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) 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:Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen associated with raw produce such as sprouts, where it encounters fluctuating nutrient, temperature, and oxidative stress. This study elucidated the global transcriptional adaptations of E. coli O157:H7 EDL933 during colonization and persistence on alfalfa sprouts under simulated commercial sprouting and refrigerated storage. RNA sequencing was used to compare STEC grown in tryptic soy broth (D0), colonizing fresh sprouts for 5 days (D5), and held during sprout refrigeration for additional 3 days (D8). Among 5,002 expressed genes, 782 and 826 were differentially expressed (DEGs; |log2FC| ≥ 2, adjusted p < 0.05) at D5 vs. D0, and D8 vs. D0, respectively, whereas 11 genes only differed in the D8 vs. D5 comparison, indicating that the STEC transcriptome was largely stabilized by the time sprouts reach maturity, with refrigeration inducing minimal further shifts. Transition of STEC from nutrient rich broth to sprout tissues induced a profound metabolic reprograming, characterized by the up-regulation of de novo amino acid biosynthesis (e.g., hisA–H, leuABCD, and ilv operons), to compensate for nitrogen limitation within sprout tissues. STEC further adapted to sprout environment by inducing high-affinity uptake systems for potassium (kdpA/B/C/F), phosphorus (pstSCAB), and sulfur (tauABCD), alongside enterobactin-mediated iron acquisition. To survive sprout microenvironment, STEC elevated the transcription of acid resistance (gadA/C, cadA/B), oxidative stress defense (sodA, katE/G), and envelope protection (degP, pspA–D, baeS) genes. Although initial colonization at D5 involved the induction of flagellar components, the transition to a sessile biofilm-associated lifestyle at D8 was marked by the repression of motility genes and the strong induction of curli fimbriae (csg) and colanic acid (wca) loci. Functional enrichment at D5 and D8, compared to D0, indicated coordinated stress regulation and translation. The major transcriptional trends observed in RNA seq were confirmed using RT-qPCR. Collectively, these findings highlight a multi tiered molecular adaptation strategy, enabling STEC adaptation and persistence in sprout environment, thus, providing insights that could be translated into STEC risk mitigation during sprout production
Project description:The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration of the infected cell. Analysis of the sequence and predicted structure alongside canonical eukaryotic serine-threonine kinases raises the questions of how it is catalytically active and how this activity is regulated. Here, we reveal that SteC is activated following the phosphorylation of a highly conserved residue, S379, by a host kinase. This induces a dramatic increase in SteC nucleotide binding affinity, providing an explanation for S379’s requirement for substrate phosphorylation and actin polymerisation. Further mutational analysis revealed HD and DGD motifs within the depleted C lobe that are important for function, and may represent non-canonical mimics of HxD and DFG motifs in eukaryotic serine/threonine kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, even though it is not required for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host.
2026-06-12 | PXD061217 | Pride
Project description:Chlamydia pecorum ST69 from Australia
Project description:Gut-resident microbes contribute to host health via multiple mechanisms. Some of the most striking gut microbiota induced effects occur in the extraintestinal tissues and are restricted to early life. The mechanisms by which gut residing bacteria induce effects on distant host tissues and why this is restricted to a period in early life are largely unknown. We found that a subset of live gut-resident bacteria spontaneously translocate from gut to extraintestinal tissues in preweaning, but not adult mice. Translocation in preweaning mice appeared physiologic as it did not induce an inflammatory response and was in part controlled by sphingosine-1-phsophate receptor (S1PR) expressing host cells and host goblet cells. One translocating strain, Lactobacillus animalisWU, contained unique coding sequences for genes in the tyrocidine-gramicidin antibiotic-synthesizing gene cluster as well as five other regions putatively producing secondary metabolites with anti-microbial activity. Lactobacillus animalisWU exhibited antimicrobial activity against the late-onset sepsis pathogen E. coli ST69 in vitro, and translocation of L. animalisWU protected preweaning mice from systemic E. coli ST69 sepsis in vivo. These observations demonstrate a previously unappreciated higher-level symbiosis with our gut microbes.