Genetic screens to identify mycobacterial porins

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DESCRIPTION (provided by applicant): Mycobacteria are a family of primarily saprophytic organisms that inhabit many ecological niches. Several of these members are highly significant human pathogens, including the causative agents of tuberculosis (M. tuberculosis, M. africanum, and M. bovis), and the opportunistic agents such as M. avium and M. intracellulare that infect primarily immunocompromised patients. A hallmark characteristic of mycobacteria is the lipid-rich cell envelope which makes these organisms highly impermeable and rendering them intrinsically resistant to many bacteriocidal compounds. To facilitate uptake of hydrophilic solutes, mycobacteria, like Gram-negative organisms, utilize porins which are water-filled channels that allow passage of hydrophilic compounds such as nutrients and antibiotics through the outer layer of the cell envelope. Thus, porins play a critical role in cell physiology, serving as gatekeepers that control access of various environmental compounds to the cell, and in the case of pathogens, antibiotics. Therefore, the identification and characterization of mycobacterial porins will not only enhance our knowledge of their physiology, but will also aid in the design of more effective therapeutics. Several mycobacterial porins have been physically characterized from whole cell extracts, the porin genes of M. smegmatis have been identified and the major porin gene, MspA, structurally characterized. However, very little is known about the porins of M. tuberculosis. No homologs to the M. smegmatis porins have been found in the M. tuberculosis genome. In addition, multiple attempts to chemically isolate sufficient amounts of porins from M. tuberculosis or M. bovis for identification have not been successful. Our long-term goal is study porin function in the slow growing mycobacteria. The immediate aims of this proposal are to study the physiology of the porins of M. smegmatis and to develop various genetic screens to identify porin genes of M. tuberculosis. Research to characterize this aspect of mycobacterial physiology could aid in the design of more effective therapeutics for the treatment of various mycobacterial infections. This would be an important contribution to reduce the public health burden of mycobacterial disease in the United States.