • Ygberg S.E.
      • Elofsson M.
      • Wolf-Watz H.
      • Rhen M.
      ), Y. pseudotuberculosis (
      • Nordfelth R.
      • Kauppi A.M.
      • Norberg H.A.
      • Wolf-Watz H.
      • Elofsson M.
      ), Shigella sp. (
      • Veenendaal A.K.
      • Sundin C.
      • Blocker A.J.
      ), and E. coli O157 (
      • Tree J.J.
      • Wang D.
      • McInally C.
      • Mahajan A.
      • Layton A.
      • Houghton I.
      • Elofsson M.
      • Stevens M.P.
      • Gally D.L.
      • Roe A.J.
      ). Two reports describe activity of the compounds in vivo (
      • Hudson D.L.
      • Layton A.N.
      • Field T.R.
      • Bowen A.J.
      • Wolf-Watz H.
      • Elofsson M.
      • Stevens M.P.
      • Galyov E.E.
      ,
      • Chu H.
      • Slepenkin A.
      • Elofsson M.
      • Keyser P.
      • de la Maza L.M.
      • Peterson E.M.
      ) and thus indicate that T3SS inhibitors have the potential to be developed into novel anti-bacterial agents (
      • Keyser P.
      • Elofsson M.
      • Rosell S.
      • Wolf-Watz H.
      ,
      • Baron C.
      ). In addition, small molecule inhibitors can be used as chemical probes to study the role of T3SS in bacterial pathogenesis (
      • Wolf K.
      • Betts H.J.
      • Chellas-Géry B.
      • Hower S.
      • Linton C.N.
      • Fields K.A.
      ,
      • Puri A.W.
      • Bogyo M.
      ,
      • Hung D.T.
      • Rubin E.J.
      ). To date, the mechanism of inhibition for the salicylidene acylhydrazides has been unclear with several mechanisms being postulated, including direct effects on the T3SS machinery or on regulatory proteins that affect T3SS expression (
      • Nordfelth R.
      • Kauppi A.M.
      • Norberg H.A.
      • Wolf-Watz H.
      • Elofsson M.
      ,
      • Veenendaal A.K.
      • Sundin C.
      • Blocker A.J.
      ,
      • Tree J.J.
      • Wang D.
      • McInally C.
      • Mahajan A.
      • Layton A.
      • Houghton I.
      • Elofsson M.
      • Stevens M.P.
      • Gally D.L.
      • Roe A.J.
      ). The activity of salicylidene acylhydrazides on Chlamydia trachomatis can be reversed by the addition of iron, suggesting a possible link to iron availability in the cell (
      • Slepenkin A.
      • Enquist P.A.
      • Hägglund U.
      • de la Maza L.M.
      • Elofsson M.
      • Peterson E.M.
      ).
      Although target-based screening can identify compounds that perturb the function of the selected protein, the activity is often lost when the compounds are tested on bacterial cells due to a lack of bacterial cell permeability (
      • Payne D.J.
      • Gwynn M.N.
      • Holmes D.J.
      • Pompliano D.L.
      ). Phenotypic screens circumvent this challenge and directly provide compounds that are active on the cellular level. The salicylidene acylhydrazides were identified using this strategy, and this is likely the underlying reason for the broad activity spectrum observed for these T3SS inhibitors. The drawback is, however, that the mode of action at the molecular level has to be studied at a later stage. Identification of the cellular targets for the salicylidene acylhydrazides constitutes a crucial step in understanding their true mode of action. Moreover, such data are essential for the structure-led design and improvement of any potential therapeutic compound. Affinity chromatography, expression-cloning technologies, and microarrays are among the many strategies that can be employed for target deconvolution (
      • Terstappen G.C.
      • Schlüpen C.
      • Raggiaschi R.
      • Gaviraghi G.
      ). To help understand the molecular mechanism by which salicylidene acylhydrazides affect the T3SS, we aimed to identify the target proteins bound by this class of novel antibacterial compounds using an affinity reagent strategy (
      • Leslie B.J.
      • Hergenrother P.J.
      ). In this study we describe synthesis of a T3SS inhibitor affinity reagent and the isolation of putative target proteins from E. coli O157. By a series of in vitro experiments, we show that the compounds directly and selectively interact with the target proteins WrbA, Tpx, and FolX. The genes encoding the target proteins were deleted individually in both E. coli O157 and Y. pseudotuberculosis, and we show that the proteins are involved in regulation of T3SS gene expression. Our work provides the first identification of the cellular targets for this group of compounds and convincing evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins.

      DISCUSSION

      The identity, function, and three-dimensional structure of the target protein of a potential therapeutic compound constitute key information in a drug development process based on structure-based design (
      • Simmons K.J.
      • Chopra I.
      • Fishwick C.W.
      ,
      • Barker J.J.
      ). The salicylidene acylhydrazide class of T3SS inhibitors was originally identified in a phenotypic bacterial reporter-gene screen (
      • Kauppi A.M.
      • Nordfelth R.
      • Uvell H.
      • Wolf-Watz H.
      • Elofsson M.
      ). This strategy provides compounds that are active on the pathogenic organism and circumvents many of the drawbacks experienced in target-based screening based on proteins obtained by genomics (
      • Payne D.J.
      • Gwynn M.N.
      • Holmes D.J.
      • Pompliano D.L.
      ). The salicylidene acylhydrazides have proved promising as T3SS inhibitors (
      • Keyser P.
      • Elofsson M.
      • Rosell S.
      • Wolf-Watz H.
      ), and to further explore the potential of this compound class we have attempted to identify putative target proteins using an affinity reagent approach (
      • Guiffant D.
      • Tribouillard D.
      • Gug F.
      • Galons H.
      • Meijer L.
      • Blondel M.
      • Bach S.
      ). In this work we have identified 16 proteins bound by the Affi-Gel-labeled compound, ME0055-Aff. These putative protein targets of the salicylidene acylhydrazides include WrbA, an NAD(P)H quinone oxidoreductase, and Tpx, a thiol peroxidase, both of which have been characterized in some detail (
      • Baker L.M.
      • Poole L.B.
      ,
      • Patridge E.V.
      • Ferry J.G.
      ), and FolX, a dihydroneopterin-tri-P-epimerase. In contrast to WrbA and Tpx, the cellular function of FolX has been poorly characterized. How many of these proteins actually contribute to the phenotype associated with the addition of salicylidene acylhydrazide compounds is not clear. It has been established that metabolism is a key factor in the regulation of expression of type III secretion in P. aeruginosa (
      • Rietsch A.
      • Mekalanos J.J.
      ). We would speculate that if the salicylidene acylhydrazides affect multiple proteins, then there could be a clear perturbation of normal metabolism, resulting in changes in gene expression and protein function. However, some interactions could have no consequence for virulence or result from weak and nonspecific binding. It is also feasible that the unlabeled compound binds additional, unidentified proteins, as the modification of ME0055 by the addition of the spacer and Affi-Gel bead could affect binding to target proteins by blocking critical functional groups. Given that we successfully confirmed binding for three of these target proteins using Far Western blotting and showed NMR chemical shift perturbations for Tpx using unlabeled ME0052, we would suggest that several of the putative target proteins are likely to be true interacting partners with the salicylidene acylhydrazides. Furthermore, we have demonstrated that WrbA, Tpx, and FolX all contribute to the normal regulation and expression of key virulence factors, specifically the T3SS and flagella, and would suggest that a combination of perturbations to normal protein activity for multiple target proteins is most likely to explain the phenotype observed upon the addition of the salicylidene acylhydrazides.
      Quantitative structure-activity relationship (QSAR) models have been computed for focused libraries (
      • Dahlgren M.K.
      • Oberg C.T.
      • Wallin E.A.
      • Janson P.G.
      • Elofsson M.
      ,
      • Dahlgren M.K.
      • Zetterström C.E.
      • Gylfe S.
      • Linusson A.
      • Elofsson M.
      ,
      • Kauppi A.M.
      • Andersson C.D.
      • Norberg H.A.
      • Sundin C.
      • Linusson A.
      • Elofsson M.
      ,
      • Dahlgren M.K.
      • Kauppi A.M.
      • Olsson I.M.
      • Linusson A.
      • Elofsson M.
      ) using a strategy based on statistical molecular design (
      • Linusson A.
      • Elofsson M.
      • Andersson I.E.
      • Dahlgren M.K.
      ). The QSAR models for the salicylidene acylhydrazides were successfully validated with an external test set, and several compounds inhibited virulence in vitro (
      • Dahlgren M.K.
      • Zetterström C.E.
      • Gylfe S.
      • Linusson A.
      • Elofsson M.
      ). The QSAR models were, however, hard to interpret, which is likely the result of the compounds being evaluated in a cell-based assay and would be consistent with the notion that they target multiple proteins. The QSAR models reflect overall properties beneficial for interaction with several proteins and other processes including cell permeability. A mode of action that includes several targets is beneficial from an antibiotic resistance perspective as the pathogen must alter several proteins and pathways to escape the drug. However, further optimization of the salicylidene acylhydrazides against multiple targets is challenging (
      • Dahlgren M.K.
      • Oberg C.T.
      • Wallin E.A.
      • Janson P.G.
      • Elofsson M.
      ,
      • Dahlgren M.K.
      • Zetterström C.E.
      • Gylfe S.
      • Linusson A.
      • Elofsson M.
      ,
      • Hillgren J.M.
      • Dahlgren M.K.
      • To T.M.
      • Elofsson M.
      ), although polypharmacology is increasingly recognized as important in drug discovery and development (
      • Morphy R.
      • Rankovic Z.
      ).
      Given that the compounds affect the function of the T3SS of several Gram-negative pathogens, the obvious implication is that the different species share common protein targets that result in similar phenotypes. At least three mechanisms of inhibition have been suggested; they are direct effects on the T3SS basal apparatus proteins (
      • Veenendaal A.K.
      • Sundin C.
      • Blocker A.J.
      ), less-direct effects on proteins that then affect T3SS expression and function (
      • Tree J.J.
      • Wang D.
      • McInally C.
      • Mahajan A.
      • Layton A.
      • Houghton I.
      • Elofsson M.
      • Stevens M.P.
      • Gally D.L.
      • Roe A.J.
      ), and possible changes in iron availability (
      • Hudson D.L.
      • Layton A.N.
      • Field T.R.
      • Bowen A.J.
      • Wolf-Watz H.
      • Elofsson M.
      • Stevens M.P.
      • Galyov E.E.
      ). With reference to the latter mechanism, our previous work indicated that for E. coli O157:H7, no operons involved in iron metabolism were affected by the addition of the salicylidene acylhydrazides and, therefore, that iron availability was not sufficient to explain the inhibition observed with this strain (
      • Tree J.J.
      • Wang D.
      • McInally C.
      • Mahajan A.
      • Layton A.
      • Houghton I.
      • Elofsson M.
      • Stevens M.P.
      • Gally D.L.
      • Roe A.J.
      ). The three target proteins we have focused on are well conserved among Gram-negative pathogens; we have shown that both Tpx and WrbA from five different pathogens are bound by the salicylidene acylhydrazides and that the compounds bind FolX from three species. Additionally, our identification of the strain that was insensitive to the effects of the salicylidene acylhydrazides showed that this strain had no genetic changes that would affect the proteins that comprise the T3SS. This “insensitive” strain contained a mutant allele of wrbA, encoding one of the proposed target proteins. Given the large number of SNPs in this strain, we do not assume or conclude that this particular non-synonymous change results in the “insensitivity,” but it is an interesting observation that this target was identified by both approaches. The major conclusion from this aspect of the study was that the T3SS was highly conserved. Other genetic changes affecting cell wall structure, ABC transporters, or efflux pumps could all account for the phenotype observed with this strain. Overall, the weight of evidence in the present study favors a mechanism based on indirect effects that lead to changes in the regulation of the T3SS.
      To understand the mechanism by which the salicylidene acylhydrazides affect individual target proteins, we focused on Tpx, a thiol peroxidase previously shown to be part of the oxidative stress defense system (
      • Baker L.M.
      • Poole L.B.
      ). One important result was the NMR chemical shift perturbation, as this showed that unlabeled compound binds to the target protein involving specific residues that cluster to a discrete area near the active site and the dimer interface. The binding site was modeled by docking the compound onto ecTpx (PDB code 3HVV), highlighting the residues identified by the chemical shift NMR experiments (Fig. 4).
      Determination of both Kd and stoichiometry were achieved using an AUC-based method. This gave a Kd of ∼50 μm and a stoichiometry of 1 ME0052 molecule per ypTpx dimer. This result correlates well with our Far Western data that showed preferential binding of ME0052-Bio to the ypTpx dimer compared with the monomer. The C61S mutant forms a less stable dimer, resulting in the majority of the protein migrating as a monomer after SDS-PAGE and less binding of the ME0052-Bio probe. In contrast, when the C61S protein was used for AUC studies, the protein was present as a dimer and gave a Kd approximately twice that of the wild type but with the same stoichiometry as the wild-type protein. The lower Kd observed for the C61S mutant is consistent with the NMR result that identified Cys-61 as being a residue involved in binding of ME0052.
      Previous work has shown that Tpx protects against exogenous hydrogen peroxide and is required for survival in macrophages (
      • Horst S.A.
      • Jaeger T.
      • Denkel L.A.
      • Rouf S.F.
      • Rhen M.
      • Bange F.C.
      ). Our data also showed that a Y. pseudotuberculosis ΔTpx mutant was as virulent against J774A macrophages as wild-type bacteria and that the mutant remained sensitive to ME0052. This result is in agreement with the E. coli transcriptomic data and supports the hypothesis that the salicylidene acylhydrazides act on multiple targets. Although deletion of tpx in E. coli O157 resulted in clear effects on gene transcription, we were initially surprised to see an up-regulation of genes associated with the T3SS. Our working hypothesis was that the function of the T3SS proteins would be directly inhibited by the addition of the compounds. However, the data from the arrays showed that in E. coli O157, Tpx function leads to repression of the T3SS. This can be attributed to the peroxidase activity of Tpx rather than any direct regulatory effect of the protein itself, as the C61S mutant displayed a transcriptional profile similar to that of the mutant with the defined deletion. However, the mechanisms by which Tpx affects gene expression are not clear. As both Tpx and WrbA function to protect against oxidative stress, it does make biological sense for their activity to be linked to activation of motility, allowing the bacteria to evade such stresses. Previous work has shown a clear link between bacterial response to numerous stresses and control of motility (
      • Nachin L.
      • Nannmark U.
      • Nyström T.
      ).
      Given that the phenotype associated with addition of the salicylidene acylhydrazide compounds is a repression of T3SS expression, the implication is that these compounds may actually increase activity of Tpx and WrbA, thereby enhancing their repressive effects on the T3SS. The addition of the salicylidene acylhydrazide compounds does not directly affect expression of tpx or wrbA (
      • Tree J.J.
      • Wang D.
      • McInally C.
      • Mahajan A.
      • Layton A.
      • Houghton I.
      • Elofsson M.
      • Stevens M.P.
      • Gally D.L.
      • Roe A.J.
      ), suggesting that the compounds might directly activate or stabilize the enzymes. The preferential binding to the Tpx dimer coupled with the stoichiometry and NMR chemical shift assignment show that the compounds bind very close to the active site of Tpx. It is possible that the binding of the compound near the active site results in a positive effect on protein activity. Future work will map the binding site of the salicylidene acylhydrazide to more target proteins, allowing any common binding site to be identified, a key step on the route to the development of this class of anti-virulence compounds.

      Acknowledgments

      We thank Dr. Roger Parton and Dr. Dan Walker for their critical reading of the manuscript. The sequencing of the ZAP 430 strain was carried out in the Sir Henry Wellcome Functional Genomics Facility at the University of Glasgow.

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