Thursday, April 25, 2024
4/25/2024
SUMMARY The goal of this project is to understand how two virulence factors from Mycobacterium tuberculosis (Mtb), CpsA and phthiocerol dimycocerosate (PDIM), impair immunity by undermining both classical and non-classical autophagy. Mtb is the causative agent of tuberculosis (TB), the leading cause of death worldwide from a bacterial infection. The main cellular niche for Mtb is macrophages and neutrophils, the very immune cells that are meant to clear infection. How Mtb survives the innate immune response to establish infection is not well understood. In the previous project period, we discovered that an exported protein, CpsA, is critically important for Mtb virulence. We showed that CpsA inhibits phagosomal recruitment of the NADPH oxidase. The NADPH oxidase makes reactive oxygen species (ROS), an important mediator of the innate immune response. In addition to its direct antimicrobial activity, ROS is required for a lysosomal trafficking pathway called LC3-associated phagocytosis (LAP), a non-classical form of autophagy. Thus, by inhibiting ROS, CpsA also inhibits LAP. We showed both in macrophages and mice that CpsA protects Mtb from the NADPH oxidase and LAP. Interestingly, CpsA physically interacts NDP52 and TAX1BP1, autophagy adaptors that function in a form of classical autophagy (xenophagy), suggesting that CpsA may also impair xenophagy. Moreover, we found that the Mtb virulence lipid, PDIM, also inhibits the NADPH oxidase. Previous studies proposed an array of roles for PDIM and showed that it protects Mtb from a poorly defined innate killing mechanism. Our data suggest that an unappreciated virulence property of PDIM is blocking the NADPH oxidase and LAP. Thus, we hypothesize that CpsA inhibits xenophagy and works in concert with PDIM to inhibit the NADPH oxidase and LAP. Further, we propose that the infectious dose of Mtb depends upon its ability to evade these innate defenses in myeloid cells that are recruited to the lungs during initial infection. Here, we will define how CpsA inhibits the NADPH oxidase and investigate whether it also impairs xenophagy by blocking NDP52 and TAX1BP1 function. We will determine whether PDIM also impairs recruitment of the NADPH oxidase to mycobacterial phagosomes and evaluate the contribution that PDIM plays towards subverting the NADPH oxidase and LAP in vivo. To determine whether CpsA promotes the establishment of infection, we will use an ultra-low dose infection model in mice. Using conditional knockout (cKO) mice, we will determine in which cells CpsA functions to inhibit the NADPH oxidase and LAP during acute and chronic infection. Our findings will provide mechanistic insight into how two key virulence factors in Mtb collaborate to undermine immunity. Our studies will reveal a cell type-specific virulence strategy of the bacilli, delineate the cell types that participate in LAP in vivo, and define host-pathogen interactions that govern the establishment of infection. Our studies will provide detailed mechanistic insight into the immune evasion strategies of one of the world's most formidable pathogens. By elucidating the mechanism of action of two crucial virulence factors in Mtb, we will advance innovative approaches to prevent and treat TB.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
