Regulatory Diversity of TUP1 in Cryptococcus neoformans

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide, while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion of the global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growth phenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1Δ strains of serotype D, the density-dependent growth phenotype was found to be absent in tup1Δ strains of serotype A which had been derived from several different genetic clusters. The serotype A H99 tup1Δ strain showed less retardation in the growth rate than tup1Δ strains of serotype D, but the mating efficiency was found to be similar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule production and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene in maintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formation and iron/copper homeostasis was observed between the wild-type and tup1Δ H99 strains. Furthermore, the H99 tup1Δ strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility to fluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has pathobiological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.The fungal Tup1 proteins function as global repressors which regulate a large number of genes associated with growth, morphological differentiation, and sexual and asexual reproduction. As a consequence, tup1 mutants are known to display numerous phenotypes (9, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive filamentous growth with no budding yeast cells and is accompanied by loss of virulence (2, 32). In Penicillium marneffei, the only dimorphic species known in the genus Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and abnormality in yeast morphogenesis (38). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, deletion of the TUP1 homologs, rcoA and rco-1, respectively, severely affects growth and sexual and asexual reproduction (12, 46).Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with two serotypes, A and D, and the function of Tup1 has been studied only for serotype D strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependent growth, sensitivity to 0.8 M KCl, and expression of genes in several other biological pathways (26). Most importantly, tup1Δ strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strains which has not been reported for any other fungal species (27).According to genome sequence data, the serotype A reference strain H99 shares 95% sequence identity with the serotype D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demonstrated in two major signaling pathways, the pheromone-responsive Cpk1 mitogen-activated protein kinase and cyclic AMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarity glycerol (HOG) pathway also showed regulatory disparity between the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A strains would have similar consequences. Surprisingly, we found striking differences in the phenotypes manifested by tup1Δ strains of the two serotypes. We report here the serotype-specific differences in TUP1 regulation between A and D strains and the novel regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans.