Kaprelyants, A. | bioRxiv Science

bioRxiv2021

Shotgun proteomic profiling of dormant, 'non-culturable' Mycobacterium tuberculosis

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by non-culturability - a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, non-culturable M. tuberculosis cells in an in vitro 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 (1379) in cells 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 (FC). 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 in vitro model of M. tuberculosis self-acidification 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 (40) among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

microbiology↗

bioRxiv2018

DrrS, a small non-coding Mycobacterium tuberculosis RNA, regulates the whole genome expression shifts consistent with adaptations for survival within host macrophages

Small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. We investigated the dynamics of expression of MTS1338, a small non-coding RNA of Mycobacterium tuberculosis, in the mouse model in vivo, regulation of its expression in the ex vivo infected macrophages, and the consequences of its overexpression in bacterial cultures. Here we demonstrate that MTS1338 significantly contributes to host-pathogen interactions. Activation of the host immune system triggered NO-inducible up-regulation of MTS1338 in macrophage-engulfed mycobacteria. Constitutive overexpression of MTS1338 in cultured mycobacteria improved their survival in vitro under low pH conditions. MTS1338 up-regulation launched a spectrum of shifts in the transcriptome profile similar to those reported for M. tuberculosis adaptation to hostile intra-macrophage environment. Using the RNA-seq approach, we demonstrate that gene expression changes accompanying MTS1338 overexpression indicate reduction in translational activity and bacterial growth, which is consistent with entering the dormant state. Taken together, our results suggest a direct involvement on this sRNA in the interplay between mycobacteria and the host immune system during infectious process.

microbiology↗