In vitro expression of vital virulent genes of clinical and environmental isolates of Cryptococcus neoformans/gattii in endothelial cells of human blood-brain barrier

Abstract

BackgroundEvaluation of the pathogenesis of clinical and environmental cryptococcal isolates to the central nervous system is necessary for understanding the risk. This study was designed to determine the in vitro expression of six important virulent genes of Cryptococcus neoformans/gattii in Human Brain Microvascular Endothelial cells (hBMEC). MethodsThe hBMEC were infected with Cryptococcus to determine invasion and survival rate at 3, 12 and 24hours by subsequent colony count of internalized yeasts. The whole RNA of the intracellular Cryptococcus was extracted to quantify the expression of CAP10, PLB1, ENA1, URE1, LAC1, and MATα genes by real-time quantitative PCR for 3 and 12hours of infection. ResultsInvasion and survival rates were higher in clinical and standard strains of C. neoformans. A significant difference was observed among the clinical and environmental isolates for the expression of CAP10, ENA1, LAC1, MATα and URE1 at 3hours, and ENA1, LAC1, MATα, PLB1 and URE1 at 12hours. Clinical isolates showed significant upregulation of all the genes except PLB1, which was higher in environmental isolates. Relative expressions at the two time-points showed statistically significant (P=0.043) changes for the clinical isolates and no significance (P=0.063) for environmental isolates. ConclusionThe C. gattii (VGI) isolates showed significantly lower invasion and survival than C. neoformans (VNI, and VNII) irrespective of their sources. Clinical isolates exhibited higher expression for the majority of the virulent genes until 12hours of infection, probably due to their better adaptation in the host system and enhanced pathogenicity than the environmental counterparts.