The Phosphate-Specific Transport System Gene pstA1 Contributes to Rifampin Tolerance in Mycobacterium tuberculosis

Tuberculosis (TB) caused an estimated 10.7 million new cases and 1.23 million deaths in 2024. Antibiotic tolerance, the ability of bacteria to survive bactericidal antibiotics without genetic resistance mutations, contributes to prolonged treatment. Targeting tolerance mechanisms could promote accelerated clearance of Mycobacterium tuberculosis (Mtb). Our previous genetic screen identified pstA1, involved in phosphate-specific import.

PstA1 has previously been implicated in Mtb virulence, as mutants lacking this gene exhibit defective survival during phosphate limitation in vitro, in macrophages, and in immunocompetent mice. We evaluated rifampin susceptibility of a pstA1 deletion mutant (ΔpstA1) using minimum inhibitory concentration (MIC) and time-kill assays, assessed survival during phosphate limitation and in macrophages, and performed differential expression analysis.

Rifampin MIC was not altered in ΔpstA1, suggesting that the difference in susceptibility was not due to antibiotic resistance. Time-kill assays revealed a shift in the mean duration of killing (2-log reduction) from 1.6 days in wild-type to 1.0 day in ΔpstA1, and complementation partially restored this phenotype. pstA1 was specifically required for Mtb survival in the absence of exogenous inorganic phosphate and was important for growth adaptation in culture without detergent and within macrophages in an interferon-γ-dependent manner. ΔpstA1 exhibited distinct transcriptional reprogramming with 58 differentially expressed genes, including altered metabolic, DNA damage repair, and secretory pathways.

These findings identify PstA1 as a contributor to rifampin tolerance and suggest that phosphate transport pathways influence antibiotic susceptibility in Mtb.