Challenge of Drosophila melanogaster with Cryptococcus neoformans and role of the innate immune response

Abstract

We found that the ingestion of Cryptococcus neoformans by Drosophila melanogaster resulted in the death of the fly but that the ingestion of Saccharomyces cerevisiae or the nonpathogenic Cryptococcus kuetzingii or Cryptococcus laurentii did not. The C. neoformans protein kinase A and RAS signal transduction pathways, previously shown to be involved in virulence in mammals, also played a role in killing Drosophila. Mutation of the Toll immune response pathway, the predominant antifungal pathway of the fly, did not play a role in Drosophila defense following ingestion of the yeast. However, the Toll pathway was necessary for the clearance of C. neoformans introduced directly into the hemolymph of D. melanogaster and for the survival of systemically infected flies.

FIG. 1.

Killing of D. melanogaster following exposure to C. neoformans. (A) Survival of wild-type D. melanogaster (OR) exposed to lawns of C. neoformans (H99) grown on different agar media (1/3 YPD, YPD, and BHI). (B) Survival of wild-type D. melanogaster (OR) exposed to lawns of C. neoformans serotypes A (ATCC 62068), B/C (ATCC 34877), and D (ATCC 36556), C. laurentii (ATCC 18803), C. kuetzingii (ATCC 42276), and S. cerevisiae (YJM 145 or YJM 237) grown on 1/3 YPD agar medium. P values are less than 0.001 for all C. neoformans strains compared to C. laurentii, C. kuetzingii, or either S. cerevisiae strain.

FIG. 2.

C. neoformans virulence factors for mammalian infection also enhance the killing of D. melanogaster. Shown is the survival of D. melanogaster (OR) flies exposed to wild-type (H99) or mutant C. neoformans. Mutations refer to disruptions in the genes encoding the PKA- or the Ras1-controlled signal transduction cascades. P values are less than 0.001 for each of the mutants compared to the parental strain, H99.

FIG. 3.

Role of starvation and D. melanogaster immune system in survival following intestinal exposure to C. neoformans. (A) Killing of wild-type (OR) flies feeding continuously on C. neoformans (H99) or feeding for 18 h per day and then starved for the remaining 6 h. (B) Killing rates for wild-type (OR) and mutant (imd; spz) flies following feeding on C. neoformans (H99) are identical. P values are less than 0.001 for the comparison of the curves in panel A.

FIG. 4.

The D. melanogaster immune response is necessary for clearing systemic C. neoformans infection. (A) Accumulation of C. neoformans within mutant (spz) or wild-type (OR) flies following injection with ∼400 CFU. CFU in surviving flies were enumerated. (B) Survival of wild-type (OR) and mutant (spz, imd, or imd; spz) flies following injection with ∼400 CFU of C. neoformans.