Transcription Factor STE12alpha Has Distinct Roles in Morphogenesis, Virulence, and Ecological Fitness of the Primary Pathogenic Yeast Cryptococcus Gattii

Cryptococcus gattii is a primary pathogenic yeast, increasingly important in public health, but factors responsible for its host predilection and geographical distribution remain largely unknown. We have characterized C. gattii STE12alpha to probe its role in biology and pathogenesis because this transcription factor has been linked to virulence in many human and plant pathogenic fungi. A full-length STE12alpha gene was cloned by colony hybridization and sequenced using primer walk and 3' rapid amplification of cDNA ends strategies, and a ste12alpha delta gene knockout mutant was created by URA5 insertion at the homologous site. A semiquantitative analysis revealed delayed and poor mating in ste12alpha delta mutant; this defect was not reversed by exogenous cyclic AMP. C. gattii parent and mutant strains showed robust haploid fruiting. Among putative virulence factors tested, the laccase transcript and enzymatic activity were down regulated in the ste12alpha delta mutant, with diminished production of melanin. However, capsule, superoxide dismutase, phospholipase, and urease were unaffected. Similarly, Ste12 deficiency did not cause any auxotrophy, assimilation defects, or sensitivity to a large panel of chemicals and antifungals. The ste12alpha delta mutant was markedly attenuated in virulence in both BALB/c and A/Jcr mice models of meningoencephalitis, and it also exhibited significant in vivo growth reduction and was highly susceptible to in vitro killing by human neutrophils (polymorphonuclear leukocytes). In tests designed to simulate the C. gattii natural habitat, the ste12alpha delta mutant was poorly pigmented on wood agar prepared from two tree species and showed poor survival and multiplication in wood blocks. Thus, STE12alpha plays distinct roles in C. gattii morphogenesis, virulence, and ecological fitness.