Background: Mycobacterium tuberculosis cell-wall associated glycoproteins are known to play an important role in host-pathogen interactions.
Methods: M.smegmatis mc2155 was used in this study. Cloning and expression was performed in pSMT3 shuttle vector. Invasion assay in HeLa cells. Survival assay, autopahgy, oxidative stress response, immunostaining by fluroscence microscope and Western blot analysis was done in mouse macrophages. Microbial adhesion to hydrocarbons (MATH) test was performed to assess bacterial hydrophocity. Bacterial susceptibility against antibiotics was done with CFU assay.
Results: Very few Mtb cell wall glycoproteins have been identified and demonstrated for their role in pathogenesis. To identify novel glycoproteins, a multi-lectin system was used to capture glycoproteins from purified Mtb cell wall and identified them by mass spectrometry analysis. This approach successfully identified a novel protein as putative acetyltransferase (ACTase). Recombinant M.smegmatis expressing the ACTase (Msm_ACTase) showed increased invasion in human epithelial cells and survival in mouse macrophages. Increased intracellular bacillary burden was a result of inhibition of autophagy and ROS production due to reduced expression of superoxide dismutase (SOD) and catalase enzymes in Msm ACTase infected macrophages when compared with control strains. Subsequent studies showed that decreased ROS production was due to over expression of ROS scavenging peroxisomal membrane protein 70 and increased expression of acylCoA oxidase, a classical marker enzyme for peroxiosmal β-fatty acid oxidation. Msm_ACTase exhibited increased production of nitric oxide and expression of iNOS in infected macrophages. Addition of inhibitors for peroxisomes and iNOS decreased the production of iNOS and peroxisomes respectively. Moreover, Msm_ACTase showed increased resistance to anti-TB drugs and to lysozyme due to the increased cell surface hydrophobicity.
Conclusion: The study reports for the first time that Msm_ACTase aids the increased intracellular bacterial survival by scavenging H2O2 due to over expression of peroxisome which gives insight to a new mechanism how the pathogen surpass the host defense in Mtb infection. The above findings may lead to identification of a potential drug target.
G. Ganguli, None