Role of Rpf Homologues in M tuberculosis Reactivation

DESCRIPTION (provided by applicant): This application outlines a career development plan consisting of combination of intensive research experience and didactic training in bacterial pathogenesis. The long-term goal is to prepare the candidate for a career as an independent investigator at an academic institution. Mycobacterium tuberculosis (Mtb) is a human pathogen of tremendous importance, responsible for an estimated 3 million deaths per year and latently infecting one third of the world s population. Despite intensive effort by numerous laboratories to elucidate the molecular mechanisms of Mtb pathogenesis, little is understood at the molecular level regarding Mtb dormancy and reactivation. This issue is of critical importance, since clinically apparent tuberculous disease is often the result of reactivation, and current chemotherapies have little activity against the latent form of disease. Much has been learned recently about adaptation to adverse environmental conditions among genera of bacteria which do not form classical, morphologically differentiated spores, and some of these adaptations may have direct relevance for the in vivo persistence of Mtb. Micrococcus luteus is, like Mtb, a bacterium with a GC-rich genome, and is able, upon extended and unagitated incubation of cultures, to enter a state of dormancy. Log phase M. luteus secrete an 16 kDa protein, resuscitation-promoting factor (Rpf), which is able to revive dormant M. luteus, and has growth-promoting effects on mycobacterial cultures as well. The Mtb genome encodes five homologues of the M luteus Rpf. This proposal outlines a series of experiments designed to investigate the functions of this gene family in Mtb. The experimental approaches include purification of recombinant his-tagged Rpf homologues, in order to examine their effects on mycobacterial growth kinetics, and to generate antisera for neutralization and expression assays. Expression of the Rpf homologues will be studied in Mtb in vitro under a variety of growth conditions, including anaerobic models of dormancy, at the protein and mRNA levels. Gene disruption of the Rpf homologues will be performed and the growth kinetics of the mutants studied in a murine model of persistent infection. The identification of genes responsive to Rpf will be explored using microarray technology and a complementation/promoter trap assay. It is anticipated that study of this family of putative bacterial pheromones will improve our understanding of the growth regulation of Mtb under various environmental conditions, and may provide insight into the important phenomena of dormancy and reactivation.