The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

Abstract

Enterohemorrhagic Escherichia coli (EHEC) adheres to intestinal epithelial cells, then stimulates the actin nucleation promoting factor N-WASP to induce localized actin assembly resulting in an actin “pedestal”, the function of which is poorly understood. EHEC also produces Shiga toxin (Stx), which penetrates the intestinal epithelium to cause a life-threatening renal and systemic disease. To assess the role of pedestal formation in colonization and disease, we utilized the murine pathogen Citrobacter rodentium, which also forms actin pedestals, and the genetically engineered C. rodentium (Φstx2dact), which additionally triggers Stx-mediated systemic disease. We found that an intestine-specific N-WASP-deficient (iNWKO) mouse suffered dramatically less colonization and disease than N-WASP-proficient littermate controls when infected with either strain. In addition, upon infection of wild type mice, mutants of C. rodentium or C. rodentium (Φstx2dact) that are specifically defective in pedestal formation demonstrated a relatively modest defect in cecal colonization and fecal shedding, but a more severe defect in colonization of the colonic mucosa. The C. rodentium (Φstx2dact) pedestal-defective mutant did not cause renal disease and, after normalizing for fecal bacterial load, was associated with a 16-fold lower risk of lethality. These findings suggest that the ability of an attaching and effacing pathogen to promote localized actin assembly contributes to virulence by promoting mucosal attachment.

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

“…High levels of colonization were also observed in colonic luminal washes and colonic homogenates following sacrifice. These titers of CR(ΦStx 2dact ) were roughly equal to the peak fecal shedding, colonic colonization, and luminal washes previously reported (Mallick et al, 2012, 2014) by intra-gastric inoculation. These data indicate that ingestion of food inoculated with mid-log phase bacteria grown without shaking results in efficient intestinal colonization.…”

“…The production of Stx by C. rodentium has been associated with slightly higher levels of intestinal colonization following inoculation by oral gavage (Mallick et al, 2012, 2014). Therefore, we also infected mice with CR(ΦStx 2dact :: kan R ), a CR(ΦStx 2dact ) mutant that does not produce Stx.…”

“…Two days prior to infection, CR(ΦStx 2dact ) and CR(ΦStx 2dact :: kan R ), described previously (Mallick et al, 2012, 2014), were streaked from frozen glycerol stock onto LB agar containing the appropriate antibiotic (i.e., 10 μg/ml chloramphenicol for CR(ΦStx 2dact ), and 10 μg/ml chloramphenicol and 25 μg/ml kanamycin CR(ΦStx 2dact :: kan R ) for single colonies. The following day, a single colony was inoculated into 40 ml LB broth (without antibiotic) in a 50 ml conical tube (VWR, catalog number 89039-662) and incubated at 37°C with 5% CO 2 , without shaking but with the cap loosened to allow for gas exchange.…”

“…To address these limitations, an alternative model, using conventional mice, was developed. The native murine AE pathogen C. rodentium (CR), which like EHEC forms attaching and effacing lesions, was lysogenized with phage Φ1720a-02, isolated from an Stx-producing E. coli strain (Mallick et al, 2012, 2014). This phage encodes and produces Stx variant Stx2dact, which is activated ~18-fold by intestinal mucus (Mallick et al, 2012; Bunger et al, 2013), resulting in high potency in mice (Teel et al, 2002; Bunger et al, 2013).…”

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

“…Similarly, a C. rodentium strain deficient for pedestal formation was outcompeted by a wild-type strain in mixed infections (34). Finally, the ability of A/E pathogens to induce pedestals in cultured cells is correlated with their ability to induce disease in humans and in animal models (31,35).…”