No abstract

SummaryEnteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC-and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knockdown of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), prolinerich region (GFP-PRR) or a -helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the a -helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted a -helical region of cortactin in actin assembly induced by EPEC and EHEC.

No abstract

SummaryEnterohaemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen that, upon infection, causes destruction of the microvilli brush border of intestinal cells. EHEC is able to recruit several host cell proteins and induce actin accumulation beneath its adherence site, forming a pedestal-like structure upon which the bacterium is firmly attached. Injection of bacterial effectors into the host cells is required to trigger the recruitment and activation of proteins, such as cortactin, neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3 complex, directly involved in the actin polymerization process. We found that cortactin, an actin-binding protein, has a pivotal role during pedestal formation by EHEC. Cortactin was found to bind directly to two important virulence factors of EHEC, Tir and EspF u, which are translocated into the host cells during infection. Binding of cortactin to these effectors is dependent upon tyrosine phosphorylation and a balance between tyrosine phosphorylation and dephosphorylation of cortactin is required to regulate pedestal formation by EHEC.