Basic Studies On Pathogens Causing Cryptococcosis

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Abstract

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 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 demages 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. We have also studied the role of MATalpha strain and MATa strain during mating. It was found that the MATalpha cells are donors and MATa cells are recipients of genetic material. This finding suggests that dioecism exists in C. neoformans although the two mating types are morphologically indistinguishable.