pH-Dependent Switching of ESX-1 Substrates in Mycobacterial Pathogenesis - PROJECT SUMMARY Pathogenic mycobacteria, including Mycobacterium tuberculosis, cause acute and chronic infections that significantly im- pact human health. Mycobacterium marinum is a non-tubercular mycobacterial pathogen and a well-established model for studying several aspects of M. tuberculosis pathogenesis, including ESX-1 secretion. Early during infection, pathogenic mycobacteria reside in and adapt to the phagosome. The pH of mycobacterial phagosomes differs based on the immunolog- ical activation state of macrophages. Although mycobacteria escape the phagosome using the ESX-1 secretion system, the mechanisms used by ESX-1 to lyse phagosomes under conditions of varied pH is a major gap in our fundamental under- standing of mycobacterial infections. The applicant’s preliminary data suggest that the ESX-1 system switches which sub- strates are secreted in response to acidic pH. The long-term goal is to understand the molecular mechanisms of mycobacte- rial pathogenesis. The objective of this proposal is to determine how ESX-1 secretion promotes lysis in response to varying pH in vitro and during infection. The central hypothesis is that pathogenic mycobacteria switch the substrates secreted by ESX-1 in response to acidic pH to lyse phagosomes in macrophages of different immunological activation states. The ra- tionale for this project is that defining how mycobacteria respond to different host cell physiologies may offer critical insight important for considering treatment and prevention of mycobacterial diseases. The central hypothesis will be tested by following these specific aims: 1) Define the genetic requirements for hemolysis at acidic pH. Under the first aim, targeted and unbiased molecular genetic approaches combined with qRT-PCR, IP followed by proteomics and ESX-1 functional assays will be used to identify the genes and mechanisms underlying lytic activity at acidic pH. 2) Measure changes in ESX- 1 secretion in response to acidic pH. The second aim will use proteomics to measure changes to the secreted proteomes of M. marinum and M. tuberculosis at acidic pH. 3) Investigate substrate switching during mycobacterial infection. The third aim will combine molecular genetics, chemical inhibitors of acidification and immune-activated infection models, and ani- mal models to determine if specific substrates are conditionally transcribed and required during M. marinum and M. tuber- culosis infection. The research proposed research is conceptually innovative because it tests the idea that ESX-1 switches substrates to lyse phagosomes associated with heterogeneous macrophage physiologies. The formation of multiple ESX-1 assemblies in response to environmental cues is conceptually innovative. There is technical innovation in the chemical inhibition of ESX-1 lytic activity, through leveraging a unique and comprehensive M. marinum strain collection for use with established proteomics work flows, and in the use of differentially activated cellular and animal models of infection. The successful completion of this proposal will be significant because it will identify seminal ESX-dependent virulence mech- anisms and provide new insight into mycobacterial infection biology by generating understanding of how mycobacteria respond to heterogeneous host cells during infection.
