ABSTRACT The goal of this project is to study the role and mechanisms by which host sphingolipids are involved in controlling the infection caused by the pathogenic fungus Cryptococcus neoformans (Cn). A rapidly emerging area of research is the study of the role of sphingolipids in the regulation of infectious diseases (Reviewed in18, 22). Most of these studies have focused on the role of microbial sphingolipids in the ability of the microbe to cause infection. Very few studies addressed if and how host sphingolipids are also involved in the regulation of microbial pathogenesis, and most of these studies have focused on bacterial or parasitic infections (reviewed in18, 22). Although some sphingolipids have been linked to antibacterial activity of phagocytic cells16, 42, 43, 59, nothing is known about the role of host sphingolipids against fungal infections. Since phagocytic cells, such as macrophages and neutrophils, are the first line of defense against Cn infection, the regulation of their cellular processes may affect their response to the fungus, and thus, determine whether they can or cannot control the development of cryptococcosis. One of the host sphingolipid-metabolizing enzymes shown to regulate immune responses is sphingomyelin synthase (SMS) encoded by two genes, SMS1 and SMS217, 30, 36, 39. SMS transfers a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, therefore producing sphingomyelin (SM) and diacylglycerol (DAG)28, 62, 65. Very interestingly, the lipids regulated by SMS have been implicated in the activation of pro-inflammatory responses, suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections. In our preliminary studies we found that: 1) inhibition of SMS activity profoundly impairs the ability of neutrophils to kill Cn cells by affecting extracellular killing in absence of phagocytosis; 2) SMS regulates production of DAG at the Golgi; 3) DAG at the Golgi regulates protein secretion through a protein kinase D (PKD)-mediated mechanism; 4) inhibition of PKD blocks extracellular killing of Cn; and 5) inhibition of either SMS or PKD activity significantly decreases secretion of antimicrobial peptite(s) such as α-defensin. Based on these observations, we hypothesize that SMS activity plays a key role in controlling Cn infection through a DAG-PKD-antimicrobial peptide(s) secretion pathway. Thus, we propose the following aims: 1) To determine the role of SMS activity during Cn infection; and 2) To define the mechanism by which SMS activity regulates the extracellular killing of Cn. By studying how host sphingolipids regulate the extracellular killing activity of neutrophils against Cn, we will provide new insights not only for a better understanding of fungal pathogenesis but also for the development of new therapeutic strategies against this and potentially other fungal microbes. The studies that we propose in this application will reveal new regulatory mechanisms involved in the killing of Cn by neutrophils. We will identify the nexus between SMS, PKD and infection.

Fungal infections have dramatically increased during the last decade and new treatment strategies are needed. This proposal focuses on a better understanding of the pathogenic processes leading to the killing of fungal microbes by the host immune cells, providing new insights into the development of better therapeutic strategies.