The N-Terminus of the Yersinia pestis yapV Autotransporter Protein Destabilizes the Entire Passenger Domain In Vitro: Implications for In Vivo Secretion

Autotransporter (AT) proteins are an important class of virulence factors from Gram-negative pathogens, including Yersinia pestis. The AT outer membrane (OM) secretion mechanism is deceptively simple, in part because major determinants for AT OM secretion are contained within the AT sequence itself. However, to date it remains unclear how the central AT 'passenger', which comprises the mature virulence protein, crosses the OM. Recent studies have demonstrated that an uneven distribution of stability within the AT passenger domain can affect its OM secretion efficiency. However, this connection has been investigated for only two of the >1500 identified AT proteins, and it remains to be determined whether there is a universal connection between AT passenger stability and OM secretion efficiency. Here we have characterized a new Yersinia pestis AT protein with which to broaden our current understanding of the connection between AT passenger domain folding and OM secretion. Yersinia pestis yapV encodes an outer membrane (OM) protein homologous to Shigella flexneri IcsA. We demonstrate that YapV is an AT protein that, like IcsA,recruits the mammalian protein neural Wiskott-Aldrich syndrome protein (N-WASP). We also designed, expressed and purified two constructs corresponding to the central functional passenger domain of YapV. While functional and rich in β-sheet structure like many other AT passengers, we show that the YapV passenger constructs do not have the typical C-terminal stable core structure identified in Bordetella pertussis pertactin or E. coli Pet, but instead have the C-terminal 75% of the passenger more resistant to proteinase K (proK) digestion than the N-terminal 25%. Furthermore, we show from in vitro analyses that the N-terminal 49 amino acids render the entire passenger domain less stable to heat and guanidine hydrochloride denaturation as well as more susceptible to proK digestion than when it is truncated. We show by in vivo folding and secretion analyses that this N-terminal peptide causes the YapV passenger to adopt a less folded conformation in the periplasm maintaining the YapV passenger in a conformation that is compatible with OM secretion. These results suggest that in addition to the gradient in stability within the passenger domain, some autotransporters can cross the OM using other unique features encoded within their passenger domain.