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 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 neuropil and nearly 10 days after infection. Our observations suggested that C. neoformans cells entered the brain by transcellular crossing of the endothelial BBB regardless of the capsular phenotype and that meningitis occurred after encephalitis. During 2004-2005, we studied the role of phospholipase B of C. neoformans in the formation of cystic lesions in the brain. It was previously believed that cystic cryptococcal lesions in the brain were due to the accumulation of polysaccharide capsule. By using PLB1 deletant and wild type strains, we showed that the PLB1 deleted strain did not form cystic lesions while the wild type produced large cysts. Immunofluorescence microscopy indicated that capsules were present in both the wild type and the PLB1 deleted strains. The PLB1 mutant reconstituted with the wild type PLB1 gene produced cystic lesions. This indicated that phospholipase damages the brain tissue to form cystic lesions. We have constructed an insertional library of C. neoformans in order to identify the genes required for capsule formation, adherance to brain endothelial cells and growth under hypoxic conditions. [unreadable] During 2005-2006, we have identified CPS1, a homolog of the Streptococcus pneumoniae type 3 polysaccharide synthase gene, to be important for pathobiology of Cryptococcus neoformans. CPS1 encodes a protein contaning a glycosyltransferase moiety and shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from C. neoformans resulted in a slight reduction of capsule size observed by India Ink preparation. The growth rate at 37C was impared and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that conserved glycosyltransferase domains are critical for the ability of strain to grow at elevated temperatures. Hyaluronan ELISA method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild type and the cps1D strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron dense layer between the outer cell wall and the capsule. These and virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans. We began screening of our 30,000 insertional mutant library for the identification of the genes necessary for C. neoformans yeast cells to form capsule, adhere to the brian endothelial cells, growth under hypoxic conditions and resistant to cobalt chloride. We have identified numerous clones that fit into each category. Sequence of the genes that contain insertions are being determined.