Claude Parsot

Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.

Shigellosis in humans is characterized by the destruction of the colonic epithelium provoked by the inflammatory response that is induced upon invasion of the mucosa by bacteria of Shigella spp. and enteroinvasive Escherichia coli (EIEC). Numerous phylogenetic analyses based on multilocus enzyme electrophoresis, ribotyping, and sequence comparison established that all members of the genus Shigella and EIEC strains belong to the species E. coli. Sequence analysis of chromosomal genes indicates that Shigella and EIEC strains belong to at least six phylogenetic groups, designated S1, S2, S3, SD1, SS, and EIEC. Informative sites used for the phylogenetic analysis of the virulence plasmid were clustered mostly in two genes (ipaD and ipgD), and a more complete view might come from the analysis of sequences of whole virulence plasmids. For the time being, there are two possible scenarios for the origin of Shigella and EIEC groups: (i) the arrival (or construction) of the virulence plasmid in an ancestral E. coli strain from which all Shigella and EIEC groups descend or (ii) multiple arrivals of the virulence plasmid(s) in different E. coli strains. The genomic sequence of five Shigella strains, including strains of S. flexneri 2a (Sf301 and 2457T), S. dysenteriae 1 (Sd197), S. boydii 4 (Sb227), and S. sonnei (Ss046), has been determined. The large number of genes deleted in Shigella and EIEC genomes, compared to the E. coli K-12 genome, is confirmed by comparative genomic hybridization analyses.

During transcription, series of approximately 9 As or Ts can direct RNA polymerase to incorporate into the mRNA nucleotides not encoded by the DNA, changing the reading frame downstream from the slippage site. We detected series of 9 or 10 As in spa13, spa33 and mxiA encoding type III secretion apparatus components. Analysis of cDNAs indicated that transcriptional slippage occurs in spa13, mxiA and spa33. Changes in the reading frame were confirmed by using plasmids carrying slippage sites in the 5' part of lacZ. Slippage is required for production of Spa13 from two overlapping reading frames and should lead to production of truncated MxiA and Spa33 proteins. Complementation of spa13 and mxiA mutants with plasmids carrying altered sites indicated that slippage in spa13 is required for assembly of the secretion apparatus and that slippage sites in spa13 and mxiA have not been selected to encode Lys residues or to produce two proteins endowed with different activities. The presence of slippage sites decreases production of Spa13 by 70%, of MxiA and Spa33 by 15% and of Spa32 (encoded downstream from spa13) by 50%. These results suggest that transcriptional slippage controls protein production by reducing the proportion of mRNA translated into functional proteins.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

The Shigella flexneri transcription activator MxiE is produced by transcriptional slippage from two overlapping open reading frames. By using plasmids encoding a mxiD-lacZ translational fusion, we showed that transcriptional slippage in mxiE influences both transcription and translation of the downstream mxiD gene encoding an essential component of the type III secretion apparatus.

Bacteria of Shigella spp. (S. boydii, S. dysenteriae, S. flexneri and S. sonnei) and enteroinvasive Escherichia coli (EIEC) are responsible for shigellosis in humans, a disease characterized by the destruction of the colonic mucosa that is induced upon bacterial invasion. Shigella spp. and EIEC strains contain a virulence plasmid of approximately 220 kb that encodes determinants for entry into epithelial cells and dissemination from cell to cell. This review presents the current model on mechanisms of invasion of the colonic epithelium by these bacteria and focuses on their pathogenicity factors, particularly the virulence plasmid-encoded type III secretion system.

Bacteria ofShigellaspp. are responsible for shigellosis in humans. They use a type III secretion system to inject effector proteins into host cells and induce their entry into epithelial cells or trigger apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes, including UbcH5, which belongs to the stem cell factor SCF^β-TrCPcomplex promoting ubiquitination of phosphorylated inhibitor of NF-κB type α (phospho-IκBα). Transfection experiments indicated that OspG can prevent phospho-IκBα degradation and NF-κB activation induced by TNF-α stimulation. Infection of epithelial cells by theS.flexneriwild-type strain, but not anospGmutant, led to accumulation of phospho-IκBα, consistent with OspG inhibiting SCF^β-TrCPactivity. Upon infection of ileal loops in rabbits, theospGmutant induced a stronger inflammatory response than the wild-type strain. This finding indicates that OspG negatively controls the host innate response induced byS.flexneriupon invasion of the epithelium.

Summary: Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of ≈15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti‐activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti‐activator (OspD1), a co‐anti‐activator (Spa15), a coactivator (IpgC) and two anti‐coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

Summary: Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion (TTS) system encoded by a 200 kb virulence plasmid to enter epithelial cells and trigger apoptosis in macrophages. This TTS system comprises a secretion apparatus, translocators and effectors that transit through this apparatus, cytoplasmic chaperones and specific transcription regulators. The TTS apparatus assembled during growth of Shigella flexneri in broth is activated upon contact with epithelial cells. Transcription of ≈ 15 genes encoding effectors, including IpaH proteins, is regulated by the TTS apparatus activity and controlled by MxiE, a transcription activator of the AraC family, and IpgC, the chaperone of the translocators IpaB and IpaC. We present evidence that MxiE is produced by a frameshift between a 59‐codon open reading frame (ORF) (mxiEa) containing the translation start site and a 214‐codon ORF (mxiEb) encoding the DNA binding domain of the protein. The mxiEa encoded N‐terminal part of MxiE is required for MxiE function. Frameshifting efficiency was ≈ 30% during growth in broth and was not modulated by the activity of secretion or the coactivator IpgC. Frameshifting involves slippage of RNA polymerase during transcription of mxiE, which results in the incorporation of one additional nucleotide in the mRNA and places mxiEa and mxiEb in the same reading frame. Frameshifting might represent an additional means of controlling gene expression under specific environmental conditions.