Efficient translocation of EspC into epithelial cells depends on enteropathogenic Escherichia coli and host cell contact.
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 degrees 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.
Vidal
Jorge E
JE
Department of Cell Biology, Cinvestav-Zacatenco, Ap. Postal 14-740, 07000 Mexico City, Mexico.
Navarro-García
Fernando
F
D001422:Bacterial Adhesion / Q000502:physiology*; D002450:Cell Communication / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D003470:Culture Media; D004705:Endocytosis / Q000235:genetics / Q000276:immunology; D004847:Epithelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D010873:Pinocytosis / Q000235:genetics / Q000276:immunology; D021381:Protein Transport / Q000235:genetics / Q000276:immunology
D003470:Culture Media; D029968:Escherichia coli Proteins; C102541:EspC protein, E coli