Cryptococcus neoformans is a neurotropic pathogen that causes fatal meningoencephalitis primarily in individuals with T-cell deficiency such as the AIDS patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. The disease is 100% fatal unless treated and even with the most effective antimycotic agents, the fatality rate is about 25%. Current estimates of annual deaths due to cryptococcosis world-wide is close to 700,000. C. neoformans is commonly found in the human environment world-wide. In previous years, we discovered that C. neoformans yeast cells invaded the brain by crossing the blood-brain barrier transcellularly. We also, reported that the product of CPS1 gene that plays an important role in association and trversal of C. neoformans yeast cells across the blood-brain barrier (BBB)is hyaluronic acid which is located at the base of the extracellular polysaccharide that surrounds the yeast cells. We also showed, in collaboration with Dr. Jong's group, that protein kinase C-alpha activation is required for the yeast to cross the BBB and CD44 is involved in the association of the yeast cells with the brain microvasular endothelial cells. In order to gain better insight into the invasion process, we have taken a genetic approach during 2008-2009 period. In collaboration with Dr. Kim at Johns Hopkins University, we screened our T-DNA nsertional mutant library in serotype D background to identify genes that play role in attachment of yeast cells to brain endothelial cells. In preliminary screen, we isolated few transposon mutants that fail to associate with human brain microvascular endothelial cells (HBMEC). Sequence analyses indicated all the mutants except one have multiple insertions. We focused on the mutant that has a single insertion in the coding region of CTS1 gene (for calcineurin temperature suppressor 1). The CTS1 gene encodes a phospholipid-binding protein containing a C2 domain and a leucine zipper motif. Literature search indicated Cts1, along with calcineurin, plays an important role in cell septation, and hyphal elongation. We obtained cts1 deletion mutants from Dr. Heitman at Duke University. We confirmed that original transposon mutant as well as deletion mutants exhibit transcytosis defect. In addition to defect in association to HBMEC cell line, these mutants are extremely sensitive to calcineurin inhibitors such as FK506 and cyclosporine A at room temperature. We complemented both the deletion mutants with the wild type CTS1 gene. Complemented strains, like wild type, did not show any growth inhibition in the presence of calcineurin inhibitors at room temperature. However, interestingly, complementation strains behave like mutants in association assays and like wild type in transcytosis assays. As association leads to transcytosis, these results are confusing and we are trying to resolve this issue. Meanwhile we are screening a deletion library in serotype A background using the same approach. Dr. Madhani at UCSF, California has constructed this deletion library. Screening is underway at JHU in Dr. Kims laboratory.

Project Start
Project End
Budget Start
Budget End
Support Year
36
Fiscal Year
2009
Total Cost
$445,609
Indirect Cost
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Li, Jingbo; Chang, Yun C; Wu, Chun-Hua et al. (2016) The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence. J Microbiol Biotechnol 26:918-27
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