Dataset Information

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

ABSTRACT: Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFN?). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.

SUBMITTER: Zhong Q

PROVIDER: S-EPMC7773185 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

Zhong Qiyun Q Roumeliotis Theodoros I TI Kozik Zuza Z Cepeda-Molero Massiel M Fernández Luis Ángel LÁ Shenoy Avinash R AR Bakal Chris C Frankel Gad G Choudhary Jyoti S JS

PLoS biology 20201230 12

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). M ...[more]

PMID: 33378358

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Project description:Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Project description:BACKGROUND: Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli are responsible for food poisoning (enteritis and enterotoxaemia) in humans in developed countries. Cattle are considered to be an important reservoir of EHEC and EPEC strains for humans. Moreover, some of the strains, belonging to the O26, O111, O118 serogroups, for example, are also responsible for digestive disorders in calves. The Translocated intimin receptor (Tir), the intimin (Eae) and the Tir-cytoskeleton coupling protein (TccP) represent three virulence factors implicated in the intimate attachment of the bacteria to the eukaryotic cell. Major variants have already been described for these genes among the different serogroups but minor variations have not often been studied. In this study, we examined the polymorphisms of the tir, eae and tccP2 genes of O26 strains (EPEC and EHEC isolated from bovines and from humans) with the aim to determine whether these polymorphisms are host specific or not. RESULTS: Of the 70 tested strains, 10 strains (14% of the strains) presented one or several polymorphisms in the tir and eae genes, which have never previously been described. Concerning tccP2 detection, 47 of the 70 strains (67% of the strains) were found to be positive for this gene. Most of the strains were found to possess tccP2 variants described in strains of serogroup O26. Nevertheless, three strains had tccP2 genes respectively described in strains of serogroup O111, O103 and O55. Moreover, none of the polymorphisms was statistically specific to the bovine or the human isolates. Nevertheless, the two major variants of tccP2 were statistically associated with the pathotype (EPEC or EHEC). CONCLUSIONS: In conclusion, tir and eae gene polymorphisms were found not to be numerous and not to be predominantly synonymous. Moreover, no difference was observed between human and bovine strains regarding the presence of polymorphisms. Finally, some tccP2 variants appeared to be pathotype specific. Further investigations need to be performed on a larger number of strains in order to confirm this specificity.

Dataset Information

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

Publications

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

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