Rocha C R, Schröppel K, Harcus D, Marcil A, Dignard D, Taylor B N, Thomas D Y, Whiteway M, Leberer E
Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Mol Biol Cell. 2001 Nov;12(11):3631-43. doi: 10.1091/mbc.12.11.3631.
The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.
人类真菌病原体白色念珠菌会根据各种外部信号从出芽酵母形态转变为极性菌丝形态。这种形态发生转换与致病性的发展有关。我们通过对酿酒酵母细胞中Ras1p和Ras2p突变导致的条件性生长缺陷进行功能互补,克隆了编码白色念珠菌腺苷酸环化酶的CaCDC35基因。此前已表明,这些Ras同源物在酵母中调节腺苷酸环化酶。白色念珠菌腺苷酸环化酶与其他真菌腺苷酸环化酶高度同源,但与哺乳动物酶的序列相似性较低。缺失CaCDC35两个等位基因的白色念珠菌细胞检测不到cAMP水平,这表明该基因编码白色念珠菌中唯一的腺苷酸环化酶。纯合突变细胞是存活的,但生长比野生型细胞慢,并且在我们体外分析的所有环境条件下都无法从酵母形态转变为菌丝形态。此外,在被巨噬细胞吞噬的突变细胞中,这种形态发生转换被完全阻断。然而,外源性cAMP可恢复形态发生转换。基于上位性实验,我们提出CaCdc35p在Ras同源物CaRas1p的下游起作用。这些上位性实验还表明,假定的转录因子Efg1p和菌丝诱导MAP激酶途径的组分在诱导形态发生转换的能力上依赖于CaCdc35p的功能。纯合cacdc35Δ细胞在粘膜小鼠模型中无法建立阴道感染,并且在全身感染的小鼠模型中无致病性。这些发现表明真菌腺苷酸环化酶和cAMP信号通路的其他调节因子可能是抗真菌药物的有用靶点。