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

UNLABELLED: Phagosome acidification is one of the challenges faced byduring 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,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 aclinical 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,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 stressedS169. Our protein aggregation capture culture filtrate proteomic approach revealed that the culture filtrates of low pH stressedS169 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 ofS169. We identified several proteins previously implicated inpersistence, including several toxin-antitoxin proteins and the chorismate mutase (Rv1885c). The observed differences identified in the characterisation of this clinical isolate in comparison to publishedH37Rv highlights the need to investigateclinical isolates for a more representative understanding of the tuberculosis stress response.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-39941-2.