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 a 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 VBNR enriched (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. We identified several proteins previously implicated in M. tuberculosis persistence, including toxin-antitoxin proteins (VapC51 and VapB10), the chorismate mutase (Rv1885c), and several uncharacterized proteins. 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. Author SummaryTuberculosis is caused by Mycobacterium tuberculosis and this pathogen can form a subpopulation of viable but non-replicating (VBNR) cells that are recalcitrant to antibiotic treatment. These persister bacteria increases the risk of treatment failure and tuberculosis recurrence following treatment. Stimulation of a persister population through triggered persister formation can be achieved by environmental stress factors such as low pH, nutrient starvation, hypoxia, and antibiotic exposure. In this study we investigate the cellular and culture filtrate proteomes of a high persister forming clinical isolate, M. tuberculosis S169, in response to acid stress. We show that following the stimulation of a VBNR subpopulation in response to acid stress, several known acid stress response proteins are more abundant in VBNR enriched cultures. Interestingly, we found that stress response proteins were less abundant. Using a protein aggregation capture approach we successfully characterized culture filtrates M. tuberculosis cultures, reducing the bacterial culture amount required for these experiments. Culture filtrates differed between actively replicating and VBNR enriched cultures. Several immunogenic proteins were identified in a higher abundance in the culture filtrates of VBNR enriched cultures.