The long term goal of this project is to understand the means by which M. tuberculosis (Mtb) regulates its response to the host environment during infection, so that improved intervention strategies and biomarkers can be developed against tuberculosis (TB) disease. Inteins are mobile protein elements that insertionally inactivate the proteins they inhabit. Autocatalytic excision of these inteins results in functional activation of their host proteins, providing an ideal mechanism for rapid post-translational gene regulation. Inteins are found in three Mtb proteins (DnaB, RecA and SufB), all of which have central roles in the process of DNA replication and recovery following exposure to the DNA damaging conditions encountered during mammalian infection. Inteins have been extensively studied at the biochemical level and have been exploited as tools for protein engineering. However, little is known about the biological roles of inteins in Mtb despite recent studies showing that prevention of intein splicing with the FDA approved anti-cancer drug cisplatin inhibits Mtb growth. This pilot project will test the hypothesis that intein splicing is modulated by host-associated environmental conditions and generate a sensitive fluorescence-based gain-of-function reporter system suitable for monitoring Mtb intein splicing within bacteria during infection. Completion of this research plan will provide critical conceptual and technical foundations that will also be needed for future studies addressing the possibility that inteins provide a rapid form of post-translational regulation in Mtb during host infection. This work is of particularly high impact because of the potential for intein splicing to establish new paradigms for gene regulation in Mtb that may control Mtb replication within the host, as well as the demonstrated role of intein splicing as a possible drug target.
Tuberculosis (TB) is a major global health problem, and new intervention strategies are urgently needed to reduce its deadly impact. Characterization of gene regulatory mechanisms in Mtb, and the means by which TB bacteria respond to their environments during infection, will contribute to our understanding of the factors that control establishment of tuberculosis infection and disease. This work will develop tools to explore the role of intein splicing in Mtb's response to host-associated environmental conditions, which has the potential to identify new biomarkers of infection, as well as targets for anti-TB therapeutics, and/or diagnostics.