The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii

The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265tps1 Delta, R265tps2 Delta, and R265nth1 Delta mutants. As in Cryptococcus neoformans, cellular trehalose was reduced in the R265tps1 Delta and R265tps2 Delta mutants, which could not grow and died, respectively, at 37 degrees C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265tps2 Delta is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans, the C. gattii R265tps1 Delta mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265tps1 Delta mutant was not due solely to its 37 degrees C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii. Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2, has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.