Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the transcription factors Zcf15 and Zcf26.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

Piperacillin/tazobactam Susceptibility Test Interpretive Criteria for Enterobacterales: Recommendations from the United States Committee on Antimicrobial Susceptibility Testing.

The in vitro susceptibility testing interpretive criteria (STIC) for TZP against Enterobacterales were recently updated by the Food and Drug Administration (FDA), Clinical & Laboratory Standards Institute (CLSI), and European Committee on Antimicrobial Susceptibility Testing (EUCAST). The United States Committee on Antimicrobial Susceptibility Testing (USCAST) also recently reviewed TZP STIC for Enterobacterales and arrived at different STIC for Enterobacterales and herein we explain our recommendations and rationale behind them. Based on our review of the available data, USCAST does not recommend TZP STIC for certain Enterobacterales species that have a moderate to high likelihood of clinically significant AmpC production (E. cloacae, C. freundii, and K. aerogenes only) or for third-generation cephalosporin-non-susceptible (3GC-NS) Enterobacterales. USCAST recommends a TZP susceptibility breakpoint of ≤ 16/4 mg/L for third-generation cephalosporin-susceptible (3GC-S) Enterobacterales but only endorses the use of extended infusion TZP regimens for patients with infections due to these pathogens.

Biofilm-associated metabolism via ERG251 in Candida albicans.

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.

Modeling Invasive Aspergillosis Risk for the Application of Prophylaxis Strategies.

The epidemiology of invasive aspergillosis (IA) is evolving. To define the patient groups who will most likely benefit from primary or secondary Aspergillus prophylaxis, particularly those whose medical conditions and IA risk change over time, it is helpful to depict patient populations and their risk periods in a temporal visual model. The Sankey approach provides a dynamic figure to understand the risk of IA for various patient populations. While the figure depicted within this article is static, an internet-based version could provide pop-up highlights of any given flow's origin and destination nodes. A future version could highlight links to publications that support the color-coded incidence rates or other actionable items, such as bundles of applicable pharmacologic or non-pharmacologic interventions. The figure, as part of the upcoming Infectious Diseases Society of America's aspergillosis clinical practice guidelines, can guide decision-making in clinical settings.

In vivo pharmacodynamic characterization of a next-generation polyene, SF001, in the invasive pulmonary aspergillosis mouse model.

SF001 is a next-generation polyene antifungal drug in development, designed to have increased specificity to fungal ergosterol, which is absent in humans, and decreased binding to cholesterol. SF001 demonstrates long-acting, potent, broad-spectrum fungicidal activity. The goal of the current study was to determine the pharmacodynamic index and target of SF001 in an immunocompromised mouse model of invasive pulmonary aspergillosis against six Aspergillus fumigatus isolates. Minimum inhibitory concentration (MIC) values ranged from 0.5 to 2.0 mg/L. Plasma and epithelial lining fluid (ELF) pharmacokinetics were performed following single intraperitoneal doses of 1, 4, 16, and 64 mg/kg. Treatment efficacy was assessed with each of the six fungal isolates using daily doses of SF001 ranging from 0.25 to 64 mg/kg/day over a 96-h treatment duration. Efficacy was assessed by A. fumigatus quantitative PCR of conidial equivalents from lung homogenates. Nonlinear regression analysis using the Hill equation demonstrated that the 24-h exposure-response relationships for both plasma and ELF area under the concentration/MIC and Cmax/MIC ratios were strong and relatively similar [coefficient of determination (R2) = 0.74-0.75). Exposure-response relationships included a median plasma 24-h Cmax/MIC target for stasis and 1-log kill endpoint of 0.5 and 0.6, respectively. The present studies demonstrated in vitro and in vivo SF001 potency against A. fumigatus. These results have potential relevance for SF001 clinical dose selection and evaluation of susceptibility breakpoints.

Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.

Candida albicans extracellular vesicles trigger type I IFN signalling via cGAS and STING.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNß production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.

Liposomal formulation of a new antifungal hybrid compound provides protection against Candida auris in the ex vivo skin colonization model.

The newly emerged pathogen, Candida auris, presents a serious threat to public health worldwide. This multidrug-resistant yeast often colonizes and persists on the skin of patients, can easily spread from person to person, and can cause life-threatening systemic infections. New antifungal therapies are therefore urgently needed to limit and control both superficial and systemic C. auris infections. In this study, we designed a novel antifungal agent, PQA-Az-13, that contains a combination of indazole, pyrrolidine, and arylpiperazine scaffolds substituted with a trifluoromethyl moiety. PQA-Az-13 demonstrated antifungal activity against biofilms of a set of 10 different C. auris clinical isolates, representing all four geographical clades distinguished within this species. This compound showed strong activity, with MIC values between 0.67 and 1.25 µg/mL. Cellular proteomics indicated that PQA-Az-13 partially or completely inhibited numerous enzymatic proteins in C. auris biofilms, particularly those involved in both amino acid biosynthesis and metabolism processes, as well as in general energy-producing processes. Due to its hydrophobic nature and limited aqueous solubility, PQA-Az-13 was encapsulated in cationic liposomes composed of soybean phosphatidylcholine (SPC), 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP), and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG 2000), and characterized by biophysical and spectral techniques. These PQA-Az-13-loaded liposomes displayed a mean size of 76.4 nm, a positive charge of +45.0 mV, a high encapsulation efficiency of 97.2%, excellent stability, and no toxicity to normal human dermal fibroblasts. PQA-Az-13 liposomes demonstrated enhanced antifungal activity levels against both C. auris in in vitro biofilms and ex vivo skin colonization models. These initial results suggest that molecules like PQA-Az-13 warrant further study and development.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: 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.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, particularly when both the immune system and antiviral therapy fail to clear the infection and enable within-host evolution. Here we describe a 486-day case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individual's virus acquired resistance, likely via the earliest known occurrence of Spike amino acid variant E484T. Recently, E484T has arisen again as a derivative of E484A in the Omicron Variant of Concern, supporting the hypothesis that prolonged infections can give rise to novel variants long before they become prevalent in the human population.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance: Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous 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 finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Utility of triazole antifungal therapeutic drug monitoring: Insights from the Society of Infectious Diseases Pharmacists: Endorsed by the Mycoses Study Group Education and Research Consortium.

Triazole antifungals (i.e., fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole) are commonly used in clinical practice to prevent or treat invasive fungal infections. Most triazole antifungals require therapeutic drug monitoring (TDM) due to highly variable pharmacokinetics, known drug interactions, and established relationships between exposure and response. On behalf of the Society of Infectious Diseases Pharmacists (SIDP), this insight describes the pharmacokinetic principles and pharmacodynamic targets of commonly used triazole antifungals and provides the rationale for utility of TDM within each agent.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

Analysis of Candida Antifungal Resistance Using Animal Infection Models.

Candida frequently produces three general disease states, including mucosal candidiasis, disseminated candidiasis, and biofilm infection (which can be present with either of the other disease states). Antifungal drug resistance is intrinsic to biofilm growth and has emerged in other disease states. Mechanistic studies have uncovered the genetic pathways governing resistance to a number of antifungal agents. However, analyzing the clinical relevance of distinct mechanisms is fundamental for broadening our knowledge of antifungal drug resistance and for delineating the potential impact of targeting these pathways medically. Also, as drug-resistant strains and biofilms represent important nosocomial problems, preclinical animal models to assess the activity of novel antifungals are of great interest. Here we describe two rodent models that mimic the most common biofilm device and disseminated candidiasis states in patients. The model systems incorporate the anatomical site, immune components, and antifungal exposures relevant for the study of antifungal resistance. The models can be used to analyze mutant strains, assess the extent of drug resistance, examine biofilm formation, test new antimicrobials, and help determine drug exposures that may be linked with clinical failure.

Novel Keto-Alkyl-Pyridinium Antifungal Molecules Active in Models of In Vivo Candida albicans Vascular Catheter Infection and Ex Vivo Candida auris Skin Colonization.

New antifungal therapies are needed for both systemic, invasive infections in addition to superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to nonsystemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2 to 16 µg/mL) against medically important yeasts and molds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce Candida albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial preclinical data suggest that molecules of this class may warrant further study and development for nonsystemic applications.

Under-oil open microfluidic systems for rapid phenotypic antimicrobial susceptibility testing.

Antimicrobial susceptibility testing (AST) remains the cornerstone of effective antimicrobial selection and optimization in patients. Despite recent advances in rapid pathogen identification and resistance marker detection with molecular diagnostics (e.g., qPCR, MALDI-TOF MS), phenotypic (i.e., microbial culture-based) AST methods - the gold standard in hospitals/clinics - remain relatively unchanged over the last few decades. Microfluidics-based phenotypic AST has been growing fast in recent years, aiming for rapid (i.e., turnaround time <8 h), high-throughput, and automated species identification, resistance detection, and antibiotics screening. In this pilot study, we describe the application of a multi-liquid-phase open microfluidic system, named under-oil open microfluidic systems (UOMS), to achieve a rapid phenotypic AST. UOMS provides an open microfluidics-based solution for rapid phenotypic AST (UOMS-AST) by implementing and recording a pathogen's antimicrobial activity in micro-volume testing units under an oil overlay. UOMS-AST allows free physical access (e.g., by standard pipetting) to the system and label-free, single-cell resolution optical access. UOMS-AST can accurately and rapidly determine antimicrobial activities [including susceptibility/resistance breakpoint and minimum inhibitory concentration (MIC)] from nominal sample/bacterial cells in a system aligned with clinical laboratory standards where open systems and optical microscopy are predominantly adopted. Further, we combine UOMS-AST with a cloud lab data analytic technique for real-time image analysis and report generation to provide a rapid (<4 h) sample-to-report turnaround time, shedding light on its utility as a versatile (e.g., low-resource setting and manual laboratory operation, or high-throughput automated system) phenotypic AST platform for hospital/clinic use.

Novel keto-alkyl-pyridinium antifungal molecules active in models of in vivo Candida albicans vascular catheter infection and ex vivo Candida auris skin colonization.

New antifungal therapies are needed for both systemic, invasive infections as well as superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to non-systemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2-16 µg/mL) against medically important yeasts and moulds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus . Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce C. albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial pre-clinical data suggest that molecules of this class may warrant further study and development.

Reinforcement amid genetic diversity in the Candida albicans biofilm regulatory network.

Biofilms of the fungal pathogen Candida albicans include abundant long filaments called hyphae. These cells express hypha-associated genes, which specify diverse virulence functions including surface adhesins that ensure biofilm integrity. Biofilm formation, virulence, and hypha-associated gene expression all depend upon the transcription factor Efg1. This transcription factor has been characterized extensively in the C. albicans type strain SC5314 and derivatives, but only recently has its function been explored in other clinical isolates. Here we define a principal set of Efg1-responsive genes whose expression is significantly altered by an efg1Δ/Δ mutation across 17 clinical isolates. This principal gene set includes 68 direct Efg1 targets, whose 5' regions are bound by Efg1 in five clinical isolates, and 42 indirect Efg1 targets, whose 5' regions are not detectably bound by Efg1. Three direct Efg1 target genes encode transcription factors-BRG1, UME6, and WOR3 -whose increased expression in an efg1Δ/Δ mutant restores expression of multiple indirect and direct principal targets, as well as biofilm formation ability. Although BRG1 and UME6 are well known positive regulators of hypha-associated genes and biofilm formation, WOR3 is best known as an antagonist of Efg1 in the sexual mating pathway. We confirm the positive role of WOR3 in biofilm formation with the finding that a wor3Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo biofilm model. Positive control of Efg1 direct target genes by other Efg1 direct target genes-BRG1, UME6, and WOR3 -may buffer principal Efg1-responsive gene expression against the impact of genetic variation in the C. albicans species.