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.