Transcriptional changes associated with chemical induction of adenylyl cyclase activity in Mtb

Goal: Assess transcriptional changes in Mtb associated with activation of adenylyl cyclase activity in the bacterium, by treatment with the Rv1625c agonist V-59 or activation of the TetOn-cAMP construct. Specifically, address changes in transcription of cholestrrol utilization genes, during growth of Mtb in cholesterol media. Method: WT, Rv1625c knockout, and Rv1625c Complement strains of Mtb were grown in cholesterol-based media and treated with V-59 or vehicle control (DMSO). V-59 is known to increase cAMP synthesis in WT, but not in Rv1625c knockout Mtb. V-59 increases cAMP synthesis above that observed in WT in the Complement strain, due to Rv1625c overexpression in this strain. Also, utilized TetOn-cAMP Mtb strain, to induce cAMP synthesis independent of V-59 and Rv1625c. Treatment with Atc induces expression of catalytic domain of Rv1264 in this strain. We grew the TetOn-cAMP strain in cholesterol-based media, treated with Atc or EtOH (vehicle control). Conclusions: Transcriptional changes to cholesterol utilization genes associated with V-59 treatment in WT Mtb were similar to those associated with TetOn-cAMP induction. The transcriptional changes associated with blockade of cholesterol degradation following V-59 treatment in WT Mtb were not observed in the Rv1625c knockout strain. The Rv1625c knockout strain had intrinsic defects in induction of cholesterol utilization genes. The Complement strain showed enhanced transcriptional changes in response to V-59 treatment. Overall design: RNA profiles of adenylyl cyclase inducing compound vs. vehicle control in Mtb during culture in cholesterol-based media for four hours