Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1

Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofact or insertion, glycosylation and pH stability. In the present study, we iden tify and characterize a component of the vesicular proton pump, Vph1p, to d etermine its role in the virulence of the AIDS-related fungal pathogen Cryp tococcus neoformans. Insertional mutagenesis and plasmid rescue were used t o identify the VPH1 gene by screening for mutants defective in laccase acti vity. Disruption of VPH1 resulted in defects in three virulence factors (ca psule production, laccase and urease expression), as well as a growth defec t at 37 degreesC, but only a small growth reduction at 30 degreesC. These e ffects were duplicated by the vacuolar (H+)-ATPase inhibitor bafilomycin A( 1). Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo-encephalitis model. Complementation of the insertional mutant with wildtype VPH1 resulted in a recovery of virulence factor expression, normal growth at 37 degreesC and restoration of full virulence. These studies est ablish the importance of the VPH1 gene and vesicular acidification in the v irulence of C. neoformans.