Unravelling of protein trafficking in Trypanosoma cruzi, the agent of Chagas disease. - Chagas disease is a parasitic neglected disease, with around 6‐7 million already infected, around 40,000 new cases per year and an at least 100 million people at risk. In Argentina an estimated 1,600,000 Chagas disease patients exist, providing unique access to parasite strains, vectors and the research infrastructure required to investigate the biology of this pathogen. The protozoan causative agent, Trypanosoma cruzi, is naturally transmitted by triatomine insects ( kissing bugs ), which are widely distributed from the southern United States to southern latitudes of South America. In the United States, an esmated 300,000 people are infected and the “endemic country” status is proposed due to “a body of evidence establishing the robust presence of T. cruzi parasites in the United States, not only among insect vectors, wildlife, and domesc animals but also among humans without travel histories who are assumed to be locally infected” (wwwnc.cdc.gov/eid/article/31/9/24‐1700_article). Understanding the molecular mechanisms underlying T. cruzi infection is thus directly relevant to U.S. public health, where increasing numbers of patients require improved, safer and more effective treatments. Due to migration from endemic regions, Chagas disease also has become a global health concern and additional cases continue to emerge in non‐endemic countries, including Australia, Japan and Spain. In this proposal we face two main challenges associated with the glycobiology of the parasite and its relevance to cell biology and in the interaction with the mammalian and insect hosts. The final purpose is not only to gain knowledge about biology of the parasite but mainly to determine targets for the development for urgently needed new chemotherapies since the available ones are from the ‘70s and highly toxic. In T. cruzi, relatively few virulence factors have been extensively characterized. Over the past 25 years, our laboratory has been at the forefront of investigating T. cruzi virulence factors, making major contributions to the characterization of two key virulence factors: trans‐sialidase (TS) and mucins. Our studies have revealed their key roles in immune modulation, membrane distribution (by using labeled sugars along with click chemistry we developed and a well‐established super‐resolution facility) and in adhesion and cell invasion. Our recent studies demonstrated that, in T. cruzi, the contractile vacuole complex (CVC) is involved in protein trafficking to the parasite surface of these two virulence factors. Here we propose to fully understand protein trafficking through CVC, in particular that mediated by Rab proteins. Because the CVC is an organelle absent in mammals, it could provide with an interesting target for chemotherapies. Unlike mammalian cells, trypanosomes exhibit a significant proportion of GPI‐anchored proteins on their surface. Its precursors are synthesized in the endoplasmic reticulum by successive transfer of monosaccharides to a phosphatidylinositol molecule. Also, our recent studies have revealed that TS and mucins, which are GPI‐anchored proteins, exhibit a distinct and ordered domain distribution on the parasite surface, forming patch regions. A possible explanation for this peculiar organization may be that the proteins have glycosylphosphatidylinositol (GPI) anchors with different lipids: mucins have alkylacylglycerol, while TS has ceramide. The role of ceramide in GPI anchoring remains poorly understood. The partitioning of proteins into distinct domains may have significant implications for parasite cell biology, infectivity, and pathogenicity. The enzyme responsible of transfer the ceramide is the ceramide remodelase. We identified the T. cruzi gene orthologue of the encoding gene in yeast. This gene was able to complement the corresponding Saccharomyces cerevisiae mutant. Based on these significant findings, we propose to investigate ceramide acquisition at the GPI-anchoring in T. cruzi, its relevan
