Leishmania amazonensis is one of the main causes of cutaneous leishmaniasis in South and Central America. The burden of this pathogen on human health contrasts with the lack of fundamental knowledge about its underlying virulence and survival mechanisms. In particular, L. amazonensis is a growing public health problem because infection with this parasite often creates diffuse non-healing lesions, and more rarely, lethal visceral disease, with increasing levels of resistance to standard therapies. This broad range of outcomes indicates that these parasites are extremely flexible and adapt to different host immune responses. L. amazonensis, and other species of the L. mexicana complex, induce the formation of specialized replication niche within infected macrophages. This parasitophorous vacuole (PV) is enlarged, occupying most of intracellular space of an infected macrophage; this unique feature is essential for parasite survival and replication. However, the mechanisms involved in biogenesis of this enlarged PV are largely unknown. The long-term objective of this project is to elucidate the cellular mechanisms that drive the biogenesis of the giant PV and allow the survival and replication of amastigotes within macrophages. Our hypothesis is that amastigotes combine multiple virulence mechanisms targeting host SUMOylation, ATP6V0D2 and the receptors CD36 and CSF1R to promote PV biogenesis, subvert host defense, and orchestrate the acquisition of essential metabolites, such as lipids. Note that all Leishmania species create PVs as safe replicative niches, therefore strategies used to L. amazonensis to generate its PV could be similarly used by different Leishmania species regardless of PV size. The rationale underlying this proposal is that a systematic study of the multiple mechanisms controlling PV biogenesis will provide a better understand how L. amazonensis successfully creates its replicative niche inside generally inhospitable macrophages. This proposal is significant because the resulting deeper mechanistic understanding of the parasite-host interaction will lead to the identification of new methods to strike at this critical, but so far indestructible, replicative niche from where amastigotes trigger the outward symptoms of leishmaniasis. The proposal is innovative because will investigate 3 novel mechanisms, identified in our earlier studies, involved in the biogenesis of the L. amazonensis amastigote enlarged PV. Aim 1 will explore the role of CD36 mediated lipid transport in delivering essential lipids to or from the PV. In Aim 2, we will study how amastigote-triggered deSUMOylation induces ATP6VOD2 expression, favoring parasite physiological demands such as increased cholesterol availability. Aim 3 will focus on the study of CSF1R, which we hypothesize is hijacked by amastigotes to support the high demand for sterols by intracellular parasites and/or to control the expression of Thrombospondin1.