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UBC Theses and Dissertations

Biogenesis of BapF : a novel acylated Bordetella autotransporter Sims, Peter Vincent

Abstract

Autotransporters are a superfamily of Gram-negative secreted proteins, composed of a Cterminal domain which forms a β-barrel in the outer membrane and plays a significant role in translocation of the N-terminal passenger domain to the cell surface. A subfamily of autotransporters is N-terminally acylated during their biogenesis, a post-translational modification demonstrated to be essential for their function. Considering the autotransporter passenger domain is secreted beyond the outer membrane, how translocation can occur in the presence of N-terminal acyl groups is undetermined. The work in this thesis describes the biogenesis of the Bordetella autotransporter F (BapF) from B. bronchiseptica RB50, which is Nterminally acylated during the initial stages of its secretion to the cell surface. The acyl groups attached to BapF at the Cys28 residue were shown to be subsequently removed by signal peptidase 1 cleavage between residues Ala34 and Ala35, indicating that BapF forms an acylated intermediate in its secretion pathway. Studying the secretion of BapF mutants in which Nterminal acylation was ablated revealed that the passenger domain was still capable of reaching the cell surface; the surface-expressed passenger domain mediated phenotypes of cellular aggregation and an increased rate of culture sedimentation, similar to that observed in E. coli cultures expressing the wild-type BapF protein. However, cells expressing these mutants appear to have damaged outer membranes, potentially due to the observed increase in fulllength protein accumulating in the periplasm. Alternatively, E. coli cells expressing a BapF mutant in which signal peptidase 1 cleavage is blocked do not exhibit obvious aggregation and sedimentation phenotypes. Yet, an independent passenger domain is clearly produced. Based on the results presented in this thesis, it can be hypothesized that sequential processing of the BapF signal peptide, producing an acylated intermediate in the secretion pathway, helps to regulate the passage of BapF through the periplasm ultimately permitting surface expression. In addition, bioinformatic and molecular analysis strongly suggest the BapF passenger domain folds into a β-propeller structure, and if proven to do so, BapF will be the first autotransporter reported with this conformation

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