Role of Rpf Homologues in M tuberculosis Reactivation

  • Tufariello, Joann M. (PI)

Project: Research project

Project Details


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.
Effective start/end date5/1/014/30/05


  • National Institute of Allergy and Infectious Diseases: $126,762.00


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