Iron supplementation potentiates oxidative stress, modulates gene expression and enhances killing of Mycobacterium tuberculosis treated with rifampicin and vitamin C.

Drug tolerance in Mycobacterium tuberculosis (Mtb) significantly undermines the success of antimycobacterial therapy. Redox active compounds such as Vitamin C (VitC) and iron can modulate drug efficacy, and in the present study we evaluated their effect on Rifampicin (RIF) mediated killing of Mycobacterium tuberculosis H37Ra (Mtb-Ra). We also studied the cellular reactive oxygen species (ROS) levels and expression of stress-response and drug target genes, and host cell cytotoxicity. ROS measurements revealed that treating Mtb-Ra with RIF and VitC led to ROS levels mostly being elevated, with iron supplementation leading to further increase. Survival analysis showed that VitC increased the killing by RIF in a dose-dependent manner, with iron potentiating this effect. Cytotoxicity studies showed that high iron and its combinations with VitC and RIF showed comparable cytotoxicity. The qRT-PCR study demonstrated RIF induced up-regulation of recA, dnaE2 and rpoB, with VitC and iron further amplifying recA and dnaE2 expression. Conversely, lexA was down-regulated in the presence of VitC and more strongly with iron, indicating induction of bacterial SOS response. Interestingly, rpoB expression, while up-regulated with RIF and VitC, was suppressed by iron. These findings suggest that VitC and iron-induced disruption of Mtb homeostasis can enhance RIF efficacy.