Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells.

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.

Muramylpeptide shedding modulates cell sensing of Shigella flexneri.

Bacterial infections trigger the activation of innate immunity through the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins are intracellular PRMs that recognize muramylpeptides contained in peptidoglycan (PGN) of bacteria. It is still unclear how Nod1 physically interacts with PGN, a structure internal to the Gram-negative bacterial envelope. To contribute to the understanding of this process, we demonstrate that, like Escherichia coli, Bordetella pertussis and Neisseria gonorrheae, the Gram-negative pathogen Shigella spontaneously releases PGN fragments and that this process can be increased by inactivating either ampG or mppA, genes involved in PGN recycling. Both Shigella mutants, but especially the strain carrying the mppA deletion, trigger Nod1-mediated NF-kappaB activation to a greater extent than the wild-type strain. Likewise, muramylpeptides spontaneously shed by Shigella are able per se to trigger a Nod1-mediated response consistent with the relative amount. Finally, we found that qualitative changes in muramylpeptide shedding can alter in vivo host responses to Shigella infection. Our findings support the idea that muramylpeptides released by pathogens during infection could modulate the immune response through Nod proteins and thereby influence the outcome of disease.

Mucosal lymphoid infiltrate dominates colonic pathological changes in murine experimental shigellosis.

BACKGROUND: Shigella species are invasive human pathogens that cause acute rectocolitis by triggering a dysregulated inflammatory reaction in the colonic and rectal mucosa. Because mice are naturally resistant to shigellosis, there is no mouse model that mimics human disease. We explore the susceptibility of intestinal flora-depleted mice to shigellosis after intragastric infection with Shigella strains. METHODS: Mice given 5 g/L streptomycin as a beverage were infected intragastrically with 1 x 108 cfu of either invasive or noninvasive Shigella strains. RESULTS: We found that invasive Shigella strains persist up to 30 days in feces, whereas the persistence of noninvasive Shigella strains was reduced. Colonization primarily involves the colon and the cecum and, to a lesser extent, the ileum. The hallmark of inflammation in the intestinal tissue is a dramatic expansion of the lymphoid follicles, in which a high apoptotic index is recorded. CONCLUSIONS: We provide a murine model in which shigellae are able to reach their natural tissue target: the colon. Moreover, the absence of polymorphonuclear leukocyte recruitment and of epithelial cell lesions reveal some aspects of shigellosis that are usually hidden by the prevalence of this cell population. This novel model may contribute to the identification of new targets for vaccines and therapies.

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri.

Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 degrees C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 degrees C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 degrees C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.

Intravenous infection of virulent shigellae causes fulminant hepatitis in mice.

Shigella spp. are pathogenic bacteria responsible for bacillary dysentery in humans. The major lesions in colonic mucosa are intense inflammation with apoptosis of macrophages and release of pro-inflammatory cytokines. The study of shigellosis is hindered by the natural resistance of rodents to oral infection with Shigella. Therefore, animal models exploit other routes of infection. Here, we describe a novel murine model in which animals receive shigellae via the caudal vein. Mice infected with 5 x 10(6) (LD(50)) virulent shigellae died at 48 h post infection, whereas animals receiving non-invasive mutants survived. The liver is the main target of infection, where shigellae induce microgranuloma formation. In mice infected with invasive bacteria, high frequency of apoptotic cells is observed within hepatic microgranulomas along with significant levels of mRNA for pro-inflammatory cytokines such as IL-1beta, IL-18, IL-12 and IFN-gamma. Moreover, in the blood of these animals high levels of IL-6 and transaminases are detected. Our results demonstrate the intravenous model is suitable for pathogenicity studies and useful to explore the immune response after Shigella infection.

Analysis of virulence and inflammatory potential of Shigella flexneri purine biosynthesis mutants.

Several Shigella flexneri mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models. To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models. With this aim, we have constructed five S. flexneri 5 (wild-type strain M90T) mutants with inactivation of one or two of the loci purEK, purHD, and guaBA, governing early or late steps of purine biosynthesis. The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis. M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, and M90T purHD purEK gave a negative result in the Sereny test. In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T guaBA purHD, which did not result in death of the animals. Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains. At 72 h postinfection, M90T guaBA purHD still induced proinflammatory cytokines and factors such as IL-1beta, IL-6, TNF-alpha, and iNOS, along with cytokines such as IL-12 and IFN-gamma. Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.

Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm.

We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol.