Accumulation of Polyphosphate Inhibits Biofilm Formation in Mycobacteria by Activating RegX3 to Reduce Intracellular c-di-GMP Levels

Abstract Mycobacterium tuberculosis can persist in the host for prolonged periods. Biofilm formation represents a key strategy that promotes bacterial persistence, enhances antibiotic resistance, and facilitates immune evasion. Thus, targeting biofilm formation holds significant therapeutic potential for tuberculosis. Exopolyphosphatase 1 (PPX1), highly conserved among mycobacteria, hydrolyzes inorganic polyphosphate. Although changes in polyphosphate levels are known to influence mycobacterial biofilm formation, the underlying mechanism remains unclear. In this study, using a Mycobacterium smegmatis model strain, we found that the level of polyphosphate in the Δppx1 mutant significantly increases, concomitant with impaired biofilm formation. Further investigation revealed that elevated polyphosphate enhances the transcriptional activity of the transcription factor RegX3, which in turn represses biofilm formation. Mechanistically, activated RegX3 directly binds to and upregulates the dcpA gene, encoding a bifunctional enzyme that lowers intracellular cyclic di-GMP levels. This reduction ultimately perturbs cell wall lipid synthesis and compromises biofilm formation. In summary, this work reveals a signaling pathway through which polyphosphate governs biofilm formation in M. smegmatis.