Abdalla, B. J. | bioRxiv Science

bioRxiv2026

Combined lactate- and phosphate-dependent cytoplasmic acidification drives Mycobacterium tuberculosis growth arrest at acidic pH

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

microbiology↗

bioRxiv2025

Mycobacterium tuberculosis growth arrest on propionate at acidic pH is suppressed by mutations in phoPR and pyrazinamide treatment

Mycobacterium tuberculosis (Mtb) arrests its growth at acidic pH, when grown on specific single carbon sources, including propionate. However, Mtb grows well on propionate at pH 7.0, supporting that propionate can support growth as a sole carbon source. To understand the basis of the propionate-driven growth arrest at acidic pH, we performed a forward genetic selection for mutants that enable growth on propionate at pH 5.7. All the selected mutants had insertions in the two-component regulatory genes phoR or phoP. We hypothesized that growth arrest at acidic pH is caused by PhoPR diverting carbon from central carbon metabolism towards lipid anabolism and that when PhoPR is inactivated, growth is promoted through metabolizing propionate by the methyl citrate cycle (MCC) into pyruvate, a permissive carbon source for growth at acidic pH. Using chemical inhibition and mutants of the MCC pathway, we demonstrate that the enhanced growth is dependent on the MCC. Furthermore, stimulating lipid synthesis via the methylmalonyl-CoA pathway by adding vitamin B12 restricts growth in the{Delta} phoPR mutant and, conversely, restricting lipid anabolism by inhibiting the triacylglycerol (TAG) synthase tgs1 enhances growth of the{Delta} phoPR mutant. Notably, CoA pools increased in the{Delta} phoPR mutant grown on propionate, directly supporting our model. Given the role of CoA metabolism in pyrazinamide sensitivity, we examined Mtb sensitivity to pyrazinamide on propionate at acidic pH and, surprisingly, observed that pyrazinamide treatment of WT Mtb suppresses growth arrest on propionate at acidic pH. In contrast, the phoPR mutant has enhanced sensitivity to pyrazinamide. Together, these findings support that propionate-driven growth arrest at acidic pH is caused by metabolic remodeling that is regulated by PhoPR and is associated with pyrazinamide sensitivity. ImportanceWhen grown on certain single carbon sources, such as propionate, Mtb arrests its growth at acidic pH and establishes a state of non-replicating persistence (NRP). To understand the genetic basis of this growth restriction, a genetic selection was performed to identify mutants unable to arrest growth at acidic pH with propionate as a sole carbon source. The selection exclusively identified mutants in the PhoPR two-component regulatory system, which functions to modulate cell envelope lipids and redox homeostasis through the upregulation of lipid synthesis at acidic pH. Using genetic and chemical inhibition studies, we demonstrate that PhoPR arrests growth at acidic pH by diverting carbon away from the methyl citrate cycle towards lipid anabolism. Surprisingly, treatment of Mtb with pyrazinamide at acidic pH on propionate, also enabled growth. Therefore, this study defines new mechanisms by which Mtb integrates environmental signaling to regulate growth, metabolism, and drug susceptibility. These findings are relevant to pathogenesis, as PhoPR is essential for growth in macrophages and animals, environments with varying pH and carbon source availability, depending on immune pressures. These data suggest that drug susceptibility may be impacted by enhanced growth and metabolic capacity of Mtb in acidic and propionate-rich environments, such as the within the macrophage or the granuloma.

microbiology↗