Project Summary Leishmaniasis is a neglected tropical disease that occurs worldwide with ineffective anti-parasite drugs and no vaccine. The disease severity is often due to an exaggerated immune response rather than uncontrolled parasite replication. Our goal is to define the mechanisms promoting immunopathology in the disease in order to develop new host-directed therapies. Our new data shows that the skin microbiome contributes to the development of more severe disease in patients and experimental models. Qualitative and quantitative changes in the bacteria present in leishmanial lesions, and high levels of Staphylococcus aureus, were associated with delayed healing. Similarly, bacterial colonization of mice infected with Leishmania leads to more severe disease without altering the parasite burden. Thus, our results indicate that disease outcome is profoundly affected by the host response and the skin microbiome. We cultured S. aureus isolates from patients and we will test the hypothesis that S. aureus promotes increased disease in a strain-specific manner. Building on preliminary data that strain-level variation in S. aureus underlies differential immunopathology in cutaneous leishmaniasis, we will use a comparative genomics approach combined with host-directed phenotyping to identify S. aureus virulence factors that promote disease. We also found that L. braziliensis patients had variable numbers of regulatory T cells (Tregs) in their lesions and that low numbers of Tregs were associated with delayed healing. We recapitulated those findings in a mouse model, showing that mice with low numbers of Tregs colonized with S. aureus and infected with L. braziliensis develop severe disease with high levels of IFN-γ and S. aureus. We will use our murine models and in vitro skin organoids to define the mechanisms leading to severe immunopathologic responses. The studies defining qualitative differences in S. aureus and the immunopathologic responses associated with S. aureus colonization will be used to identify potential targets for microbial- and host-directed therapies. Pentavalent antimony is the standard of care for CL in Brazil, but it is often ineffective. Our findings strongly support the concomitant use of host-directed therapies to improve outcomes. However, to ensure that such therapies do not lead to an increase in S. aureus burden, we aim to develop therapies that limit host immunopathologic responses while limiting S. aureus burden and virulence. To accomplish this, we will test a combination of host-directed therapies with a consortium of skin commensal bacteria that directly inhibit S. aureus, and in an alternative approach, test a pan-caspase inhibitor that blocks cell death but also controls S. aureus. Together, these studies will uncover how S. aureus worsens disease caused by Leishmania, information vital for developing new leishmaniasis treatments, and will also define how S. aureus, a major cause of skin and soft tissue infectio...