ppe51 variants promote non-replicating Mycobacterium tuberculosis to grow at acidic pH by selectively promoting glycerol uptake

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acidic growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we conducted forward genetic screens that identified several Mtb mutants that could grow under these restrictive conditions. All of the mutants were mapped to the ppe51 gene and resulted in three amino acid substitution - S211R, E215K, and A228D. Expression of the PPE51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, enhanced acid growth (eag) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting ppe51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced in virulence in activated macrophages as compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the WT or S211R variants in the presence of glycerol, supporting the eag substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis.