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ABSTRACT Candida albicans is among the most significant human fungal pathogens. However, the vast majority of C. albicans studies have focused on a single clinical isolate and its marked derivatives. We investigated natural variation among clinical C. albicans isolates in gene regulatory control of biofilm formation, a process crucial to virulence. The transcription factor Efg1 is required for biofilm-associated gene expression and biofilm formation. Previously, we found extensive variation in Efg1-responsive gene expression among 5 diverse clinical isolates. However, chromatin immunoprecipitation sequencing analysis showed that Efg1 binding to genomic loci was uniform among the isolates. Functional dissection of strain differences identified three transcription factors, Brg1, Tec1, and Wor1, for which small changes in expression levels reshaped the Efg1 regulatory network. Brg1 and Tec1 are known biofilm activators, and their role in Efg1 network variation may be expected. However, Wor1 is a known repressor of EFG1 expression and an inhibitor of biofilm formation. In contrast, we found that a modest increase in WOR1 RNA levels, reflecting the expression differences between C. albicans strains, could augment biofilm formation and expression of biofilm-related genes. The analysis of natural variation here reveals a novel function for a well-characterized gene and illustrates that strain diversity offers a unique resource for elucidation of network interactions. IMPORTANCE Clinical isolates of all pathogens vary in the strength of traits linked to disease. In this study, we focused on variation in a pathogenicity trait of the fungal pathogen Candida albicans, biofilm formation. This trait is under the control of the cell type regulator Efg1. Expression of Efg1 is known from previous studies to be repressed by a second cell type regulator, Wor1. However, we found that natural variation in biofilm formation and biofilm-related gene expression was driven by collaboration between Efg1 and Wor1. Our findings show that analysis of natural isolates can reveal unexpected features of gene function, even for well-studied genes.

ABSTRACT Engineered conditional gene expression is used in appraisal of gene function and pathway relationships. For pathogens like the fungus Candida albicans, conditional expression systems are most useful if they are active in the infection environment and if they can be utilized in multiple clinical isolates. Here, we describe such a system. It employs the RBT5 promoter and can be implemented with a few PCRs. We validated the system with RBT5 promoter fusions to two genes that promote filamentation and polarized growth, UME6 and HGC1, and with efg1Δ/Δ mutants, which are defective in an activator of filamentous growth. An RBT5 promoter fusion to either gene enabled filamentous growth of an efg1Δ/Δ mutant of strain SC5314 in iron-limited media, including RPMI with serum and yeast extract-peptone-dextrose with bathophenanthrolinedisulfonic acid. The RBT5-UME6 fusion promoted filamentation of efg1Δ/Δ mutants in RPMI with serum of four other clinical C. albicans isolates as well. In a mouse model of disseminated candidiasis, the RBT5-UME6 fusion promoted filamentation of the SC5314 efg1Δ/Δ mutant in kidney tissue, an indication that the RBT5 promoter is active in the iron-limited host environment. The RBT5 promoter expands the conditional expression toolkit for C. albicans genetics. IMPORTANCE Genetic strategies have been vital for mechanistic analysis of biological processes. Here, we describe a genetic tool for the fungal pathogen Candida albicans.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.