VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Escherichia coli</jats:named-content>(EAEC). These proteins include the 17-kDa Skp chaperone and the 37-kDa VirK protein. We found that the<jats:italic>virK</jats:italic>gene is present in different<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Enterobacteriaceae</jats:named-content>. VirK bound to misfolded conformations of the Pet passenger domain, but it did not bind to the folded passenger domain or to the β domain of Pet. Assays with an EAECΔ<jats:italic>virK</jats:italic>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 Δ<jats:italic>skp</jats:italic>mutant. VirK was not required for the insertion of porin proteins into the outer membrane but assisted with insertion of the Pet β 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 β-domain insertion into the outer membrane.</jats:p>

The Periplasmic Chaperone Network of Campylobacter jejuni: Evidence that SalC (Cj1289) and PpiD (Cj0694) Are Involved in Maintaining Outer Membrane Integrity

“…We have shown that a virK mutant in C. jejuni NCTC 11168 displays decreased growth, enhanced motility and biofilm formation, and a strongly hydrophobic cell surface – the latter phenotype shared only with the peb4 mutant ( Figures 1A–C,E ). This supports the hypothesis that VirK in C. jejuni may play a more general role in OM or cell surface biogenesis than reported in E. coli , where VirK is necessary, and potentially specific, for Pet toxin secretion (Tapia-Pastrana et al, 2012), a system absent in C. jejuni. …”

“…In E. coli VirK is thought to be a periplasmic chaperone for the plasmid-encoded toxin (Pet), an autotransporter produced by enteroaggregative E. coli (Tapia-Pastrana et al, 2012). While VirK in E. coli is periplasmic, Novik et al (2009) showed that the C. jejuni VirK homolog is associated with the inner membrane on the cytoplasmic face, and so may act as a chaperone prior to Sec-mediated export.…”

“…The role of PPIase domains in such chaperones is not always clear, as the E. coli PpiD parvulin-like domain is catalytically inactive (Weininger et al, 2010) as is one of the two parvulin domains in SurA itself. Another potential chaperone, VirK, is a 37 kDa periplasmic protein that may have a role in autotransporter assembly and toxin export in E. coli (Tapia-Pastrana et al, 2012). Finally, HtrA (DegP) is a chaperone and proteolytic enzyme that degrades unfolded proteins in the periplasm (Ge et al, 2014).…”

A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

“…Blot overlay assays were performed as previously reported (43) with a few modifications. Purified proteins (EspC, His-NH 2 , His-Middle, His-COOH, His-EspCΔMiddle, and His-EspA) (1 µg) were separated by SDS-PAGE and transferred to PVDF membranes.…”

Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence

“…However, this discrepancy in results is not hard to reconcile if we observe the variable cytotoxic effect elicited by other class-I SPATEs [56, 64, 65, 67, 95] (discussed below), which also have distinctive degradation patterns on -spectrin—the target being linked to the cytotoxic effects [56, 64, 65]. Consequently, amino acid variation among SPATEs, protease doses, time of exposure, mammalian cell types, and bacterial strains from where SPATEs are purified [108, 109] may have influenced the strength of protease activity.…”