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ABSTRACT Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

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 The biological function of a gene is often probed through its interactions with other genes. This general approach has been especially useful to build knowledge about poorly understood genes upon the bedrock of well-characterized genes. Genetic interaction analysis requires the construction of strains with mutations in two or more genes. Single-gene mutants of microbial pathogens are generally validated through introduction of a wild-type copy of the affected gene to create a complemented or reconstituted strain, followed by testing for restoration of a wild-type phenotype. This practice, formalized as one of Falkow’s “molecular Koch’s postulates” ensures that the phenotype of the mutant depends upon the known mutation. However, multigene mutants are seldom validated because of the labor required to assemble multiple genomic segments into a vector that can be introduced into the mutant strain. We present here an approach, concatemer assembly for rescue of mutant abilities (CARMA), that circumvents this impediment through an in vivo recombinational assembly strategy that does not require cloning at all. Our results show that CARMA allows genetic rescue of two double-gene mutant strains of the fungal pathogen Candida albicans. IMPORTANCE Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype.

ABSTRACT We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

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.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Genotype-phenotype relationships can vary extensively among members of a species. One cause of this variation is circuit diversification, the alteration of gene regulatory relationships among members of a species. Circuit diversification is thought to be a starting point for the circuit divergence or rewiring that occurs during speciation. How widespread is circuit diversification? Here we address this question with the fungal pathogen Candida albicans, which forms biofilms rich in distinctive hyphal cells as a prelude to infection. Our understanding of the biofilm/hyphal regulatory network comes primarily from studies of one clinical isolate, strain SC5314, and its marked derivatives. We used CRISPR-based methods to create mutations of four key biofilm transcription factor genes-BCR1, UME6, BRG1, and EFG1 -in SC5314 and four additional clinical isolates. Phenotypic analysis revealed that mutations in BCR1 or UME6 have variable impact across strains, while mutations in BRG1 or EFG1 had uniformly severe impact. Gene expression, sampled with Nanostring probes and examined comprehensively for EFG1 via RNA-Seq, indicates that regulatory relationships are highly variable among isolates. Our results suggest that genotype-phenotype relationships vary in this strain panel in part because of differences in control of BRG1 by BCR1, a hypothesis that is supported through engineered constitutive expression of BRG1. Overall, the data show that circuit diversification is the rule, not the exception, in this biofilm/hyphal regulatory network.