Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-α activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKC alpha in HBMEC. During C. neoformans infection, phosphorylation of PKC alpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKC alpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKC alpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKC alpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKC alpha and downstream actin filament activity during the fungal invasion into HBMEC.