Elucidating the Role of Extracellular Polyphosphate in Mycobacterium tuberculosis Antibiotic Tolerance. - Project Summary Infection with the intracellular pathogen Mycobacterium tuberculosis (Mtb) causes one of the most devastating infectious diseases globally: tuberculosis (TB). Mtb exhibits a remarkable capacity to persist within the host for extended periods by adapting to the hostile intracellular environment and evading immune responses. Furthermore, the rise of multidrug-resistant Mtb strains has further complicated treatment efforts. A key factor in the development of drug resistance is Mtb's ability to tolerate a range of stresses. This tolerance is vital for its survival under conditions of stress, whether from host cell defenses or antibiotic treatment. The bacterium's metabolic flexibility enables it to reprogram its metabolic pathways in response to stresses, enhancing its resilience. Polyphosphate (polyP), a polymer of phosphate, is essential for Mtb's response to stresses, helping to adjust its metabolism for enhanced survival in host cells. This adaptation is linked to the bacterium's virulence, persistence, latency, and antibiotic tolerance. While much research has focused on the roles of intracellular polyP in these processes, the specific contributions of cell surface and extracellular polyphosphate (hereafter referred to as extracellular polyP) remain poorly understood. My research shows that Mtb increases the accumulation of extracellular polyP, in addition to intracellular polyP, in response to isoniazid (INH), a first-line TB antibiotic. Gallein, a small molecule inhibitor of bacterial polyphosphate kinases (PPKs), the enzymes that synthesize polyP, prevents the accumulation of both intracellular and extracellular polyP, reduces cell envelope thickening, decreases key metabolites involved in envelope biosynthesis, and increases Mtb's susceptibility to INH. Here, we propose to investigate the metabolic pathways in Mtb that regulate extracellular polyP during antibiotic tolerance, which will us to explore the potential of targeting the extracellular polyP pathway as a novel therapeutic strategy for treating drug-tolerant Mtb. In Aim 1, we will focus on selectively knocking down genes that encode polyP-regulating enzymes and those encoding metabolites altered by the INH and gallein. We will examine the impact of reduced expression of these genes on Mtb's utilization of extracellular polyP, cell envelope formation, metabolism, and growth in both in vitro culture and human macrophages in the presence of antibiotics. From this aim, we will identify additional components of metabolic pathways altered by reduced expression of genes that encode polyP-regulating enzymes and those encoding metabolites altered by the antibiotic INH and gallein, and how that impact utilization of extracellular polyP during antibiotic tolerance. In addition, this will allow us to exploit these metabolic vulnerabilities for therapeutic targeting. In Aim 2, we will investigate whether the utilization of extracellular polyP to protect Mtb during antibiotic-induced stress is a conserved mechanism across different antibiotics or specific to certain ones. We will treat Mtb strains generated in Aim 1 with gallein, either alone or in combination with other frontline TB antibiotics rifampin, ethambutol, and pyrazinamide, and examine extracellular polyP levels, cell envelope integrity, metabolism, and bacterial growth in both in vitro culture and macrophages as in Aim 1. This aim will clarify whether disrupting extracellular polyP pathways enhances the efficacy of all or some TB antibiotics and identify potential drug combinations that could overcome Mtb's tolerance mechanisms.
