Abstract
Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.
Highlights
From the ‡Mycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695014, India; §Mass Spectrometry and Proteomic Core Facility, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695014, India
In accordance with earlier reports we found that nonreplicating persistent stage 1 (NRP1) was attained at 192 h (8th day) and the nonreplicating persistent stage 2 (NRP2) or enduring hypoxia was attained at 336 h (14th day) [9, 34]
nonreplicating persistence stage 2 (NRP2) mimics the in vivo situation where Mycobacterium tuberculosis (MTB) attains dormancy inside the macrophages [5]
Summary
From the ‡Mycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695014, India; §Mass Spectrometry and Proteomic Core Facility, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695014, India. Proteomics of Dormant and Reactivated M. tuberculosis conditions MTB is found to undergo drastic changes in its energy and metabolic status [10, 11]. Wayne’s dormancy model has proven to be a very effective and simple method to understand the molecular mechanisms in dormant bacteria, and to discover novel therapeutic agents [8]. They identified 122 ATP-binding proteins of which roughly 60% were reported to be essential for the in vitro survival [14]. Extracellular proteins of nutrient-starved MTB were analyzed by Albrethsen et al They identified 1176 proteins, of which 230 were up-regulated, and 208 were downregulated [25]
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