The long-term objective of this project is to elucidate the roles, mechanisms, and functions of inositol-phosphoryl ceramide synthase 1 (Ipc1) in cell regulation and fungal pathogenesis, focusing on Cryptococcus neoformans. Cryptococcus neoformans is an opportunistic pathogen that primarily infects immunocompromised patients. Infection initiates in the lung following inhalation, spreads hematogenously to the brain, and results in meningoencephalitis. It is the most common cause of fungal meningitis worldwide (8, 56). Since the lung is the port of entry for C. neoformans, phagocytosis by alveolar macrophages represents the first host immune response against the fungal organism. If phagocytosis succeeds by killing the organism there will be no disseminated disease. On the other hand, C. neoformans possesses the ability to produce virulence factors, which protect the fungal cells from the phagocytosis and/or the killing by the host alveolar macrophages. Therefore, the determination of how this opportunistic pathogen interacts with alveolar macrophages will lead to the understanding of the molecular mechanism(s) by which C. neoformans disseminates and, thus, produces disease. Ipc1 is a fungus-specific enzyme encoded by the essential IPC1 gene (also called AUR1). It regulates the formation of complex sphingolipids (essential components of the membranes), the levels of phytoceramide and sphingoid bases (implicated in stress responses), and diacylglycerol (a wellestablished mitogen and activator of protein kinase C). Therefore, the regulation of Ipc1 might represent an important factor in the maintenance of crucial cellular functions. In the case of a fungal opportunistic pathogen (such as C. neoformans), the impairment of Ipcl activity could exert a significant impact on host-fungus interactions, either by modification of the production of virulence factors (fungal-factors) or/and by modification of the immune responses (host-factors). In our previous studies, we regulated the expression of the essential IPC1 gene by using the GAL7 promoter. We found that C. neoformans is no longer pathogenic in an animal model of cryptococcal meningitis when Ipc1 is down-regulated (54). In our ongoing studies to understand how Ipc1 regulates Ohyto Ipc1 pathogenicity of C. neoformans, we find that down-regulation of Ipc1 increases phagocytosis whereas up-regulation of lpc1 decreases phagocy:tosis by the host alveolar macrophages. In preliminary epistasis analysis, we identified, through differential display RT-PCR, a novel protein, inhibitory protein 1 (IPI), which exerts antiphagocytic property, and whose level of expression is regulated by lpc1. Based on these observations, we HYPOTHESIZE that Ipc1 regulates the pathogenicity of C. neoformans in part by affecting its phagocytosis through the expression of lPI. Therefore we will:
Specific Aim 1. To determine the mechanisms by which Ipc1 regulates IPI. Since Ipcl regulates in opposing directions the levels of two bioactive molecules, phytoceramide and DAG, and activates the transcription of IPI, we hypothesize that IPI expression is modulated by a transcription factor, which could be regulated by the level of phytoceramide or DAG. Therefore, in this specific aim we will: A) evaluate the transcriptional activation of IPI by Ipc1; and B) determine the effect of phytoceramide and DAG on this mechanism of regulation. Our hypothesis predicts that Ipcl regulates IPI at the transcriptional level and these studies will allow us to establish whether the lipids modulated by Ipc1 regulate this molecular mechanism through a transcription factor.
Specific Aim 2. To establish the role of IPI in the regulation of pathogenicity of C. neoformans. Since IPI exerts an anti-phagocytic effect, we hypothesize that the increase of IPI by Ipc1 protects fungal cells from alveolar macrophages. Therefore in this specific aim we will: A) establish the effect of modulation of IPI on phagocytosis by alveolar macrophages; and B) determine the role of IPI on virulence of C. neoformans in the animal models. Our hypothesis predicts that IPI represents a virulence factor, protecting the fungal cells against phagocytosis by alveolar macrophages. Using our animal models, these studies will allow us to determine the impact of IPI on the initiation and the consequent dissemination of the infection from the lung to other organs and tissues.
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