Shotgun proteomic profiling of dormant, ‘non-culturable’Mycobacterium tuberculosis

Abstract

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by 'non-culturability' – a state of the inability of the cells to grow on solid media. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, 'non-culturable' M. tuberculosis cells in a model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions. This approach revealed the accumulation of a significant number of proteins after 4 months of storage in dormancy; among them, 468 proteins were significantly different from those in the actively growing cells and bore a positive fold change. Differential analysis revealed the proteins of the pH-dependent regulatory system phoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages, especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current model of M. tuberculosis reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins with antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored in the dormant state were annotated.