High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis.
ABSTRACT Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, utilize a plasmid encoded type III secretion system (T3SS) to promote infection by delivering Yersinia outer proteins (Yops) into the cytosol of mammalian cells. This T3SS is absolutely required for Yersinia virulence, which makes T3SS an attractive target in the development of novel therapeutics for treatment of plague and other Yersinia infections. In this study, a new method for high throughput screening (HTS) of small molecules for the ability to inhibit type III secretion (T3S) in Y. pestis has been developed. In comparison with screening assays employed by others, this method is very simple and rapid, and thus well suited for examining very large compound sets. Using this method, we screened a diverse collection of libraries at the US National Screening Laboratory. The initial examination of 70,966 compounds and mixtures from 13 libraries resulted in 431 primary hits. Strong positive indications of inhibition were observed at a rate of 0.01%, while moderate and weak but potentially meaningful signals were observed at rates of 0.056% and 0.54% respectively. Further characterizations were conducted on selected primary hits in Y. pestis. Of the eight compounds examined in secondary assays, four show good promise as leads for structure activity relationship studies. They are a diverse group, each having chemical scaffolds not only distinct from one another, but also distinct from previously described candidate T3S inhibitors.
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ABSTRACT: Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.PLoS ONE 12/2013; 8(12). DOI:10.1371/annotation/e6ae3cd6-3d55-4ec6-8437-19572280e260 · 3.53 Impact Factor
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ABSTRACT: Antibiotic resistance in pathogens requires new targets for developing novel antibacterials. The bacterial type III secretion system (T3SS) is an attractive target for developing antibacterials as it is essential in the pathogenesis of many Gram-negative bacteria. The T3SS consists of structural proteins, effectors, and chaperones. Over 20 different structural proteins assemble into a complex nanoinjector that punctures a hole on the eukaryotic cell membrane to allow the delivery of effectors directly into the host cell cytoplasm. Defects in the assembly and function of the T3SS render bacteria non-infective. Two major classes of small molecules, salicylidene acylhydrazides and thiazolidinones, have been shown to inhibit multiple genera of bacteria through the T3SS. Many additional chemically and structurally diverse classes of small molecule inhibitors of the T3SS have been identified as well. While specific targets within the T3SS of a few inhibitors have been suggested, the vast majority of specific protein targets within the T3SS remain to be identified or characterized. Other T3SS inhibitors include polymers, proteins, and polypeptides mimics. In addition, T3SS activity is regulated by its interaction with biologically relevant molecules, such as bile salts and sterols, which could serve as scaffolds for drug design. © 2015 John Wiley & Sons A/S.Chemical Biology & Drug Design 01/2015; 85(1):30-42. DOI:10.1111/cbdd.12422 · 2.51 Impact Factor
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ABSTRACT: The type III secretion system (T3SS) is a major virulence factor in many Gram-negative bacterial pathogens and represents a particularly appealing target for antimicrobial agents. Previous studies have shown that the plant phenolic compound p-coumaric acid (PCA) plays a role in inhibiting the T3SS expression of the phytopathogen D. dadantii 3937. This study screened a series of derivatives of plant phenolic compounds and identified that trans-4-hydroxycinnamohydroxamic acid (TS103) has an 8-fold higher inhibitory potency on the T3SS of D. dadantii compared to PCA. The effect of TS103 on regulatory components of the T3SS was further elucidated. Our results suggest that TS103 inhibits HrpY phosphorylation, and leads to reduced levels of hrpS and hrpL transcripts. In addition, through reducing RNA levels of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the posttranscriptional level via the rsmB-RsmA regulatory pathway. Finally, TS103 inhibits hrpL transcription and mRNA stability, which leads to reduced expression of HrpL-regulon genes, such as hrpA and hrpN. To our knowledge, this is the first of an inhibitor which affects the T3SS through both the transcriptional and posttranscriptional pathways in the soft-rot phytopathogen D. dadantii 3937.Molecular Plant Pathology 07/2014; 16(2). DOI:10.1111/mpp.12168 · 4.49 Impact Factor