Medicina Pulmonar e Respiratória

Defining New Drug Targets Through Protein-Protein Interaction: Interaction of Resuscitation Promoting Factors with SucA of TCA Cycle in M. tuberculosis H37Rv

Abstract

Ravi Kr Gupta, Swati Srivastava, Brahm S Srivastava and Ranjana Srivastava

Upon infection with Mycobacterium tuberculosis, only a small percentage causes active infection while the rest goes into latent infection. These latent bacilli can reactivate under immunocompromised conditions and cause active disease. Little is known about the mechanism by which the mycobacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation promoting factors (Rpf) from Micrococcus luteus and M. tuberculosis has been shown to stimulate growth of dormant mycobacteria as well as reactivation of chronic tuberculosis in mice. Rpf is present as a single, essential gene in M. luteus and five homologues (rpfA-E) in M. tuberculosis. The ability to stimulate culturability and resuscitation appears to be related to muralytic activity of Rpf and they have been identified as peptidoglycan glycosidases. Rpf B has earlier been shown to interact and synergize with Rpf-interacting protein A (RipA), an endopeptidase to cleave bonds in bacterial peptidoglycan suggesting distinct role of Rpf B in cell wall hydrolysis. In order to further understand the role of other Rpfs in resuscitation of dormant mycobacteria, we used an E. coli two hybrid system to identify SucA of TCA cycle as an interacting partner of Rpf from M. tuberculosis (Rpf A, C and D) and M. luteus (RpfM). The in vivo protein-protein interaction was confirmed by M-PFC system in mycobacterial host, in vitro by FRET analysis. An enhanced expression of SucA and Rpf genes was observed during resuscitation phase. We hypothesize that during transition from nonculturable to resuscitation phase mycobacteria cleaves its hard breaking cell wall by endopeptidase RipA interacting with Rpf B and increases its metabolic energy generation by evoking TCA cycle, interacting with Rpf A, C and D and could serve as prospective target along with Rpfs and RipA for development of new anti-tuberculosis drugs preventing reactivation of dormant bacilli.