Pathogenic yeast Cryptococcus neoformans can disseminate through the blood stream and cause devastating meningitis. At present, cryptococcal meningitis is the most common fungal infection of the central nervous system (CNS) and also the most frequent neurological complication in AIDS patients. The mechanism that transversal of C. neoformans across the blood-brain barrier (BBB) to cause the CNS infection is largely unknown. The goal of this project is to continue investigating how C. neoformans enters into human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In the last grant period, we found that C. neoformans could induce morphological changes in HBMEC via cytoskeleton reorganization. We demonstrated that C. neoformans CPS1 encoded hyaluronic acid synthase. We also verified that CPS1 was required for C. neoformans binding to HBMEC using an in vitro BBB model. Furthermore, our studies showed that CD44 was the primary receptor on HBMEC for C. neoformans adhesion. Upon C. neoformans binding to the HBMEC, host CD44 translocated to the membrane rafts and surrounded the yeast entry site. Either CPS1 deletion in C. neoformans or CD44- knockout on HBMEC significantly impaired the yeast infection. We also observed that yeast binding and/or invasion was considerably reduced in the filipin-, GF109203X-, cytochalasin D- treated HBMEC. Filipin extracts cholesterol and caveolin on the membrane rafts, GF109203X is a Protein Kinase C (PKC) inhibitor, and cytochalasin D is an F-actin disrupting reagent. Dominant-negative PKCa also inhibited yeast invasion into HBMEC. The results suggest that the integrity of membrane rafts, functional PKCa, and F-actin were necessary for yeast invasion. Based on the above observations, we hypothesize that CD44-elicited signals and induced cytoskeleton reorganization are required for yeast entry into HBMEC. We will explore the mechanisms of C. neoformans invasion by the following Aims: (1) To determine the CD44-elicited signaling during the C. neoformans invasion, (2) To examine how C. neoformans induces cytoskeleton reorganization on HBMEC and its relationship to yeast infection, and (3) To evaluate the role of CD44 during C. neoformans invasion in mouse models. In our previous grant period, we have demonstrated the adhesion step of pathogen-host interaction. In this grant period, we will further characterize the molecular events at the internalization step. These studies are related to the clinical observations that a significant number of patients suffer severe meningitis and eventually succumb to this pathogen. The information derived from the studies is expected to be helpful in the development of novel strategies to prevent cryptococcal meningitis and its associate morbidity.

Public Health Relevance

The goal of this project is to continue investigating how C. neoformans invades into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In the last grant period, we identified and characterized a novel virulence factor CPS1, which is required for the adhesion to the HBMEC. We also demonstrated that host CD44 is the primary receptor for its adhesion. In this grant proposal, we will explore how C. neoformans internalizes into the HBMEC. The information derived from the studies is expected to be helpful in the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Pathogenic Eukaryotes Study Section (PTHE)
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Wong, May
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Children's Hospital of Los Angeles
Los Angeles
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Zhang, Bao; Yu, Jing-Yi; Liu, Li-Qun et al. (2015) Alpha7 nicotinic acetylcholine receptor is required for blood-brain barrier injury-related CNS disorders caused by Cryptococcus neoformans and HIV-1 associated comorbidity factors. BMC Infect Dis 15:352
Sena, Laura A; Li, Sha; Jairaman, Amit et al. (2013) Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity 38:225-36
Huang, Sheng-He; Wang, Lin; Chi, Feng et al. (2013) Circulating brain microvascular endothelial cells (cBMECs) as potential biomarkers of the blood-brain barrier disorders caused by microbial and non-microbial factors. PLoS One 8:e62164
Jong, Ambrose; Wu, Chun-Hua; Gonzales-Gomez, Ignacio et al. (2012) Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection. J Biol Chem 287:15298-306
Tseng, Hsiang-Kuang; Liu, Chang-Pan; Price, Michael S et al. (2012) Identification of genes from the fungal pathogen Cryptococcus neoformans related to transmigration into the central nervous system. PLoS One 7:e45083
Long, Min; Huang, Sheng-He; Wu, Chun-Hua et al. (2012) Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells. J Biomed Sci 19:19
Chi, Feng; Bo, Tao; Wu, Chun-Hua et al. (2012) Vimentin and PSF act in concert to regulate IbeA+ E. coli K1 induced activation and nuclear translocation of NF-ýýB in human brain endothelial cells. PLoS One 7:e35862
Huang, Sheng-He; Wu, Chun-Hua; Chang, Yun C et al. (2012) Cryptococcus neoformans-derived microvesicles enhance the pathogenesis of fungal brain infection. PLoS One 7:e48570
Huang, Sheng-He; Long, Min; Wu, Chun-Hua et al. (2011) Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3). J Biol Chem 286:34761-9
Chi, Feng; Wang, Lin; Zheng, Xueye et al. (2011) Meningitic Escherichia coli K1 penetration and neutrophil transmigration across the blood-brain barrier are modulated by alpha7 nicotinic receptor. PLoS One 6:e25016

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