Polyphosphate and Transcriptional Changes During Persister Formation in Mycobacterium tuberculosis

Objectives: Extrapulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a major public health issue impacting at least 3 people in every 100,000 in the U.S., due in part to HIV positive patients where there is synergistic susceptibility to infection. TB requires six-to-nine months of combination anti-tubercular drug therapy to cure the disease. One possible reason for the long TB treatment is due to Mtb persisters, phenotypically antibiotic-tolerant cells that remain genetically identical to antibiotic-sensitive cells in the population. Starvation is known to induce the Mtb persister state, increasing persister frequency by 10-fold more than exponentially growing cultures. Starved cells synthesize polyphosphate (polyP), a polymer of inorganic P linked by high-energy phosphoanhydride bonds. PolyP is important for “reprogramming” the cell for starvation conditions. We hypothesize that changes in the amount of polyP storage in the cell determines Mtb persister frequency.
Methods: To test this, we performed growth curve experiments to measure polyP storage and changes in expression of genes associated with polyP synthesis and degradation. Wild-type Mtb cultures increased polyP storage in the cell over time that corresponded with changes in polyP kinase and exophosphatase gene expression. Our lab has also determined that a phosphate-specific transport mutant, ΔpstA1, stores more polyP during exponential growth than wild-type. Furthermore, we performed growth curve experiments to monitor polyP storage and polyP kinase/phosphatase gene expression to determine the cause for the increased intracellular polyP.
Results: The ΔpstA1 mutant had increased polyP kinase 1 and decreased exopolyphosphatase 2 gene expression compared to wild-type, suggesting that the increased storage of polyP is modulated by both polyP kinase and exopolyphosphatase production.
Conclusions: Deletion of exopolyphosphatase 2 in the wild-type and ΔpstA1 mutant backgrounds induced in a growth defect for the Δppx2 mutant but not the ΔpstA1Δppx2 mutant. Future work will identify how changes in polyP storage affect Mtb persister formation.