Combining host and pathogen-directed approaches to kill Mtb by exploiting its dependence on cholesterol metabolism

About This Grant

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is a leading cause of death from infection. Curative drug regimens are long, plagued by resistance and often toxic. In addition to using antimycobacterial drugs, adjunctive host directed therapy (HDT) aims to modulate host immune and inflammatory responses to reduce tissue damage, treatment duration and relapse rate. Here we will explore a unique way to combine pathogen-directed and host-directed therapy by exploiting the contrasting ways that Mtb and macrophages, the predominant niche for Mtb, handle cholesterol, Mtb's preferred carbon source. Mtb-infected macrophages form droplets filled with cholesterol esters and triacylglycerides that sustain the bacterium and induce an antibiotic-tolerant state. However, Mtb's degradation of cholesterol generates a toxic intermediate, propionyl-CoA. Mtb avoids propionyl-CoA accumulation by assimilating propionyl-CoA into virulence lipids (PDIMs and sulfolipids). Inhibition of the essential Mtb enzyme phosphopantetheinyl transferase (PptT) blocks synthesis of PDIMS and sulfolipids by inhibiting charging of the acyl carrier proteins required for their synthesis. We found that anti-mycobacterial agents that inhibit PptT kill Mtb in association with propionyl-CoA accumulation and CoA depletion. Moreover, we found that propionyl-CoA directly inhibits Mtb CoaBC in vitro, an essential enzyme for CoA biosynthesis. This likely explains why propionyl-CoA accumulation is toxic. Here, we will harness the PptT–PDIM–propionyl-CoA– CoaBC axis to kill intracellular Mtb by the cumulative effects of inhibition of Mtb's PptT and the host macrophage's sterol-O-acyltransferase (SOAT), an enzyme crucial for conversion of free cholesterol to its esters. Inhibition of SOAT will lead to accumulation of free cholesterol rather than its storage as esters. Mtb's metabolism of free cholesterol will exacerbate killing of Mtb by PptT inhibitors and the consequent prevention of propionyl- CoA incorporation into lipids, leading to the blockade of CoA synthesis. Inhibition of both SOAT isoforms (1 and 2) can prevent lipid droplet formation, and chemical inhibition of SOAT and its knockdown by RNA interference have been shown to restrict intracellular bacterial growth. However, chemical inhibition of SOATs 1 + 2 has resulted in toxicity due to lack of isoform specificity. Herein, we will use a new series of SOAT1 inhibitors that are selective for the SOAT1 isoform, which predominates in macrophages. We will test the efficacy of SOAT1 inhibition on intracellular growth of Mtb in conditions of lipid droplet induction that lead to different ratios of cholesterol esters and triacylglycerides. We will treat macrophages with SOAT1 inhibitor and lipid droplet inducer, infect them with Mtb, treat with our highly potent and selective PptT inhibitors and monitor killing of Mtb.