A novel calcium binding protein in Mycobacterium tuberculosis--potential target for trifluoperazine

Phenothiazines have been reported for anti-mycobacterial activity by inhibiting calcium binding proteins, potassium transport processes of phagolysosomes, NADH dependent oxygen consumption by M. tuberculosis membranes and DNA, and lipid synthesis of the bacterium. Thioridazine (TZ), chloropromazine (CPZ) and trifluoperazine (TFP) belong to the class of phenothiazines widely used as neuroleptic drugs. Trifluoperazine, a calmodulin antagonist in eukaryotes, binds to a similar protein containing prototypical EF hand to bind to calcium in M. tuberculosis. Calmodulin, a calcium binding protein, plays a critical role in regulating the activities of several enzymes in response to intracellular calcium levels. Since calmodulins are best characterized in eukaryotes as opposed to prokaryotes, the presence of calmodulin-like activity in M. tuberculosis, the causative agent of tuberculosis, is unknown. We have provided biochemical evidence that M. tuberculosis recombinant (r) Rv1211 gene product stimulates the activities of heterologous calcium-deficient NAD-kinase and bovine brain phosphodiesterase (PDE), much like the eukaryotic calmodulins. Further we have shown that EGTA, a calcium chelator, inhibits rRv1211-stimulated NAD-kinase and PDE activities. We have also shown that trifluoperazine interferes with the activation of NAD-kinase and PDE activities by Rv1211. Using a bioinformatics approach, we have shown that Rv1211 contains one prototypical calcium-binding EF-hand motif, a characteristic feature of calmodulins. Based on these data, we conclude that Rv1211 encodes a protein with calmodulin-like activity (CAMLP) in the human pathogen M. tuberculosis and acts as a potential target for trifluoperazine.