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.
Highlights
Mycobacterium tuberculosis (Mtb) is a most successful pathogen that may persist in a dormant state in a human body for decades and can reactivate to the active stage of disease after a long period of time (Flynn and Chan, 2001)
We have developed a model of the transition of Mtb cells into the dormant state based on the gradual acidification of the culture medium (Shleeva et al, 2011)
In our study we explore proteomic profiling of Mtb dormant cells after 4 and 13 months of storage using 2D electrophoresis followed by MALDI-TOF analysis to characterize proteins of dormant cells that stored well after such a long period of time
Summary
Mycobacterium tuberculosis (Mtb) is a most successful pathogen that may persist in a dormant state in a human body for decades and can reactivate to the active stage of disease after a long period of time (Flynn and Chan, 2001). Dormancy has been defined as a reversible state of low metabolic activity in which cells could survive for a long time without replication (Young et al, 2005). All known proteomic studies of dormant Mtb cells were performed on “short-term” models, such as the hypoxic Wayne model (formation of non-replicative form due to gradual depletion of oxygen in the growth medium) (Wayne, 1994) and the Loebel model based on starvation of cells in PBS buffer (Loebel et al, 1933), where the time of stress does not exceed 6 weeks. In our study we explore proteomic profiling of Mtb dormant cells after 4 and 13 months of storage using 2D electrophoresis followed by MALDI-TOF analysis to characterize proteins (if any) of dormant cells that stored well after such a long period of time. The 2D electrophoresis method for proteome characterization was used in this study since this method allows the determining of intact proteins in the presence of the products of protein degradation which is highly possible after long storage
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