Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in

ABSTRACT Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inor-ganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here thatM. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identifyM. tuberculosis Rv1026 as a novel exopoly-phosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found thatM. tuberculosis poly(P) accumulation leads to slowed growth and reduced suscepti-bility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electronmicroscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation rel-ative to the empty vector control. Transcriptomic andmetabolomic analysis revealed a metabolic downshift of the ppx2 knock-down characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role inM. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tol-erance through altered cell wall permeability. IMPORTANCE The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, i