Steps Towards Unraveling the Driving Force of BrkA Passenger Domain Translocation: The Role of Passenger Domain Length in Secretion Efficiency

Abstract

Autotransporters are a unique type of secretion system utilized by gram-negative bacteria to secrete proteins across the bacterial outer membrane. Specifically, Bordetella pertussis employs the BrkA autotransporter to provide resistance to host serum killing and to mediate host cell surface attachment. BrkA protein structure has been characterized with a signal peptide, a passenger domain which is secreted, and a translocation domain that remains anchored in the outer membrane. The translocation of the passenger domain requires energy; however no ATP hydrolysis or proton gradients are involved. Previous literature has proposed a model for autotransporter passenger domain translocation in which an increase in free energy due to the disruption of the passenger’s hydrating shell during translocation drives its secretion. However, no exact mechanism has been described for the driving force of BrkA translocation. Therefore, this study aimed to explore the potential application of the free energy model on BrkA passenger domain translocation by creating three brkA constructs, each with different passenger domain lengths, and measuring their relative surface display efficiencies. Various lengths of BrkA passenger domain were created with primers designed to amplify only desired regions of the brkA gene, while intentionally removing unamplified sequences. After confirming correct deletions via restriction enzyme digest and Nanopore sequencing, western blotting was used to investigate BrkA expression. After several attempts, BrkA expression from plasmid constructs with altered passenger domains was not detected. Despite difficulties elucidating the exact cause of this unsuccessful detection, this paper sets a foundation for understanding the importance of the passenger domain in BrkA stability and expression. Understanding the driving force of BrkA translocation may provide insight into the mechanism of bacterial pathogenesis and potentially lead to new therapeutic targets.