Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions and can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans biofilm formation on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine if the in vitro biofilm transcriptional regulatory network also plays a role in OPC, we carried out a systematic genetic interaction analysis in a mouse model of C. albicans OPC. Whereas each of the six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1ΔΔ, bcr1ΔΔ, and rob1ΔΔ) and one heterozygous mutant (tec1Δ/TEC1) have reduced infectivity in the mouse model of OPC. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors during OPC. Using epistasis analysis, we have formulated a genetic circuit, [EFG1+BRG1]→TEC1→BCR1, that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation, such as efg1ΔΔ, rob1ΔΔ, and brg1ΔΔ filament, during oral infection and that reduced filamentation does not correlate with infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections during infection are distinct from those observed in vivo. IMPORTANCE The pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that the network of transcription factors that regulates in vitro biofilm formation has similarities and differences during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.