Proteomic insights into a M. tuberculosis clinical isolate with an increased propensity to form viable but non-replicating subpopulations during acid stress

Phagosome acidification is one of the challenges faced by Mycobacterium tuberculosis during infection. This intracellular pathogen is known to adapt to its stressful environment through stress response pathways and by secreting proteins to modify the host immune response for survival and proliferation. However, M. tuberculosis also holds the potential to form viable but non-replicating (VBNR) and antibiotic tolerant persisters in response to environmental stress, including acid stress. In this study we used an in vitro acid stress model to stimulate the formation of a VBNR subpopulation in a M. tuberculosis clinical isolate with an increased propensity to form VBNR bacteria. Mass spectrometry-based proteomics was used to characterize the cellular proteome and culture filtrate proteome of actively replicating (pH 6.5) and acid stressed (pH 4.5) cultures. We show that in response to acid stress, M. tuberculosis S169 increases the expression of known stress response proteins, including the methyltransferase Rv1405c and the acid stress response two-component regulatory protein TcrX. Interestingly, we found that the dormancy response regulon components were less abundant in acid stressed M. tuberculosis S169. Our protein aggregation capture culture filtrate proteomic approach revealed that the culture filtrates of low pH stressed M. tuberculosis S169 contained less proteins than that of actively replicating cultures. In response to acid stress, several lipoproteins and proteases were significantly more abundant in the culture filtrates of M. tuberculosis S169. We identified several proteins previously implicated in M. tuberculosis persistence, including several toxin-antitoxin proteins and the chorismate mutase (Rv1885c). The observed differences identified in the characterisation of this clinical isolate in comparison to published M. tuberculosis H37Rv highlights the need to investigate M. tuberculosis clinical isolates for a more representative understanding of the tuberculosis stress response.