RESUMEN
Despite the autotransporter (AT) moniker, AT secretion appears to involve the function of periplasmic chaperones. We identified four periplasmic proteins that specifically bound to plasmid-encoded toxin (Pet), an AT produced by enteroaggregative Escherichia coli (EAEC). These proteins include the 17-kDa Skp chaperone and the 37-kDa VirK protein. We found that the virK gene is present in different Enterobacteriaceae. VirK bound to misfolded conformations of the Pet passenger domain, but it did not bind to the folded passenger domain or to the ß domain of Pet. Assays with an EAECΔvirK mutant and its complemented version showed that, in the absence of VirK, Pet was not secreted but was instead retained in the periplasm as proteolytic fragments. In contrast, Pet was secreted from a Δskp mutant. VirK was not required for the insertion of porin proteins into the outer membrane but assisted with insertion of the Pet ß domain into the outer membrane. Loss of VirK function blocked the EAEC-mediated cytotoxic effect against HEp-2 cells. Thus, VirK facilitates the secretion of the AT Pet by maintaining the passenger domain in a conformation that both avoids periplasmic proteolysis and facilitates ß-domain insertion into the outer membrane.
Asunto(s)
Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/metabolismo , Toxinas Bacterianas , Línea Celular , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Hepatocitos/efectos de los fármacos , Humanos , Peso Molecular , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Pliegue de ProteínaRESUMEN
The plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli is a 104-kDa autotransporter protein that exhibits proteolytic activity against the actin-binding protein alpha-fodrin. Intracellular cleavage of epithelial fodrin by Pet disrupts the actin cytoskeleton, causing both cytotoxic and enterotoxic effects. Intoxication requires the serine protease activity of Pet and toxin endocytosis from clathrin-coated pits. The additional events in the intracellular trafficking of Pet are largely uncharacterized. Here, we determined by confocal microscopy that internalized Pet is transferred from the early endosomes to the Golgi apparatus and then travels to the endoplasmic reticulum (ER). Pet associates with the Sec61p translocon before it moves into the cytosol as an intact, 104-kDa protein. This translocation process contrasts with the export of other ER-translocating toxins, in which only the catalytic A subunit of the AB toxin enters the cytosol. However, like intoxication with these AB toxins, Pet intoxication was inhibited in a subset of mutant CHO cell lines with aberrant activity in the ER-associated degradation pathway of ER-to-cytosol translocation. This is the first report which documents the cell surface-to-ER and ER-to-cytosol trafficking of a bacterial non-AB toxin.