Identification of Secretion Determinants of theBordetella pertussisBrkA Autotransporter

Abstract

The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "␣-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "␤-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal ␣-domain and a 30-kDa C-terminal ␤-domain. We have previously shown that a recombinant form of the ␤-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31-to 39-amino-acid region found immediately upstream of the ␤-domain that was essential for surface expression. This 31-to 39-amino-acid linker region, together with the ␤-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.

Structure-Function Analysis of the Enteroaggregative Escherichia coli Plasmid-encoded Toxin Autotransporter Using Scanning Linker Mutagenesis

“…In homologous proteins, this region has previously been designated the autotransporter junction region and has been suggested to play a role in secretion (25)(26)(27)(28). Ohnishi et al (26) have shown that nonsecreted deletion mutants in the junction region of SSP, an autotransporter from Serratia marcescens, could be rescued for secretion when the junction region is supplied in trans.…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…Oliver et al (364,365) recently discovered an autochaperone domain in BrkA. This well-conserved domain, found upstream of the translocation unit, is present within the passenger domains of several autotransporters, like AIDA-I, IgA1 protease, Ag43, Hap, and IcsA, but was not detected in some autotransporters such as TcfA and Hia (365).…”

“…However, the passenger domain is not necessarily cleaved but may also remain intact as a large protein with a membrane-bound C-terminal domain and an N-terminal domain extending into the external milieu, e.g., Hia or Hsr (367,465). Nevertheless, the cleavage between the passenger domain and the translocation unit can occur either well upstream of the linker region or within the predicted ␣-helical region, like BrkA (364). In this latter case, BrkA remains steadfastly associated with the bacterial outer membrane, raising the possibility that the linker region could also act as an anchor for the passenger domain.…”

“…BrkA is synthesized as a 103-kDa protein that is processed into a 73-kDa passenger domain and a 30-kDa ␤-domain. N-terminal sequencing of the 73-kDa passenger domain has determined the signal sequence to be 42 amino acids long (364). Cleavage of BrkA after amino acid 731 yields the outer membrane-resident ␤-domain (187).…”

“…The next domain is the passenger domain (also called the ␣-domain, N-passenger domain, or N-domain), which confers the diverse effector functions of the various autotransporters. The last main domain, located at the C-terminal end of the protein, is the translocation unit (also called the ␤-domain, helper domain, C-domain, transporter domain, or autotransporter domain), consisting of a short linker region with an ␣-helical secondary structure and a ␤-core that adopts a ␤-barrel tertiary structure when embedded in the outer membrane (317,364,365,475), facilitating translocation of the passenger domain through the outer membrane.…”