Institution: Pontifical Catholic University of PR

Email: yaliz_loperena@pucpr.edu

The main goal of this research is to better understand the mechanisms that drive the pathogen/host interaction between Cryptococcus neoformans and Cryptococcus gattii complex (C.neo/gat) with macrophages, by establishing a possible crosstalk between these two cells. Cryptococcosis is a fungal infection of the lungs and brain that affects both immunocompromised and immune-competent individuals caused by C. neo/gat. Currently, there is little understanding of the yeast pathogenicity to humans, although its main virulence factor is its capsule. In response of changes in their environment, C. neo/gat has the ability to adapt to dry conditions outside the host, while inside it can develop the capability to produce an enlarge capsule called titan cells, a mechanism that limits the attack of macrophages. Therefore, we are proposing to study how macrophages may be the key players on the development of titan cells in the yeast complex and also how C.neo/gat capsule affects macrophages polarization. It is hypothesized that macrophages secreted products interact with Gpr5 cascade, activating Rim101 and therefore, provoke titanization, which in consequence induces polarization of macrophage to a M2 phenotype. Our preliminary data shows that the macrophage conditioned media induce titanization, in both C. neo/gat species, more dominantly in C. gattii. Also, preliminary data shows that the expression of GPR-5 and cAMP increase between species, indicating and important role of these proteins in the process. In order to achieve this, two specific aims have been developed. In specific aim 1, J774 macrophages will be grown tin starving media for 24 and 48 h and used to monitor the cytokines present via a cytokine profile. This assay will allow for the detection of 40 different cytokines, chemokines and grow factors. For specific aim 2, macrophages will be co-culture with regular, mixed, and enriched titan cells from C.neo/gat. Then, the expression of polarization markers will be performed via flow cytometry. Fluorescent microscopy will be used to determine if the titan cell capsule is interacting with PRP such as TLR-2 on the surface of macrophages via co-localization. The results obtained in this proposal will be the first step of the long goal of this research to better understand the cellular process behind the titan cell development which may explain the pathogenesis of the C. neoformans vs C. gattii. This new improved virulence factor comes as a challenge to the immune system and studying the mechanism involved in titan cell formation can give us insight new treatment strategies for cryptococcosis.