1 VirK is a Periplasmic Protein Required for Efficient Secretion of Pet from 2

Despite the autotransporter (AT) moniker, AT secretion appears to involve the function of 23 periplasmic chaperones. We identified four periplasmic proteins that specifically bound to 24 plasmid-encoded toxin (Pet), an AT produced by enteroaggregative Escherichia coli (EAEC). 25 These proteins include the 17 kDa Skp chaperone and the 37 kDa VirK protein. We found that 26 the virK gene is present in different Enterobacteriaceae. VirK bound to misfolded conformations 27 of the Pet passenger domain, but it did not bind to the folded passenger domain or to the β 28 domain of Pet. Assays with an EAEC∆virK mutant and its complemented strain showed that, in 29 the absence of VirK, Pet was not secreted but was instead retained in the periplasm as proteolytic 30 fragments. In contrast, Pet was secreted from a ∆skp mutant. VirK was not required for the 31 insertion of porin proteins into the outer membrane but assisted with insertion of the Pet β 32 domain into the outer membrane. Loss of VirK function blocked the EAEC-mediated cytotoxic 33 effect against HEp-2 cells. Thus, VirK facilitates secretion of the AT Pet by maintaining the 3