Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections.
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ABSTRACT:
SUBMITTER: Etienne-Mesmin L
PROVIDER: S-EPMC3157389 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.
| S-EPMC5357955 | biostudies-literature
Project description:The ppk1 gene encodes polyphosphate kinase (PPK1), which is the major catalytic enzyme that Escherichia coli utilizes to synthesize inorganic polyphosphate (polyP). The aim of this study was to explore the role of PPK1 in the pathogenesis of Enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). An isogenic in-frame ppk1 deletion mutant (Δppk1) and ppk1 complemented mutant (Cppk1) were constructed and characterized in comparison to wild-type (WT) EHEC O157:H7 strain EDL933w by microscope observation and growth curve analysis. Survival rates under heat stress and acid tolerance, both of which the bacteria would face during pathogenesis, were compared among the three strains. LoVo cells and a murine model of intestinal colitis were used as the in vitro and in vivo models, respectively, to evaluate the effect of PPK1 on adhesion and invasion during the process of pathogenesis. Real-time reverse-transcription PCR of regulatory gene rpoS, adhesion gene eae, and toxin genes stx1 and stx2 was carried out to corroborate the results from the in vitro and in vivo models. The ppk1 deletion mutant exhibited disrupted polyP levels, but not morphology and growth characteristics. The survival rate of the Δppk1 strain under stringent environmental conditions was lower as compared with WT and Cppk1. The in vitro assays showed that deletion of the ppk1 gene reduced the adhesion, formation of attaching and effacing (A/E) lesions, and invasive ability of EHEC O157:H7. Moreover, the virulence of the Δppk1 in BALB/c mice was weaker as compared with the other two strains. Additionally, mRNA expression of rpoS, eae, stx1 and stx2 were consistent with the in vitro and in vivo results. In conclusion: EHEC O157:H7 requires PPK1 for both survival under harsh environmental conditions and virulence in vivo.
| S-EPMC8578739 | biostudies-literature
Project description:The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37 degrees C, a nutrient-rich environment, high osmolarity, and the presence of Mn(2+). Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933DeltasepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.
| S-EPMC94797 | biostudies-literature
Project description:Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.
| S-EPMC5829826 | biostudies-literature
Project description:The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.
| S-EPMC1087320 | biostudies-literature
Project description:Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.
| S-EPMC3172284 | biostudies-literature
Project description:Global regulation of the effects of deletion of phage cro gene in EHEC
2016-09-29 | GSE86271 | GEO
Project description:Transcriptional analysis of the effects of the deletion of the sRNAs glmY and glmZ in EHEC
2015-02-01 | GSE63336 | GEO
Project description:A total of 21 (4.3%) enterohemorrhagic E. coli strains were isolated by biochemical tests and identification of the eae(+)stx1(+)stx2(+) genotype from 490 stool samples obtained from calves with diarrhea during 1-year period from a major farm in Tehran, Iran. All of the strains showed resistance to ampicillin, ciprofloxacin, trimethoprim, streptomycin, chloramphenicol and tetracycline, while 19% showed resistance to gentamicin. Out of 21 EHEC strains, 11 (53%) harbored class 1 integron. Two different amplification products, which were approximately 750 and 1,700 bp in size, were obtained from amplified variable regions (in-F/in-R primers) in 3 (14.3%) and 4 (19%) of the EHEC isolates, which corresponded to dfrA7(dihydrofolate reductase type I) and dfrA1/aadA1(dihydrofolate reductase/aminoglycoside adenyltransferase) resistance gene cassettes, respectively, and this was confirmed by sequencing. Genotyping analysis revealed a total of 16 pulsotypes that corresponded to 16 isolates with the similarity indices of 62% and 30% for the most and least similar isolates, respectively, 9 of which harbored class 1 integron. Analysis of pulsotypes showed an extensive diversity among the isolates harboring integron, which is indicative of a lack of any significant genetic relatedness among the isolates. No obvious relation could be deduced between integron content and special pulsotypes. The little data available on the genotyping patterns of EHEC isolates from cattle and their resistance gene contents emphasize the need to establish genotyping databases in order to monitor and source track the source of emergence and spread of new resistant and integron-carrying genotypes.
| S-EPMC4197144 | biostudies-literature
Project description:The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose.ImportanceEnteric pathogens have to be crafty when interpreting multiple environmental cues to successfully establish themselves within complex and diverse gut microenvironments. Differences in oxygen tension and nutrient composition determine the biogeography of the gut microbiota and provide unique niches that can be exploited by enteric pathogens. EHEC is an enteric pathogen that colonizes the colon and causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome worldwide. It has a very low infectious dose, which requires it to be an extremely effective pathogen. Hence, here we show that EHEC senses multiple sugar sources and oxygen levels to optimally control the expression of its virulence repertoire. This exquisite regulatory control equips EHEC to sense different intestinal compartments to colonize the host.
| S-EPMC5120142 | biostudies-literature