C. neoformans is a major opportunistic infection in AIDS. Melanin production is unique to pathogenic species of Cyptococcus and has been associated with virulence in three separate studies comparing lethality of Mel+ and Mel- strains of this organism in mice. However, the association between melanin formation and virulence has not been established using fungal strains produced or characterized by molecular biological methods. Preliminary work has been done by the investigator which has resulted in the purification of the enzyme diphenol oxidase responsible for melanin formation and cloning of the respective structural gene, CNLAC 1, CNLAC 1 deleted mutants of C. neoformans have been produced and a Mel- mutant has been complemented to Mel+ with CNLAC 1. The present proposal seeks to test the hypothesis that CNLAC 1 is the gene responsible for the association between melanin synthesis and virulence in an animal model.
The first aim will be to construct and fully characterize a gene deletant of CNLAC 1 in C. neoformans and a CNLAC 1 complemented mutant of Mel- C. neoformans strain. These experiments will thus provide two sets of Mel+ and Mel- mutants that will be similar except for a selective difference in the ability to produce CNLAC 1 transcripts.
The second aim will be to construct isogenic CNLAC 1 and cnlac 1 sets of C. neoformans strains. Stable mating types and the previous production of congenic sets of C. neoformans strains allow the investigator to decrease the chance of irrelevant mutations between pairs of mutants and their controls by backcrossing out unidentified mutations after transformations. In addition, a third set of Mel+ and Mel- mutants will be produced by backcrossing JEC 82 (Mel-) to its congenic Mel+ parents.
The third aim will be to assess the contribution of the CNLAC 1 gene to the virulence of C. neoformans using an intratracheal mouse model. Mice will be followed to determine time of death from Cryptococcal meningoencephalitis and assessed by periodic organ cultures to determine differences in virulence between each three test strains and its control.
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