One-Year Old Dormant, “Non-culturable” Mycobacterium tuberculosis Preserves Significantly Diverse Protein Profile

Abstract

For adaptation to stressful conditions, Mycobacterium tuberculosis (Mtb) is prone to transit to a dormant, non-replicative state, which is believed to be the basis of the latent form of tuberculosis infection. Dormant bacteria persist in the host for a long period without multiplication, cannot be detected from biological samples by microbiological methods, however, their "non-culturable" state is reversible. Mechanisms supporting very long capacity of mycobacteria for resuscitation and further multiplication after prolonged survival in a dormant phase remain unclear. Using methods of 2D electrophoresis and MALDI-TOF analysis, in this study we characterized changes in the proteomic profile of Mtb stored for more than a year as dormant, non-replicating cells with a negligible metabolic activity, full resistance to antibiotics, and altered morphology (ovoid forms). Despite some protein degradation, the proteome of 1-year-old dormant mycobacteria retained numerous intact proteins. Their protein profile differed profoundly from that of metabolically active cells, but was similar to the proteome of the 4-month-old dormant bacteria. Such protein stability is likely to be due to the presence of a significant number of enzymes involved in the protection from oxidative stress (katG/Rv1908, sodA/Rv3846, sodC/Rv0432, bpoC/Rv0554), as well as chaperones (dnaJ1/Rv0352, htpG/Rv2299, groEL2/Rv0440, dnaK/Rv0350, groES/Rv3418, groEL1/Rv3417, HtpG/Rv2299c, hspX/Rv2031), and DNA-stabilizing proteins. In addition, dormant cells proteome contains enzymes involved in specific metabolic pathways (glycolytic reactions, shortened TCA cycle, degradative processes) potentially providing a low-level metabolism, or these proteins could be "frozen" for usage in the reactivation process before biosynthetic processes start. The observed stability of proteins in a dormant state could be a basis for the long-term preservation of Mtb cell vitality and hence for latent tuberculosis.

Prolonged infection triggered by dormant Mycobacterium tuberculosis: Immune and inflammatory responses in lungs of genetically susceptible and resistant mice

“…This resolves a complicated problem of transportation of genetically manipulated virulent strains between research teams. In addition, we recently characterized the proteome of in vitro attenuated M. tuberculosis, which provides the grounds for further dissection of physiological changes that occur during mycobacterial acidification [27].…”

“…In addition, we recently characterized the proteome of in vitro attenuated M . tuberculosis , which provides the grounds for further dissection of physiological changes that occur during mycobacterial acidification [ 27 ].…”

Immunoreactive Proteins of Dormant Mycobacterium tuberculosis Cells

“…Cytosolic and membrane fraction proteins were extracted from M. tuberculosis cells obtained during acidification of culture medium, as well as from active multiplying Mtb cells. The protein profiles were obtained via 2D electrophoresis [4]. Despite the long-term storage of dormant cells without division, the proteome of such cells retained a large quantity of proteins, but individual proteins differed from the proteome of active cells.…”

“…Apparently, the interaction between the pathogen and the host, in particular, the interaction with the host immune system, plays a significant role in this process [2]. Therefore, it is relevant to reveal a repertoire of immunogenic proteins in dormant forms (DFs) of Mtb, which differ considerably by their biochemical composition from those detected in active multiplying bacteria [3,4].…”

Corynebacterium jeikeium Dormant Cell Formation and Photodynamic Inactivation

“…In this study, we ascribe conditions needed for corynebacterial cells to transition to a state of dormancy, which was accompanied by: (1) the formation of stress-resistant forms intended for longterm survival that have been identified in many spore-forming and non-spore-forming bacteria (Zhang, 2004;Mulyukin et al, 2010Mulyukin et al, , 2014Lennon and Jones, 2011) and (2) the acquisition of an NC state during the prolonged incubation of post-stationary cultures, which was previously described for Micrococcus luteus , Rhodococcus rhodochrous (Shleeva et al, 2002) and mycobacteria (M. tuberculosis and M. smegmatis) (Shleeva et al, 2003(Shleeva et al, , 2004(Shleeva et al, , 2011(Shleeva et al, , 2015Kudykina et al, 2011). The dormant forms of mycobacteria, which form when the external medium undergoes gradual acidification, can be distinguished from active bacteria based on their distinct proteomic (Trutneva et al, 2018(Trutneva et al, , 2020 and metabolomic (Nikitushkin et al, 2020) profiles.…”