Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

Laurie Pinaud,Armelle Phalipon,Robin Friedman,Mariana L Ferrari,François-Xavier Campbell-Valois,Nico Jehmlich,Philippe J Sansonetti,Martin Von Bergen

doi:10.1371/journal.pone.0186920

Abstract

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

Highlights

The virulence of several Gram-negative pathogenic bacteria, such as Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC), depends on expression of a Type Three Secretion System (T3SS)
To define the pool of potential T3SA substrates, we listed the basic properties of genes annotated on the virulence plasmid pWR100 from S. flexneri strain M90T [17]. This indicated that the virulence plasmid encodes 31 known or suspected T3SA substrates, 48 cytoplasmic, periplasmic or membrane proteins, and 28 annotated proteins with unknown localisation (Fig 1)
We have identified five novel T3SA substrates encoded on S. flexneri virulence plasmid pWR100: Orf13, Orf131a, Orf176, Orf86 and Orf48

Summary

Introduction

The virulence of several Gram-negative pathogenic bacteria, such as Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC), depends on expression of a Type Three Secretion System (T3SS). Genes in specific virulence-associated loci that encode proteins suspected to be T3SA substrates can be singled out from sequenced genomes and their translocation into host cells confirmed using various assays (reviewed in [2,3]) These assays rely on the fusion of putative substrates to an enzyme, such as the adenylate cyclase Cya or the β-lactamase TEM-1, whose activity can be quantified [4,5]. We have exploited label-free MS combined with the extensive knowledge of the genetic circuitry regulating activity of the Shigella T3SA and β-lactamase assays to study the T3SA secretome encoded on the virulence plasmid of S. flexneri Using this approach, we have confirmed the secretion of most known or suspected T3SA substrates and identified five novel substrates that may constitute additional molecular weapons in the Shigella arsenal

Materials and methods

Results

Discussion