Project description:Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a notorious foodborne pathogen capable of causing severe gastrointestinal infections in humans. The bovine rectoanal junction (RAJ) has been identified as a primary reservoir of STEC O157:H7, playing a critical role in its transmission to humans through contaminated food sources. Despite the relevance of this host-pathogen interaction, the molecular mechanisms behind the adaptation of STEC O157:H7 in the bovine RAJ and its subsequent infection of human colonic epithelial cells remain largely unexplored. This study aimed to unravel the intricate dynamics of STEC O157:H7 in two distinct host environments: bovine RAJ squamous epithelial (RSE) cells and human colonic epithelial cells. Comparative transcriptomics analysis was employed to investigate the differential gene expression profiles of STEC O157:H7 during its interaction with these cell types. The bacterial cells were cultured under controlled conditions to simulate the microenvironments of both bovine RAJ and human colonic epithelial cells. Using high-throughput RNA sequencing, we identified key bacterial genes and regulatory pathways that are significantly modulated in response to each specific host environment. Our findings reveal distinct expression patterns of virulence factors, adhesion proteins, and stress response genes in STEC O157:H7 grown in bovine RAJ cells as opposed to human colonic epithelial cells. Additionally, the comparative analysis highlights the potential role of certain genes in host adaptation and tissue-specific pathogenicity. Furthermore, this study sheds light on the potential factors contributing to the survival and persistence of STEC O157:H7 in the bovine reservoir and its ability to colonize and cause disease in humans.
Project description:Purpose: In this work, we evaluated the role of two indicative species, Citrobacter werkmanii (CW) and Escherichia albertii (EA), in the virulence of two DEC pathotypes, Shiga toxin-producing (STEC) and enteroaggregative (EAEC) Escherichia coli. Methods: To determine the effect of supernatant obtained from CW and EA cultures in STEC strain 86-24 and EAEC strain 042 gene expression, a RNA-seq analysis was performed. T84 cells were infected with DEC strains in the presence or absence of supernatant from EA and IL-8 secretion was evaluated. The effect of supernatant from EA on the growth and adherence of STEC and EAEC to T84 cells was also evaluated. Finally, we studied the participation of long polar fimbriae (Lpf) in STEC and plasmid-encoded toxin (Pet) in EAEC during DEC infection in the presence of supernatant from EA. Results: RNA-seq analysis revealed that several virulence factors in STEC and EAEC were up-regulated in the presence of supernatants from CW and EA. Interestingly, an increase in the secretion of IL-8 was observed in T84 cells infected with STEC or EAEC in the presence of a supernatant from EA. Similar results were observed with the supernatants obtained from clinical strains of E. albertii. Supernatant from EA had no effect on the growth of STEC and EAEC, or on the ability of these DEC strains to adhere to intestinal epithelial cells. Finally, we found that Pet toxin in EAEC was up-regulated in the presence of a supernatant from EA. In STEC, using mutant strains for Lpf fimbriae, our data suggested that these fimbriae might be participating in the increase of IL-8 induced by STEC on intestinal epithelial cells in the presence of a supernatant from EA. Conclusion:Supernatant obtained from an indicative species of DEC-positive diarrhea could modulate gene expression in STEC and EAEC, and IL-8 secretion induced by these bacteria. These data provide new insights into the effect of gut microbiota species in the pathogenicity of STEC and EAEC.
Project description:The Salmonella effector SteC is the only protein kinase encoded by Salmonella pathogenicity island 2 that is secreted through the type III secretion system. SteC is known to trigger actin rearrangement via the phosphorylated MEK pathway, and our previous experiments demonstrated that the migration process of macrophages found during Salmonella infection is dependent on the rearrangement of the host cell actin backbone and the action of SteC.To further investigate the target of SteC in the host, we constructed a SteC-RAW264.7 cell line and performed phosphomics analysis using 4D-FastDIA to identify the direct substrates of SteC that trigger macrophage migration and lead to cytoskeletal rearrangement.
Project description:Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms in humans. Raw milk products are often incriminated as vectors for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs); highlight STEC surface proteins associated with adhesion; and evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the z-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The z-potential values of the strains and MFGs were between -3.7 and -2.9 mV and between -12.2 more or less 0.14 mV, respectively. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human GI tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.
Project description:Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 2: full replicated control screen. Conditions as for Experiment 1 (E-MTAB-5923), but no spinach root present. Two BAC clones (BAC2B5 & BAC2B24) used for additional positive controls.