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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS047599-08
Application #
8269941
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Wong, May
Project Start
2004-01-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$392,000
Indirect Cost
$147,000
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
052277936
City
Los Angeles
State
CA
Country
United States
Zip Code
90027
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
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
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
Chi, Feng; Wang, Lin; Zheng, Xueye et al. (2011) Recruitment of ýý7 nicotinic acetylcholine receptor to caveolin-1-enriched lipid rafts is required for nicotine-enhanced Escherichia coli K1 entry into brain endothelial cells. Future Microbiol 6:953-66
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
Huang, Sheng-He; Wu, Chu-Hua; Jiang, Shibo et al. (2011) HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities. Biochem J 438:457-66
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
Chi, Feng; Wang, Ying; Gallaher, Timothy K et al. (2009) Identification of IbeR as a stationary-phase regulator in meningitic Escherichia coli K1 that carries a loss-of-function mutation in rpoS. J Biomed Biotechnol 2009:520283
Jong, Ambrose; Wu, Chun-Hua; Prasadarao, Nemani V et al. (2008) Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation. Cell Microbiol 10:1854-65

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