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Pore-forming ability and topological structure of the C-terminal domain of the Bordetella Pertussis Autotransporter BrkA Shannon, Jennifer Louise


One of the many virulence factors of Bordetella pertussis, the causative agent of whooping cough, is BrkA, which is involved in adherence and mediates resistance to antibody-dependent killing by complement. BrkA is a 103 kDa outer membrane protein that is proteolytically processed into a 73 kDa N-terminal domain and a 30 kDa C-terminal domain. It is also a member of the autotransporter family of proteins. With autotransporters, translocation of their N-terminal domains across the outer membrane is hypothesized to occur through a pore formed by their Cterminal domains, which adopt an amphipathic 6-barrel structure. However, neither pore-forming ability nor this topology has ever been shown for any of the autotransporters. To test pore formation by the BrkA C-terminal domain, black lipid bilayer experiments were performed, where insertion of pore-forming proteins into an artificial membrane causes measurable jumps in conductance. A His-tagged fusion form of the protein formed channels with an average size of 3.0 nS. Similar results were obtained with the fusion protein that was eluted from an SDS-PAGE gel. The addition of the 73 kDa BrkA N-terminal protein that had been purified in a similar way to the fusion protein, showed no increases in conductance; channels were again observed after addition of the BrkA C-terminal protein to the system. Similar results were obtained with protein from a clone containing a vector without the brkA insert. These results show that the C-terminal autotransporter domain of BrkA is capable of forming a pore. BrkA C-terminal protein that was purified from the outer membrane also formed channels. However, the protein could not be completely separated from other proteins that when extracted from a vector-only clone showed some pore-forming activity, although to a lesser extent. This data suggests that the BrkA Cterminal domain does form a pore in the outer membrane. To determine the topology of the BrkA C-terminal domain, a transposon-based method of generating 31 a.a. tags was employed because it produces random in-frame insertions. Four different insertions were obtained, but only two were in-frame. The anti-31 a.a. tag antibody worked in Western blots, but did not work in immunofluorescence, so the tag could not be localized. The results obtained from expression experiments confirmed the position of a transmebrane strand and a loop region, which agreed with my proposed model.

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