Coevolution of -Regulated Hyper-Inflammation and Mycobacterial Resistance to Oxidative Killing Through Adaptation to Hydrogen Peroxide

Mycobacterium tuberculosis () is highly resistant to host oxidative killing. We hypothesized that the evolutionary adaptation of M. smegmatis to hydrogen peroxide (HO) would endow the nonpathogenic Mycobacterium persistent in a host. In the study, we screened a highly HO-resistant strain (mc114) via evolutionary HO adaptation . The MIC of mc114 to HO is 320 times that of wild-type mc155. Mouse infection experiments showed that mc114, similar to , was persistent in the lungs and caused high lethality in mice with restricted responses of NOX2, ROS, IFN-γ, decreased macrophage apoptosis, and overexpressed inflammatory cytokines in the lungs. Whole-genome sequencing analysis revealed that mc114 harbored 29 single nucleotide polymorphisms in multiple genes; one of them was on the gene that caused FurA deficiency-mediated overexpression of KatG, a catalase-peroxidase to detoxify ROS. Complementation of mc114 with a wild-type gene reversed lethality and hyper-inflammatory response in mice with rescued overexpression of KatG and inflammatory cytokines, whereas NOX2, ROS, IFN-γ, and macrophage apoptosis remained reduced. The results indicate that although FurA regulates KatG expression, it does not contribute significantly to the restriction of ROS response. Instead, FurA deficiency is responsible for the detrimental pulmonary inflammation that contributes to the severity of the infection, a previously nonrecognized function of FurA in mycobacterial pathogenesis. The study also indicates that mycobacterial resistance to oxidative burst results from complex mechanisms involving adaptive genetic changes in multiple genes. Mycobacterium tuberculosis () causes human tuberculosis (TB), which has killed more people in human history than any other microorganism. However, the mechanisms underlying pathogenesis and related genes have not yet been fully elucidated, which impedes the development of effective strategies for containing and eradicating TB. In the study, we generated a mutant of M. smegmatis (mc114) with multiple mutations by an adaptive evolutionary screen with HO. One of the mutations in caused a deficiency of FurA, which mediated severe inflammatory lung injury and higher lethality in mice by overexpression of inflammatory cytokines. Our results indicate that FurA-regulated pulmonary inflammation plays a critical role in mycobacterial pathogenesis in addition to the known downregulation of NOX2, ROS, IFN-γ responses, and macrophage apoptosis. Further analysis of the mutations in mc114 would identify more genes related to the increased pathogenicity and help in devising new strategies for containing and eradicating TB.