Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis

Cryptococcus neoformans var. neoformans is an important opportunistic funga l pathogen of patients whose immune system has been compromised due to vira l infection, antineoplastic chemotherapy, or tissue transplantation. As man y as 13% of all AIDS patients suffer a life-threatening crytococcal infecti on at some time during the course of their HIV disease. To begin to underst and the molecular basis for virulence in Crytococcus neoformans var. neofor mans serotype A, we have employed signature-tagged mutagenesis (STM) to ide ntify mutants with altered virulence in a mouse model. The critical paramet ers of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed togethe r in a single animal with each participating in the infection and that ther e is no apparent interaction between a virulent strain and an avirulent str ain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecu lar analyses of these mutants indicated that 19 (49%) contained an insertio n in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal inser tion sites of those mutants that did not have an integration event in the a ctin promoter revealed an approximately random distribution among the chrom osomes. Individual challenge of the putative mutants in a mouse model revea led five hypovirulent mutants and one hypervirulent mutant.