Utilizing a Two-Hybrid System to Determine Possible Roles and Functions of Hypothetical Proteins in Chlamydia trachomatis

McKinney, Megan

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Utilizing a Two-Hybrid System to Determine Possible Roles and Functions of Hypothetical Proteins in Chlamydia trachomatis

McKinney, Megan

Abstract

Chlamydia trachomatis, an obligate intracellular pathogen, is the leading cause of sexually transmitted bacterial infections and preventable blindness worldwide. A major challenge to understanding many aspects of the basic biology and pathogenesis for C. trachomatis is the portion (~32%) of proteins that are functionally unknown. In order to address this issue, the paradigm that interacting proteins frequently participate in the same biological role was applied. To discover protein interactions the first ever large-scale bacterial adenylate cyclase-based two-hybrid (BACTH) system using C. trachomatis proteins was utilized. These large-scale studies in other organisms have been conducted to determine global protein interactions (interactome) in order to provide a comprehensive appreciation and prediction for the biological role or function of proteins. An interactome was constructed by screening 50 chlamydial proteins (18,000 possible interactions) and only 333 interactions occurred (1.85% interaction rate). Of those, interactions between hypothetical and functionally annotated proteins involved in type III secretion (T3S) system, inclusion membrane formation, and secretion were discovered. The BACTH system focuses on a large screen of binary interactions and determining possible roles of hypothetical proteins. This approach is expected to generate a more comprehensive understanding of the chlamydial protein interactome, further build the proteomic map of C. trachomatis, and allow for insight into the possible role of functionally unknown proteins. In addition to a large- scale screen with chlamydial proteins, a smaller BACTH screen was used to determine the binding partner of the kinase protein, RsbW, which is involved in the Rsb phosphoregulatory pathway of C. trachomatis. A library was constructed containing all known Rsb pathway proteins along with hypothesized target proteins of RsbW. Through BACTH screening, both RsbU and Sigma 66 exhibited interactions with the RsbW protein. This suggests that RsbU and RsbW may form a complex during their phosphorylation activities, while Sigma 66 could be the target protein of RsbW.