Efficient Translocation of EspC into Epithelial Cells Depends on Enteropathogenic Escherichia coli and Host Cell Contact

Abstract

EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown.We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4°C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.

Pet secretion, internalization and induction of cell death during infection of epithelial cells by enteroaggregative Escherichia coli

“…Moreover, EspC, an autotransporter homologous to Pet secreted by EPEC, is favoured in DMEM (Vidal & Navarro-Garcia, 2006); it is important to mention that EPEC T3SS and EspC are regulated by Ler (Elliott et al, 2000). The combination of LB and DMEM allowed us to discover that tryptone is a factor that favours the secretion of Pet by EAEC.…”

Bacterial Macroscopic Rope-like Fibers with Cytopathic and Adhesive Properties

“…Enterotoxin and cytotoxin activities on rat jejunal segments and cultured epithelial cells have been demonstrated for EspC (12,13). Cytotoxicity is more efficient when EspC is translocated during contact of EPEC with epithelial cells (14), a process that apparently also requires the participation of the T3SS (15). Recently, it was also found that EspC conferred enhanced lysozyme resistance to EPEC (16).…”

“…Recently, it was also found that EspC conferred enhanced lysozyme resistance to EPEC (16). EspC and EspP display enzymatic activities, and although EspC cleaves spectrin (also called fodrin) and hemoglobin, EspP of EHEC cleaves pepsin A, and human coagulation factor V (2,11,14,(17)(18)(19). The EspP protein was also shown to influence gut colonization in calves and contributes to adherence of EHEC to bovine primary rectal cells (20).…”

EspC Promotes Epithelial Cell Detachment by Enteropathogenic Escherichia coli via Sequential Cleavages of a Cytoskeletal Protein and then Focal Adhesion Proteins

“…Protein concentrations were estimated by the method of Bradford. Equivalent amounts were boiled for 7 min, analyzed on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), and electrotransferred to nitrocellulose membranes for immunoblot analyses essentially as previously described (26), using anti-fodrin (MAB1622; Chemicon International), anti-FAK (C20) (sc-558; Santa Cruz Biotechnology), rabbit anti-phospho-FAK (Tyr397) [pFAK-(Tyr397)] (sc-11765-R; Santa Cruz Biotechnology), and anti-paxillin (H-114) (sc-5574; Santa Cruz Biotechnology) antibodies.…”

“…We have shown that EspC is not efficiently internalized under nonphysiological conditions (i.e., as a purified protein) because no receptor is involved in its uptake and because no intracellular traffic is required. However, the physiologically secreted EspC by EPEC, which is enhanced in tissue culture medium and by cell contact, is efficiently internalized during the interaction of EPEC and epithelial cells (26). Recently, we also showed that during EPEC infection, EspC is secreted from the bacteria by the type V secretion system (T5SS), and then it is efficiently translocated into the epithelial cells by the type III secretion system (T3SS) translocon (27).…”