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UBC Theses and Dissertations

The requirement of putative autochaperone motifs for autotransporter passenger domain folding Gagnon, Elizabeth

Abstract

Protein secretion plays an essential role in the virulence of Gram-negative bacterial pathogens. Gram-negative bacteria have evolved multiple specialized secretion pathways in order to navigate proteins across the Gram-negative cell envelope. The simplest and most widespread secretion pathway is the type V secretion system and autotransporters (ATs) (Va) represent the largest class of secreted proteins in Gram-negative bacteria. ATs are structurally characterized by the presence of three distinct domains; an N-terminal signal sequence that targets the N-terminus of the polypeptide to the inner membrane, a passenger domain that often possesses β-helix structure and carries out the virulence function(s) and a conserved C-terminal translocation unit (TU) consisting of a β-domain that forms a porin-like structure in the outer membrane (OM) through which the passenger domain is thought to be extruded and an α-helical linker region that joins the β-domain to the C-terminus of the passenger domain. At the Cterminus of the majority of AT passenger domains is a conserved region termed the autochaperone (AC). The importance of conserved AC residues for passenger domain secretion and folding has been demonstrated for numerous ATs, yet the exact role of the AC region in OM translocation and passenger domain folding has yet to be clarified. In this study, the requirement of conserved C-terminal passenger domain motifs for the acquisition of passenger domain secondary structure and the interchangeability of these conserved AC motifs was investigated. A combination of far-UV CD spectroscopy and limited proteolysis with trypsin of full-length and AC-deleted Vag8, Ag43 and Smp passenger variants revealed that the requirement of conserved C-terminal AC motifs for the acquisition of passenger domain secondary structure varies among ATs. A cell wall fraction assay, in which the ability of BrkA, Ag43 and Smp AC regions to rescue BrkA AC-deleted passenger folding was tested, indicated that an AC region with similar structure to the cognate AC is necessary to rescue passenger domain folding. Altogether, the results of this study highlight the involvement of multiple factors in passenger domain folding and the likely variation that exists in the mechanism of passenger folding at the bacterial cell surface.

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