Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells.
BACKGROUND
Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown.


METHODS
In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated.


RESULTS
We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network.


CONCLUSIONS
These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.
Department of Microbiology, Southern Medical University, Guangzhou, China.
Min
M
Long
Sheng-He
SH
Huang
Chun-Hua
CH
Wu
Gregory M
GM
Shackleford
Ambrose
A
Jong
D008841:Actin Cytoskeleton; D001812:Blood-Brain Barrier / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002494:Central Nervous System Infections / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000378:metabolism / Q000472:pathogenicity*; D004705:Endocytosis; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005372:Filipin / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering
C497483:CD44 protein, human; D051242:Caveolin 1; D018960:Hyaluronan Receptors; D034741:RNA, Small Interfering; D005372:Filipin