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Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens causing severe human infections including hemorrhagic colitis and hemolytic uremic syndrome, particularly in children. Ruminants are the main reservoir of EHEC which colonize their intestinal tract through a mechanism involving the bacterial adhesin intimin. Vaccination of cattle has shown efficacy in reducing EHEC O157:H7 shedding in feces. However, most of these vaccines rely on purified proteins and/or adjuvants, making them expensive and not used by breeders. This study introduced the development of a new type of vaccine based on Outer Membrane Vesicles (OMVs) carrying the C-terminal domain of intimin (Int280). A vaccine which combines OMVs carrying luminal Int280 and OMVs displaying surface-exposed Int280 was produced using two addressing systems based on PelB peptide signal and Lpp-OmpA hybrid protein, respectively. Dot blot experiments on OMVs combined with FAS assay with bacteria confirmed the correct localization of the fusion proteins and the functionality of Lpp-OmpA-Int280, respectively. As a proof of concept, the efficiency of the mixed vaccine was tested in a mouse model using the pathogen Citrobacter rodentium which shares a similar intimin-based adhesion mechanism with EHEC. Intraperitoneal vaccination of mice, at two-week intervals with 1 μg of the mixture of OMV-Int280, elicited a strong anti-intimin IgG response. Interestingly, we observed a shortened C. rodentium fecal shedding duration in immunized mice compared to the control unvaccinated group, with significant reduction of C. rodentium colonization from day 14 (q < 0.0001) to day 18 (q = 0.0068). This OMV-Int280 vaccine therefore represents a promising candidate for the control of EHEC intestinal carriage and fecal shedding in ruminants. •Outer membrane vesicles (OMVs) enriched with intimin C-terminal domain were used as vaccine.•Two addressing systems successfully exported Int280 to the lumen or surface of OMVs.•Int280 was addressed to the outer membrane surface of E. coli and was functional.•The OMV-Int280 based vaccine induced a seroconversion against intimin in mice.•OMV-Int280 immunized mice had a shortened duration of C. rodentium fecal shedding.

Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force. Catch bonds have a role in bacterial adhesion and infection by uropathogenic E. coli. Here, the authors report crystal structures, molecular dynamics simulations, ligand binding analysis and cell tracking to characterise the catch bond interaction between the adhesin FimH and carbohydrate receptors.

Background In the present study, we aimed to determine the frequency of the csgA, fimH, mrkD, foc, papaGI, papGII and papGIII genes, to provide and to design fimbrial adhesin gene (FAG) patterns and profiles for the isolated uropathogenic Escherichia coli (UPEC) strains. Methods The enrollment of 108 positive urine samples was performed during seven months, between January 2022 and July 2022. The UPEC strains were confirmed through the standard microbiological and biochemical tests. The antimicrobial susceptibility test was performed through the Kirby–Bauer disc diffusion method. Molecular screening of FAGs was done through the polymerase chain reaction technology. The statistical analyses including chi square and Fisher’s exact tests were performed to interpret the obtained results in the present study. Results As the main results, the antimicrobial resistance (AMR) patterns, multi- (MDR) and extensively drug-resistance (XDR) patterns and FAG patterns were designed and provided. fimH (93.3%), csgA (90.4%) and papG (37.5%) ( papGII (30.8%)) genes were recognized as the top three FAGs, respectively. Moreover, the frequency of csgA-fimH gene profile was identified as the top FAG pattern (46.2%) among the others. The isolates bearing csgA-fimH gene profile were armed with a versatile of phenotypic AMR patterns. In the current study, 27.8%, 69.4% and 1.9% of the UPEC isolates were detected as extended-spectrum ß-lactamases (ESBLs) producers, MDR and XDR strains, respectively. Conclusions In conclusion, detection, providing and designing of patterns and profiles in association with FAGs, AMR feature in UPEC strains give us an effective option to have a successful and influential prevention for both of UTIs initiation and AMR feature.

M cells in immunity The mucosal immune system plays a major role in protecting mucosal surfaces against pathogens and also in promoting co-habitation with commensal microflora. To evoke the mucosal immune response, antigens on the mucosal surface must first cross the impermeable epithelial barrier into lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is thought to be mediated mainly by M cells, specialized epithelial cells in the Peyer's patches. A study of the mechanisms underlying antigen transcytosis by M cells shows that glycoprotein-2, expressed on the apical side of intestinal M cells, is the transcytotic receptor for bacteria expressing the FimH antigen. As M cells are considered a promising target for various oral vaccinations, this work points to glycoprotein-2-dependent transcytosis as a possible vaccine target. To evoke the mucosal immune system, which forms the largest part of the entire immune system, antigens on the mucosal surface must be transported across the epithelial barrier. The molecular mechanisms promoting this antigen uptake, called antigen transcytosis and mediated by specialized epithelial M cells, remain largely unknown. Here, glycoprotein 2, specifically expressed by M cells, is reported to serve as a transcytotic receptor for mucosal antigens. The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens 1 , 2 , 3 . To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer’s patches 4 . This function, called antigen transcytosis, is mediated by specialized epithelial M cells 5 , 6 . The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium ( S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer’s patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases 7 , 8 , the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Various bacteria are suggested to contribute to colorectal cancer (CRC) development 1 – 5 , including pks + Escherichia coli , which produces the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells 6 . However, it remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells to cause harm. Here, using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC 7 , we demonstrate that the oncogenic potential of pks + E. coli critically depends on bacterial adhesion to host epithelial cells, mediated by the type 1 pilus adhesin FimH and the F9 pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. We also show that allelic switching of FimH strongly influences the genotoxic potential of pks + E. coli and can induce a genotoxic gain-of-function in the probiotic strain Nissle 1917. Adhesin-mediated epithelial binding subsequently allows the production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic routes for the development of anti-adhesive therapies aimed at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC. The oncogenic potential of pks + Escherichia coli depends critically on bacterial adhesion to host epithelial cells mediated by the type 1 pilus adhesin FimH and the F9 pilus adhesin FmlH.

Bacterial biofilms represent a promising opportunity for engineering of microbial communities. However, our ability to control spatial structure in biofilms remains limited. Here we engineer Escherichia coli with a light-activated transcriptional promoter (pDawn) to optically regulate expression of an adhesin gene (Ag43). When illuminated with patterned blue light, long-term viable biofilms with spatial resolution down to 25 μm can be formed on a variety of substrates and inside enclosed culture chambers without the need for surface pretreatment. A biophysical model suggests that the patterning mechanism involves stimulation of transiently surface-adsorbed cells, lending evidence to a previously proposed role of adhesin expression during natural biofilm maturation. Overall, this tool—termed “Biofilm Lithography”—has distinct advantages over existing cell-depositing/patterning methods and provides the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom–up approaches to microbial consortia design.

Uropathogenic Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent β-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains. The pilus type 1 of uropathogenic E. coli must resist mechanical forces to remain attached to the epithelium. Here the authors use single-molecule force spectroscopy to demonstrate a hierarchy of mechanical stability among the pilus domains and show that the oxidoreductase DsbA also acts as a folding chaperone on the domains.

Escherichia coli is the leading cause of urinary tract infection, one of the most common bacterial infections in humans. Despite this, a genomic perspective is lacking regarding the phylogenetic distribution of isolates associated with different clinical syndromes. Here, we present a large-scale phylogenomic analysis of a spatiotemporally and clinically diverse set of 907 E. coli isolates, including 722 uropathogenic E. coli (UPEC) isolates. A genome-wide association approach identifies the (P-fimbriae-encoding) papGII locus as the key feature distinguishing invasive UPEC, defined as isolates associated with severe UTI, i.e., kidney infection (pyelonephritis) or urinary-source bacteremia, from non-invasive UPEC, defined as isolates associated with asymptomatic bacteriuria or bladder infection (cystitis). Within the E. coli population, distinct invasive UPEC lineages emerged through repeated horizontal acquisition of diverse papGII -containing pathogenicity islands. Our findings elucidate the molecular determinants of severe UTI and have implications for the early detection of this pathogen. Escherichia coli is a major cause of urinary tract infection. Here, Biggel et al. provide a phylogenomic analysis of 907 clinical E. coli isolates and identify the P-fimbriae-encoding locus associated with invasive uropathogenic E. coli isolates.

Adaptation of avian pathogenic E. coli (APEC) to changing host environments including virulence factors expression is vital for disease progression. FdeC is an autotransporter adhesin that plays a role in uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells. Expression of fdeC is known to be regulated by environmental conditions in UPEC and Shiga toxin-producing E. coli (STEC). The observation in a previous study that an APEC strain IMT5155 in which the fdeC gene was disrupted by a transposon insertion resulted in elevated adhesion to chicken intestinal cells prompted us to further explore the role of fdeC in infection. We found that the fdeC gene prevalence and FdeC variant prevalence differed between APEC and nonpathogenic E. coli genomes. Expression of the fdeC gene was induced at host body temperature, an infection relevant condition. Disruption of fdeC resulted in greater adhesion to CHIC-8E11 cells and increased motility at 42 °C compared to wild type (WT) and higher expression of multiple transporter proteins that increased inorganic ion export. Increased motility may be related to increased inorganic ion export since this resulted in downregulation of YbjN, a protein known to supress motility. Inactivation of fdeC in APEC strain IMT5155 resulted in a weaker immune response in chickens compared to WT in experimental infections. Our findings suggest that FdeC is upregulated in the host and contributes to interactions with the host by down-modulating motility during colonization. A thorough understanding of the regulation and function of FdeC could provide novel insights into E. coli pathogenesis.

Background. Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins— potentially critical for control efforts—remain undefined. Methods. Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967—2009) E. coli isolates representing sequence type ST131 and 853 recent (2010—2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. Results. Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%—52%). Conclusions. Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multi-drug-resistant E. coli epidemic.