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Structure/function studies of the Bordetella pertussis autotransporter protein BrkA : Oliver, David C.

Abstract

The autotransporter secretion system represents a fundamental strategy that Gramnegative bacteria have evolved to deliver an array of functionally diverse proteins to the cell surface. As the name implies, the autotransporter secretion system does not employ specific accessory factors; all of the information necessary for movement of a substrate polypeptide across the two membranes of the cell envelope is encoded within a single gene product. Autotransporters are modular multidomain proteins consisting of an N - terminal signal peptide, a passenger domain that contains the effector function(s) to be delivered to the cell surface, and a C-terminal domain termed the translocation unit. The signal peptide directs translocation across the inner membrane and the translocation unit facilitates export across the outer membrane. How this seemingly simple protein secretion strategy functions is largely unknown. This study addresses the secretion mechanism of the BrkA protein, a known virulence factor of Bordetella pertussis (the causative agent of whooping cough) and a putative autotransporter protein. Using a combination of genetic, biochemical, bioinformatic and cell biological approaches, BrkA was shown here to be a bona fide autotransporter protein and was established as a model system for studying autotransporter secretion. Structural and functional dissection of BrkA revealed a multidomain architecture consisting of a signal peptide, a passenger domain that contains the BrkA effector functions (serum resistance and adherence), and a C-terminal translocation unit. Significantly, a region termed the junction that is located at the C-terminus of the BrkA passenger domain was identified to be required for passenger folding during secretion. The conservation of this domain in a functionally diverse group of autotransporter proteins suggested that it plays an important role in secretion. The demonstration that the junction region mediated BrkA passenger folding when supplied in trans as a separate polypeptide suggests that it can function as an intramolecular chaperone. Further dissection of the BrkA junction revealed a sub-region that is not required for passenger folding but is required for secretion of a "folding competent" native BrkA passenger. These findings have been integrated with our current knowledge of autotransporter secretion to generate a working model of BrkA secretion that may be applicable to other autotransporter proteins.

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