Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 μm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

Bacterial Macroscopic Rope-like Fibers with Cytopathic and Adhesive Properties

“…1A). These dimensions are well beyond those of other bacterial filamentous superstructures such as flagella (20 nm wide, 5-10 m long) (22), type 4 pili (20 m long), or T3SS needles (12 nm wide and 600 nm to several m long) (7,9,23,24). We were able to reproducibly see these highly flexible and sticky fibers, albeit of different dimensions on a day-to-day basis (supplemental Fig.…”

“…Ultrathin longitudinal and transversal sections of ropes were used for structural analysis and immunodetection of EspP with primary rabbit anti-EspP antibodies (produced in this study against purified EHEC EspP) or preimmune sera and anti-rabbit IgG gold-labeled conjugates as previously described (9).…”

“…To demonstrate host protein-EspP binding, the immobilized EspP protein derived from the ropes was incubated with fibronectin (5 g/ml) and reacted with rabbit antifibronectin serum (Sigma) (1:20,000) and secondary antibody. Immunoblots were developed with HyGLO chemiluminescent HRP antibody detection reagent (Denville) (9).…”

“…Maximum LDH release (high control) was determined by adding 100 l/well of 2% Triton X-100. For immunofluorescence microscopy, anti-EspP antibodies were added to 2% formalin-fixed samples for 1 h in 10% horse serum followed by the Alexa Fluor-conjugated secondary antibody and then visualized using an Axio Imager1.0 Zeiss microscope as previously described (8,9). The cytopathic effect exerted by ropes or EspP monomers were compared visually counting the number of rounded HeLa cells under fluorescence microscopy.…”

Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Slaughtered Qurban Animal in Jakarta Province

“…The Stxs are AB5 toxins that inhibit protein synthesis in the host cell. This process may lead to an apoptotic cell death, especially to a damage of renal glomerular endothelial cells (Melton-Celsa et al, 2012;Xicohtencatl-cortes et al, 2007). Stx2 is the toxin type most related to hemolytic uremic syndrome (HUS).…”