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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Cell. Infect. Microbiol. | doi: 10.3389/fcimb.2018.00404

An experimental pipeline for initial characterization of bacterial type III secretion system inhibitor mode of action using enteropathogenic Yersinia

Jessica M. Morgan1, Jocelyn M. Delgado1,  Hanh N. Lam1, Justin Luu1, Sina Mohammadi2,  Ralph R. Isberg2,  Helen Wang3 and  Victoria Auerbuch1*
  • 1University of California, Santa Cruz, United States
  • 2Tufts University School of Medicine, United States
  • 3Uppsala University, Sweden

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia.

Keywords: T3SS, type III secretion system, Yersinia, Virulence blocker, High through put screening

Received: 30 Jul 2018; Accepted: 26 Oct 2018.

Edited by:

Matthew C. Wolfgang, University of North Carolina at Chapel Hill, United States

Reviewed by:

Stephanie R. Shames, Kansas State University, United States
Deborah Anderson, University of Missouri, United States  

Copyright: © 2018 Morgan, Delgado, Lam, Luu, Mohammadi, Isberg, Wang and Auerbuch. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Victoria Auerbuch, University of California, Santa Cruz, Santa Cruz, 95064, California, United States, vastone@ucsc.edu