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bioRxiv2025

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

Tuberculosis (TB) caused an estimated 10.8 million new cases and 1.25 million deaths in 2023. Antibiotic tolerance, the ability of bacteria to survive exposure to bactericidal antibiotics without genetic resistance mutations, contributes to the need for prolonged treatment. Targeting antibiotic tolerance mechanisms could promote accelerated clearance of Mycobacterium tuberculosis (Mtb), thereby improving medical adherence. Our previous forward genetic screen for rifampin tolerance genes identified pstA1, which is involved in phosphate-specific import. The ABC-type transporter permease PstA1 has previously been implicated in Mtb virulence, as mutants lacking this gene exhibit defective survival during phosphate limitation in vitro, in infected macrophages, and in immunocompetent mice. The minimum inhibitory concentration (MIC) of rifampin was not altered in a pstA1 deletion mutant ({Delta}pstA1), suggesting this difference in susceptibility is not due to antibiotic resistance. Consistent with a role in rifampin tolerance, 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 {Delta}pstA1, and complementation partially restored this phenotype. We found that pstA1 is specifically required for Mtb survival in the absence of exogenous inorganic phosphate and is important for adaptation to growth in culture without detergent, and within macrophages in an interferon-{gamma}-dependent manner. Differential expression analysis revealed that {Delta}pstA1 exhibited substantial transcriptional reprogramming with 58 differentially expressed genes, including altered expression of metabolic, DNA damage repair, and secretory pathways. PstA1 represents a novel drug target, and inhibitors could serve as adjunctive therapies to shorten treatment times, reducing opportunities for drug resistance emergence. ImportanceTuberculosis remains one of the worlds deadliest infectious diseases, causing over a million deaths annually. Current treatment requires months of antibiotic therapy, and poor adherence to these lengthy regimens contributes to the emergence of drug-resistant strains that are increasingly difficult to treat. A major barrier to shorter treatment is antibiotic tolerance, which allows bacteria to survive drug exposure without genetic resistance mutations. This study identifies the phosphate transport component PstA1 as a critical factor enabling Mycobacterium tuberculosis to tolerate the frontline antibiotic rifampin. Bacteria lacking pstA1 are eliminated more rapidly by rifampin, demonstrating that this transporter actively promotes bacterial survival during treatment. These findings suggest that drugs targeting PstA1 could be combined with standard antibiotics to accelerate bacterial clearance, potentially shortening treatment duration and improving patient adherence. Such adjunctive therapies targeting tolerance mechanisms represent a promising strategy to combat tuberculosis and reduce the global burden of drug-resistant disease.

microbiology↗

bioRxiv2024

Mycobacterium dormancy and antibiotic tolerance within the retinal pigment epithelium of ocular tuberculosis

Tuberculosis (TB) is a leading cause of death among infectious diseases worldwide due to latent TB infection, which is the critical step for the successful pathogenic cycle. In this stage, Mycobacterium tuberculosis resides inside the host in a dormant and antibiotic-tolerant state. Latent TB infection can lead to a multisystemic diseases because M. tuberculosis invades virtually all organs, including ocular tissues. Ocular tuberculosis (OTB) occurs when the dormant bacilli within ocular tissues reactivate, originally seeded by hematogenous spread from pulmonary TB. Timely and accurate diagnosis as well as efficient chemotherapies are crucial in preventing poor visual outcomes of OTB patients. Histological evidence suggests that retinal pigment epithelium (RPE) cells play a central role in immune privilege and in the protection from the antibiotic effects, making them an anatomical niche for invading M. tuberculosis. RPE cells exhibit high tolerance to environmental redox stresses, allowing phagocytosed M. tuberculosis bacilli to maintain viability in a dormant state. However, the microbiological and metabolic mechanisms determining the interaction between the RPE intracellular environment and phagocytosed M. tuberculosis are largely unknown. Here, liquid chromatography mass spectrometry (LC-MS) metabolomics was used to illuminate the metabolic state within RPE cells reprogrammed to harbor dormant M. tuberculosis bacilli and enhance the antibiotic tolerance. The results have led to propose a novel therapeutic option to synthetically kill the dormant M. tuberculosis inside the RPE cells by modulating the phenotypic state of M. tuberculosis, thus laying the foundation for a new, innovative regimen for treating OTB. ImportanceUnderstanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with M. tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure OTB. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug tolerant state, thereby blunting the efficacy of anti-TB chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that intracellular environment within RPE cells is enriched with greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

microbiology↗