A systems approach to decipher a role of transcription factor RegX3 in the adaptation of Mycobacterium tuberculosis to hypoxic stress.

Two-component systems (TCSs) are required for the ability of Mycobacterium tuberculosis to respond to stress. The paired TCS, SenX3-RegX3 is known to respond to phosphate starvation and acid stress. The other stress conditions under which RegX3 is required for M. tuberculosis to mount an appropriate response, remain incompletely understood. Here we have employed genome-wide microarray profiling to compare gene expression in a ΔregX3 mutant with the wild-type under phosphate stress, in order to gain information on the probable RegX3 regulon. We pulled out a set of 128 hypoxia-associated genes, which could potentially be regulated by RegX3, by overlapping the gene set downregulated at least twofold in ΔregX3 with the gene set reported in the literature to be associated with the response to hypoxia. We identified potential RegX3 binding inverted repeats at the loci of 41 of these genes, in silico. We also observed that ΔregX3 was attenuated in terms of its ability to withstand hypoxia, and this was reversed upon complementation with regX3, corroborating a role of RegX3 in the response of M. tuberculosis to hypoxia. We validated the binding of RegX3 at the upstream regions of a selected set of these genes. Electrophoretic mobility shift assays (EMSAs) confirmed that RegX3 binds to the upstream regions of the hypoxia-associated genes Rv3334, whiB7, Rv0195, Rv0196 and Rv1960c. Gene expression analyses showed that the expression of these genes is regulated by RegX3 under hypoxia. We also show that the expression of whiB7, Rv3334 and Rv0195 in macrophage-grown M. tuberculosis, is dependent on RegX3. Finally, we show that attenuation of survival of ΔregX3 under hypoxia is partly reversed upon overexpression of either Rv0195 or Rv3334, suggesting that the RegX3-Rv0195 and the RegX3-Rv3334 axis are involved in the adaptation of M. tuberculosis to a hypoxic environment.