Targeting Type III Secretion in Yersinia pestis

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, 01655, USA.
Antimicrobial Agents and Chemotherapy (Impact Factor: 4.48). 12/2008; 53(2):385-92. DOI: 10.1128/AAC.00670-08
Source: PubMed


Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance
to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for
the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined
a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for
further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations.
Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial
growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent
cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity
relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but
also distinct from previously described candidate type III secretion inhibitors.

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    • "centrations indicated . The same volume of dimethylsulphoxide ( DMSO ) was added as a control as 100 μM of compounds . Photographs were taken 24 h post infiltration . Fig . S5 Structures of the small - molecule inhibitors used in this study . Not shown are compounds 6 ( see Felise et al . , 2008 ; com - pound #1 , TTS29 ) , 7 , 8 , 15 and 16 ( see Pan et al . , 2009 ; com - pounds #2 , 1 , 3 and 3 - diproprionate , respectively ) . Table S1 Differentially expressed genes in all comparisons . Table S2 Promoter cloning and quantitative real - time polymerase chain reaction ( qRT - PCR ) primers used in this study ."
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    ABSTRACT: The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.
    Full-text · Article · Jul 2013 · Molecular Plant Pathology
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    • "Two independent high throughput screens for inhibitors of the Yersinia type III secretion system have used bioluminescent bioreporters. The first screen monitored changes in yopE transcription with a PyopE::luxAB reporter [6], while the second used a lux operon driven by a constitutive promoter to monitor bacterial growth [7]. Other groups have engineered luxCDABE reporters to be under the transcription control of promoters of virulence genes to monitor expression patterns of these genes [8]–[10]. "
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    ABSTRACT: Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux(PtolC) and Lux(PcysZK). These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux(PtolC)) and monitor intracellular survival (Lux(PtolC) and Lux(PcysZK)) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.
    Full-text · Article · Oct 2012 · PLoS ONE
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    • "The current strategies for targeting Yersinia pestis and other type III secretion system-utilizing pathogens rely mostly on phenotypic screens [3], [9], [48], [49], [50], [51], [52]. The strategy uses a library of random compounds or a smaller set of molecules and the readout is a blockage of secretion of a defined substrate into the host. "
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    ABSTRACT: Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50) values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.
    Full-text · Article · May 2011 · PLoS ONE
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