| Summary: | 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. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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