ABSTRACTThe genusCryptococcusincludes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to beCryptococcus neoformansandCryptococcus gattii. We compared the interaction of murine macrophages with threeC. gattiispecies complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and oneC. neoformansspecies complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies ofC. neoformansandC. gattiispecies complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with eitherC. gattiiorC. neoformans. Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.