Interaction of CarD with RNA polymerase mediates Mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis.
Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
Leslie A
LA
Weiss
Phillip G
PG
Harrison
Bryce E
BE
Nickels
Michael S
MS
Glickman
Elizabeth A
EA
Campbell
Seth A
SA
Darst
Christina L
CL
Stallings
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D050296:Microbial Viability; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000254:growth & development / Q000472:pathogenicity / Q000502:physiology*; D017354:Point Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012293:Rifampin / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*; D014774:Virulence
D000900:Anti-Bacterial Agents; D050505:Mutant Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin