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Structure/function studies on the autotrasporter BrkA and investigation of the role of periplasmic chaperones in BrkA secretion Yue, Jody

Abstract

BrkA is a virulence factor in Bordetella pertussis that belongs to the ATI subfamily of autotransporter proteins in Gram negative bacteria. ATI members have: (i) an N-terminal signal peptide for secretion across the inner membrane, (ii) a surface expressed passenger domain, and (iii) a C-terminal translocation unit for passenger secretion across the outer membrane. There are many questions about ATI secretion across the periplasm and the outer membrane. Studies on the ATI member IcsA suggests rapid periplasmic transit, and that DegP's chaperone activity is involved. Comparison of passenger size with estimated and known pore sizes of transporter domains suggest that the passenger is too large for translocation across the outer membrane in a folded state. The mechanism by which an ATI passenger might maintain an unfolded state in the periplasm while resisting proteolysis is unknown. BrkA secretion in Escherichia coli was used to investigate periplasmic transit and outer membrane translocation of an ATI passenger. Previous studies revealed the junction domain in BrkA, which functioned in passenger folding. A highly conserved subdomain (region 3) was identified by ClustalW alignment of the junction, and was dispensable for in vitro folding. This subdomain might function in secretion by keeping the passenger unfolded for translocation by binding to a periplasmic chaperone. The role of region 3 in BrkA surface expression and in vivo folding was tested by trypsin accessibility and immunofluorescence microscopy, and limited proteolysis assays, respectively. In parallel studies, the involvement of periplasmic chaperones in BrkA secretion was investigated by trypsin accessibility assays on Skp, DegP, and SurA knockout strains expressing BrkA. Residues A⁶⁸¹ -E⁶⁹³ of region 3 were important for secretion of a folding-competent BrkA passenger, but were dispensable for in vivo folding. We termed A⁶⁸¹-E⁶⁹³ the "hydrophobic secretion facilitation" (HSF) domain, after the term coined by Velarde and Nataro for the corresponding region in the ATI member EspP, which was important for passenger secretion. SurA, a peptidyl prolyl isomerase (PPIase) that functions in porin secretion, was implicated in BrkA secretion. The PPIase activity of SurA was dispensable, suggesting that SurA's chaperone activity is the necessary activity for BrkA secretion.

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