Escherichia coli Secretion of a Variant in the Bordetella pertussis Autotransporter Protein BrkA Containing a Dual Polyhistidine-Tagged 112 Amino Acid Insert Between Asp-57 And Ala-58 of its Passenger Domain

Bordetella pertussis is a Gram-negative bacterium which expresses the Bordetella resistance to killing A (BrkA) protein. BrkA mediates autotransporter functionality and undergoes a processing event to produce a cleaved and uncleaved protein. Despite advancements in the functionality of different regions of this autotransporter, there is a lack of understanding of the mechanistic dynamics driving BrkA translocation. This study aimed to generate a sizable amino acid insert into BrkA passenger domain, analyze the structural layout of the protein, and to determine surface-expression status, regardless of the increase in molecular weight. We hypothesized that inserting a 112 amino acid spacer into the N-terminal domain will not affect BrkA surface expression, but may modulate processing events in an Escherichia coli model. We constructed pSERC, containing two 6x histidine tags within a 336 base pair insert in the brkA passenger region. The amino acid sequence corresponding to the insert revealed passenger residue repetition and potential for a protease cleavage site. Finally, we confirmed that the 112 amino acid insert-containing BrkA passenger retained surface expression in an E. coli model and a processing event. Our findings report that BrkA translocation is retained following a substantial amino acid insertion, and further suggests a potential avenue for studying BrkA surface expression and the complex translocation pathway that the passenger follows from cytoplasm to outer-membrane.