RNA-Seq analysis of the T3SA regulon in Shigella flexneri reveals two new chromosomal genes upregulated in the on-state.

Shigella spp. are enterobacteria that invade human colonic mucosal cells using their Type Three Secretion Apparatus (T3SA). Shigella spp. possess a large plasmid that encodes most of its virulence factors and has been the focus of seminal work that defined the T3SA regulon. Thus, a global assessment of the transcriptional response regulated by the T3SA has been lacking. Herein we used RNA-Seq to identify genes that are differentially expressed when the T3SA is active (on-state) versus inactive (off-state). The quality of the RNA-Seq dataset was validated by its correlation with a prior microarray study. Using novel insights about the expression of non-coding regions, bioinformatic tools and experimentations, we demonstrated the existence of six operons and evidence that ipaH2.5 is a pseudogene. In addition, 86 chromosomal genes were downregulated in the on-state including several non-coding transcripts corresponding to short antisense RNA embedded in the 16S and 23S RNA genes, and 40 coding transcripts, whose cognate proteins were highly connected at the genetic and biochemical levels. Finally, we identified two novel chromosomal genes dubbed gem1 and gem3, which were upregulated in the on-state similarly to genes belonging to the T3SA regulon. The latter findings were validated on biological triplicates by droplet digital PCR. To our knowledge gem1 and gem3 are the first chromosomal members of the T3SA regulon that have no homologs on the plasmid. Our approach provides a path to optimizing RNA-Seq studies in case of bacterial models that had previously been the subject of medium to large scale studies.

Enterobacteria and host resistance to infection.

Enterobacteriaceae are a large family of Gram-negative, non-spore-forming bacteria. Although many species exist as part of the natural flora of animals including humans, some members are associated with both intestinal and extraintestinal diseases. In this review, we focus on members of this family that have important roles in human disease: Salmonella, Escherichia, Shigella, and Yersinia, providing a brief overview of the disease caused by these bacteria, highlighting the contribution of animal models to our understanding of their pathogenesis and of host genetic determinants involved in susceptibility or resistance to infection.

A Tale about Shigella: Evolution, Plasmid, and Virulence.

Shigella spp. cause hundreds of millions of intestinal infections each year. They target the mucosa of the human colon and are an important model of intracellular bacterial pathogenesis. Shigella is a pathovar of Escherichia coli that is characterized by the presence of a large invasion plasmid, pINV, which encodes the characteristic type III secretion system and icsA used for cytosol invasion and cell-to-cell spread, respectively. First, we review recent advances in the genetic aspects of Shigella, shedding light on its evolutionary history within the E. coli lineage and its relationship to the acquisition of pINV. We then discuss recent insights into the processes that allow for the maintenance of pINV. Finally, we describe the role of the transcription activators VirF, VirB, and MxiE in the major virulence gene regulatory cascades that control the expression of the type III secretion system and icsA. This provides an opportunity to examine the interplay between these pINV-encoded transcriptional activators and numerous chromosome-encoded factors that modulate their activity. Finally, we discuss novel chromosomal genes icaR, icaT, and yccE that are regulated by MxiE. This review emphasizes the notion that Shigella and E. coli have walked the fine line between commensalism and pathogenesis for much of their history.

icaR and icaT are Ancient Chromosome Genes Encoding Substrates of the Type III Secretion Apparatus in Shigella flexneri.

Shigella is an Escherichia coli pathovar that colonizes the cytosol of mucosal cells in the human large intestine. To do this, Shigella uses a Type III Secretion Apparatus (T3SA) to translocate several proteins into host cells. The T3SA and its substrates are encoded by genes of the virulence plasmid pINV or by chromosomal genes derived thereof. We recently discovered two chromosomal genes, which seem unrelated to pINV, although they are activated by MxiE and IpgC similarly to some of the canonical substrates of the T3SA. Here, we showed that the production of the corresponding proteins depended on the conservation of a MxiE box in their cognate promoters. Furthermore, both proteins were secreted by the T3SA in a chaperone-independent manner through the recognition of their respective amino-terminal secretion signal. Based on these observations, we named these new genes icaR and icaT, which stand for invasion chromosome antigen with homology for a transcriptional regulator and a transposase, respectively. icaR and icaT have orthologs in commensal and pathogenic E. coli strains belonging mainly to phylogroups A, B1, D and E. Finally, we demonstrated that icaR and icaT orthologs could be activated by the coproduction of IpgC and MxiE in strains MG1655 K-12 (phylogroup A) and O157:H7 ATCC 43888 (phylogroup E). In contrast, the coproduction of EivF and YgeG, which are homologs of MxiE and IpgC in the E. coli T3SS 2 (ETT2), failed to activate icaR and icaT. IMPORTANCEicaR and icaT are the latest members of the MxiE regulon discovered in the chromosome. The proteins IcaR and IcaT, albeit produced in small amounts, are nonetheless secreted by the T3SA comparably to canonical substrates. The high occurrence of icaR and icaT in phylogroups A, B1, D, and E coupled with their widespread absence in their B2 counterparts agree with the consensus E. coli phylogeny. The widespread conservation of the MxiE box among icaR and icaT orthologs supports the notion that both genes had already undergone coevolution with transcriptional activators ipgC and mxiE- harbored in pINV or a relative- in the last common ancestor of Shigella and of E. coli from phylogroups A, B1, D, and E. The possibility that icaR and icaT may contribute to Shigella pathogenesis cannot be excluded, although some of their characteristics suggest they are fossil genes.

The T3SS of Shigella: Expression, Structure, Function, and Role in Vacuole Escape.

Shigella spp. are one of the leading causes of infectious diarrheal diseases. They are Escherichia coli pathovars that are characterized by the harboring of a large plasmid that encodes most virulence genes, including a type III secretion system (T3SS). The archetypal element of the T3SS is the injectisome, a syringe-like nanomachine composed of approximately 20 proteins, spanning both bacterial membranes and the cell wall, and topped with a needle. Upon contact of the tip of the needle with the plasma membrane, the injectisome secretes its protein substrates into host cells. Some of these substrates act as translocators or effectors whose functions are key to the invasion of the cytosol and the cell-to-cell spread characterizing the lifestyle of Shigella spp. Here, we review the structure, assembly, function, and methods to measure the activity of the injectisome with a focus on Shigella, but complemented with data from other T3SS if required. We also present the regulatory cascade that controls the expression of T3SS genes in Shigella. Finally, we describe the function of translocators and effectors during cell-to-cell spread, particularly during escape from the vacuole, a key element of Shigella's pathogenesis that has yet to reveal all of its secrets.