Candida albicans Double Mutants Lacking both EFG1 and WOR1 Can Still Switch to Opaque

Candida albicans, a pervasive opportunistic pathogen, undergoes a unique phenotypic transition from a “white” phenotype to an “opaque” phenotype. The switch to opaque impacts gene expression, cell morphology, wall structure, metabolism, biofilm formation, mating, virulence, and colonization of the skin and gastrointestinal (GI) tract. Although the regulation of switching is complex, a paradigm has evolved from a number of studies, in which, in its simplest form, the transcription factors Efg1 and Wor1 play central roles. When EFG1 is upregulated under physiological conditions, it represses WOR1, an activator of white-to-opaque switching, and the cell expresses the white phenotype; when EFG1 is downregulated, WOR1 is derepressed and activates expression of the opaque phenotype. Deletion of either EFG1 or WOR1 supports this yin-yang model of regulation. Here, we demonstrate that this simple model is insufficient, since strains in which WOR1 and EFG1 are simultaneously deleted can still be induced to switch en masse from white to opaque. Opaque cells of double mutants (efg1(−/−) wor1(−/−)) are enlarged and elongate, form an enlarged vacuole, upregulate mCherry under the control of an opaque-specific promoter, form opaque cell wall pimples, express the opaque phenotype in lower GI colonization, and, if MTL homozygous, form conjugation tubes in response to pheromone and mate. These results can be explained if the basic and simplified model is expanded to include a WOR1-independent alternative opaque pathway repressed by EFG1. IMPORTANCE The switch from white to opaque in Candida albicans was discovered 33 years ago, but it is still unclear how it is regulated. A regulatory paradigm has emerged in which two transacting factors, Efg1 and Wor1, play central roles, Efg1 as a repressor of WOR1, which encodes an activator of the transition to the opaque phenotype. However, we show here that if both EFG1 and WOR1 are deleted simultaneously, bona fide opaque cells can still be induced en masse. These results are not compatible with the simple paradigm, suggesting that an alternative opaque pathway (AOP) exists, which can activate expression of opaque and, like WOR1, is repressed by EFG1.