The multicopper laccase is a major virulence factor in Cryptococcus neoformans. Its expression regulation is complex. We presented molecular evidence to show that laccase expression was induced by high concentrations of exogenous copper. Melanin production and laccase enzymatic activity increased dramatically in response to the addition of copper to the media. Reverse transcription-PCR amplification of the laccase gene LAC1 mRNA revealed that the induction occurred at the transcriptional level, which required the copper-responsive factor-encoding gene CUF1. Disruption of CUF1 demolished the activation of LAC1 transcription by copper, whereas the reconstituted strain restored the phenotypic defects. Furthermore, copper induction was shown to be independent of derepression by glucose starvation, a well-established activation factor for laccase expression. These results demonstrate a role of the copper-responsive factor gene CUF1 in the expression of laccase in C. neoformans.

The multicopper oxidase laccase is widespread in fungi and has great industrial importance. One puzzle regarding laccase production in the basidiomycetous yeast Cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37°C). In this paper, we report identification of a nitrogen metabolite repression-related gene, TAR1, which is responsible for laccase repression. Disruption of TAR1 results in a significant increase in the level of LAC1 mRNA at 37°C. The putative protein Tar1 shares a moderate level of similarity with the nitrogen metabolite repressors Nmr1 and NmrA from Neurospora crassa and Aspergillus nidulans, respectively. Likewise, Tar1 has a negative role in the utilization of nitrate. Furthermore, the structure of Tar1 is unique. Tar1 lacks the long C-terminal region of Nmr1 and NmrA. It contains the canonical Rossmann fold motif, GlyXXGlyXXGly, whereas Nmr1 and NmrA have variable residues at the Gly positions. Interestingly, the promoter region of TAR1 contains three TTC/GAA repeats which are likely the heat shock factor (Hsf) binding sites, implying that Hsf has a role in laccase inhibition. TAR1 mediation of temperature-associated repression of LAC1 suggests a novel mechanism of laccase regulation and a new function for Nmr proteins. Our work may be helpful for industry in terms of promotion of laccase activity.