Coevolution of furA-Regulated Hyper-Inflammation and Mycobacterial Resistance to Oxidative Killing through Adaptation...

Mycobacterium tuberculosis (Mtb) is highly resistant to host oxidative killing. We hypothesized that the evolutionary adaptation of M. smegmatis to hydrogen peroxide (H₂O₂) would endow the nonpathogenic Mycobacterium persistent in a host. In the study, we screened a highly H₂O₂-resistant strain (mc²114) via evolutionary H₂O₂ adaptation in vitro. The MIC of mc²114 to H₂O₂ is 320 times that of wild-type mc²155. Mouse infection experiments showed that mc²114, similar to Mtb, 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 mc²114 harbored 29 single nucleotide polymorphisms in multiple genes; one of them was on the furA gene that caused FurA deficiency-mediated overexpression of KatG, a catalase-peroxidase to detoxify ROS. Complementation of mc²114 with a wild-type furA 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. IMPORTANCE Mycobacterium tuberculosis (Mtb) causes human tuberculosis (TB), which has killed more people in human history than any other microorganism. However, the mechanisms underlying Mtb 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 (mc²114) with multiple mutations by an adaptive evolutionary screen with H₂O₂. One of the mutations in furA 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 mc²114 would identify more genes related to the increased pathogenicity and help in devising new strategies for containing and eradicating TB.