Year: 2019
Type: article
Abstract: Mycobacterium tuberculosis is an intracellular pathogen that resides primarily in host macrophages. Proteins that reside on the surface of the bacillus or are secreted into environment are ideally positioned to interact with host cell components and are therefore candidate virulence factors and immunogenic antigens. These proteins are actively transported from the cytoplasm to the cell envelope or extracellular space by specific export pathways. In this work, we describe the initial characterization of the twin-arginine translocation (Tat) export pathway in mycobacteria. The Tat pathway exports folded proteins, including virulence factors, in a number of bacteria. Proteins targeted for Tat-export carry N-terminal signal sequences that have a conserved twin-arginine motif, referred to as R-R-x-φ-φ(φ = uncharged residue). We provide evidence that the Tat pathway is functional in Mycobacterium smegmatis and required for the export of mycobacterial β-lactamases, including BlaC of M. tuberculosis. We demonstrate that BlaC can be used as a reporter to exclusively identify Tat-exported fusion proteins that promote resistance to the β-lactam antibiotic carbenicillin. We describe using the BlaC reporter with a M. tuberculosis genomic expression library to identify random Tat signal sequence-BlaC fusions that promote Tat export in M. smegmatis. Using this approach, we identified eleven M. tuberculosis Tat signal sequences shown to direct the export of the BlaC reporter through the Tat pathway, including the known phospholipase C virulence factors. In addition, we identified a protein lacking the typical twin-arginine motif, suggesting that our current understanding of what defines a Tat signal sequence is limited, and demonstrating the effectiveness of our experimental system. We describe ongoing evaluation to determine the specificity and requirements for export of the authentic full length M. tuberculosis proteins. Finally, we demonstrate the novel finding that BlaC can be used as a reporter for the identification of proteins exported by M. tuberculosis during growth in macrophages. This may lead to the identification of a subset of proteins exported exclusively within the intracellular environment. This work has important implications in determining the role that protein export plays in M. tuberculosis, as well as increasing the understanding of how the Tat pathway functions in bacteria.
DOI: 10.17615/5wec-bs21
Author: McDonough, Justin Andrew
Institution: University of North Carolina at Chapel Hill
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2952855139