Cryptococcus neoformans is a neurotropic pathogen that causes fatal meningoencephalitis primarily in individuals with T-cell deficiency such as the AIDS patients. The disease is 100% fatal unless treated. C. neoformans is a heterothallic yeast that occurs in two mating types MATalpha and MATa. The yeast cells are encapsulated with a polysaccharide which mainly consists of glucuronoxylomannan. The polysaccharide capsule has been determined to be the major virulence factor of C. neoformans which allows the yeast to resist host defenses. However, the essential role of the capsule in allowing it to resist host defenses during initial lung infection is not clearly understood. In 2001-2002,we studied the fate of Cap- cells and Cap+ cells of the same genetic background in mice after intratracheal inoculation. The Cap+ cells persisted in the lung of C.B-17 mice and disseminated to the brain, whereas the Cap- cells grew poorly in the lung and were infrequently detected in the brain. T-cell mediated immunity was required to control growth in the lung and the brain. T-cells were also required for optimal inhibition of growth of Cap- cells in the lung but not for maintaining control of the fungal burden in the brain. These observations indicated that the capsule plays an important role in lung infection and dissemination to the brain. In 2002-2003,we studied the mechanism by which Cap+ and Cap- strains of C. neoformans cross the blood-brain-barrier (BBB). We used human brain microvascular endothelial cells (HBMEC) as the in vitro model of the human BBB in order to investigate the cryptococcal invasion of HBMEC. Exposure of HBMEC to C. neoformans triggered an extensive formation of microvilli-like membrane protrusions within 15-30 min. Acapsular as well as encapsulated C. neoformans cells adhered to and traversed transcellularly across the HBMEC. Histopathology of the mouse brain obtained after an intravenous challenge with C. neoformans supported our observations in the in vitro model. By three hours post injection, C. neoformans cells were observed either within the endothelial cells or localized adjacent to the brain capillary vessels in the neuropil. C. neoformans was observed in the brain parenchyma by 22 hr post injection while no association of C. neoformans with the choroid plexus was detected even after 10 days. Meningeal involvement was observed only after establishment of yeast cells in nuropil and nearly 10 days after infection. Our observations suggested that C. neoformans cells entered the brain by trancellular crossing of the endothelial BBB regardless of the capsular phenotype and that meningitis occured after encephalitis. During the same period, we also studied the role of the pheromone receptor in the pathobiology of C. neoformans. We isolated the CPRa gene that encodes a putative 7-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRa expressed functional diversity. Deletion of the gene drastically reduced mating efficiency but did not abolish the mating. The gene expression of CPRa was found to be developmentally regulated and was not affected by the deletion of the transcriptional regulator, STE12a. CPRa was involved in sensing different environmental conditions and its expression was markedly increased by shifting cultures from liquid to solid media. CPRa also played a significant role in virulence by regulating size of the capsule in the mouse brain. Our results suggested that pheromone receptors of C. neoformans are important for sensing pheromones as well as the environmental cues not associated with mating. It is probable that the CPR gene in both mating type strains plays a critical role in virulence by assisting the cryptococcal cells in sensing ligands and transmission of signals to the down stream genes that are required for survival and growth of the fungus in host tissue. During the year 2003-2004, we have analyzed STE12alpha, a transcriptional activator, involved in mating as well as in virulence. Using site directed mutagenesis, we delineated the roles of the homeodomain and the C2H2 zinc finger regions of STE12 alpha. The homeodomain region was found to be important for DNA binding ability, mating frequency and haploid fruiting capability. The zinc finger, on the other hand, was found to be important for the production of capsule in vivo and virulence. We have also studied the function of the CAP59 gene in capsule formation and found it to be involved in the extracellular trafficking of capsular glucuronoxylomannan.