A CLC-type Chloride Channel Gene is Required for Laccase Activity and Virulence in Cryptococcus Neoformans

Laccase is a major virulence factor required for infection caused by the human pathogenic yeast Cryptococcus neoformans. However, cellular processes involved in the regulation and expression of laccase remain largely unknown in C. neoformans. Here we report the identification of a chloride channel gene CLC-A which is essential for laccase activity in C. neoformans. CLC-A shares homology to CLC-type voltage-gated chloride channels from other organisms; for example, 63% homology to GEF1, a chloride channel gene from Saccharomyces cerevisiae. A clc-a mutant, Mlac3, generated by insertional mutagenesis as well as a targeted Deltaclc-a mutant produced undetectable laccase in a liquid assay and produced no melanin on asparagine agar containing norepinephrine. Mlac3 was complemented with wild-type CLC-A which restored laccase activity and melanin biosynthesis. The clc-a mutants also showed reduced synthesis of another important virulence factor, capsule, and showed reduced growth at elevated pH. In addition, the clc-a mutation resulted in attenuated virulence in a mouse cryptococcosis model that was restored by complementation with wild-type CLC-A, indicating that the chloride channel plays an important role in the virulence of the organism. Further analysis revealed that the basis for absent laccase expression in the clc-a mutant was a laccase transcriptional defect that could be restored by adding exogenous copper. In conclusion, our findings show that CLC-A plays a role in the expression of two important virulence factors, capsule and laccase expression, which are required for virulence of the fungal pathogen.