Truncated Hemoglobin, HbN, Is Post-translationally Modified in Mycobacterium tuberculosis and Modulates Host-Pathogen Interactions during Intracellular Infection

Mycobacterium tuberculosis (Mtb) is a phenomenally successful human pathogen having evolved mechanisms that allow it to survive within the hazardous environment of macrophages and establish long term, persistent infection in the host against the control of cell-mediated immunity. One such mechanism is mediated by the truncated hemoglobin, HbN, of Mtb that displays a potent O2-dependent nitric oxide dioxygenase activity and protects its host from the toxicity of macrophage-generated nitric oxide (NO). Here we demonstrate for the first time that HbN is post-translationally modified by glycosylation in Mtb and remains localized on the cell membrane and the cell wall. The glycan linkage in the HbN was identified as mannose. The elevated expression of HbN in Mtb and M. smegmatis facilitated their entry within the macrophages as compared with isogenic control cells, and mutation in the glycan linkage of HbN disrupted this effect. Additionally, HbN-expressing cells exhibited higher survival within the THP-1 and mouse peritoneal macrophages, simultaneously increasing the intracellular level of proinflammatory cytokines IL-6 and TNF-α and suppressing the expression of co-stimulatory surface markers CD80 and CD86. These results, thus, suggest the involvement of HbN in modulating the host-pathogen interactions and immune system of the host apart from protecting the bacilli from nitrosative stress inside the activated macrophages, consequently driving cells toward increased infectivity and intracellular survival. Background:Mycobacterium tuberculosis HbN detoxifies nitric oxide and protects its host under nitrosative stress. Results: The HbN remains glycosylated and membrane-localized in M. tuberculosis and modulates host-pathogen interactions. Conclusion: The HbN facilitates intracellular infection and cell survival by evading the immune system of the host. Significance: This study unravels new knowledge about function(s) of HbN in biology and pathogenesis of M. tuberculosis.