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 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—the raw data are available via ProteomeXchange with identifier PXD028849. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically 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 displaying antigenic properties makes it possible to distinguish immunoreactive proteins 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.

Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn–porphyrins

“…In the present study, dormant forms of Mtb were obtained as a result of gradual environmental acidification in the late stationary phase (40–60 days from the moment of inoculation) under aerobic conditions 18 . Such dormant forms were characterized by morphological, biochemical and physiological changes in comparison with actively grown bacteria 18 as well as by differences on transcriptomic and proteomic level 19 , 20 . The appearance of dormant forms was monitored by the decrease in uracil incorporation in the culture, as well as the development of “non-culturability” (decrease in CFU number up to zero 18 ).…”

MftG is crucial for ethanol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

“…Intriguingly, the MFT gene locus seems to be derepressed by long-chain fatty acid-CoA esters in M. smegmatis, which bind to the repressor mftR (10). Furthermore, a comparison of the proteome of active and dormant cells revealed that mftD (Rv0694) was upregulated in the dormant state, which is associated with the persistence of M. tuberculosis in macrophages (11). Lastly, a study on the impact of mycofactocin inactivation (∆mftD) on M. tuberculosis showed increased growth on glucose containing media as well as a decreased number of mycobacterial cells in a mouse model after the onset of hypoxia (12).…”