Candida albicans Forms a Specialized “Sexual” as Well as “Pathogenic” Biofilm

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO₂ and at 37°C. Mating frequencies are negligible in seeded patches of both a/α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or α/α) white cells form a specialized “sexual biofilm.”     

Biofilm Formation by and Antifungal Susceptibility of Candida Isolates from Urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC₅₀ of <1 μg/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

The White Cell Response to Pheromone Is a General Characteristic of Candida albicans Strains

For Candida albicans, evidence has suggested that the mating pheromones activate not only the mating response in mating-competent opaque cells but also a unique response in mating-incompetent white cells that includes increased cohesion and adhesion, enhanced biofilm formation, and expression of select mating-related and white cell-specific genes. On the basis of a recent microarray analysis comparing changes in the global expression patterns of white cells in two strains in response to α-pheromone, however, skepticism concerning the validity and generality of the white cell response has been voiced. Here, we present evidence that the response occurs in all tested media (Lee's, RPMI, SpiderM, yeast extract-peptone-dextrose, and a synthetic medium) and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone is therefore a general characteristic of MTL-homozygous strains of C. albicans.     

Biofilm formation by Candida albicans on various prosthetic materials and its fluconazole sensitivity: a kinetic study

Candida albicans has the ability to colonize various materials used in prostheses. In this report, we have studied the kinetics of biofilm formation on prosthetic materials and their susceptibility to fluconazole at various stages of development. Results indicated that C. albicans efficiently adheres to and colonizes polystyrene, polyvinylchloride, silicon, and polycarbonate surfaces. Candida albicans biofilm formation was observed to be both strain- and substrate dependent. Adhesion of cells to solid substrates was found sufficient to induce fluconazole resistance. Drug susceptibility at different stages of biofilm growth showed that Candida biofilms on these substrates are highly resistant to fluconazole. The study focuses on the limitations of fluconazole to combat biofilm-related infections and emphasizes the need for better therapeutic strategies against prosthesis-associated C. albicans infections.     

Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMl 1640 (RPMI) at 37°C and 5% CO₂ causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO₂ and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resuming in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiaeα-agglutinin, encoded by AGα1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for a gglutinin-l ike s equence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AGα1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AGb1. These repeats are also present on the nucleotide level as a highly conserved 108bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and. In some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.     

Adaptations of the Secretome of Candida albicans in Response to Host-Related Environmental Conditions

The wall proteome and the secretome of the fungal pathogen Candida albicans help it to thrive in multiple niches of the human body. Mass spectrometry has allowed researchers to study the dynamics of both subproteomes. Here, we discuss some major responses of the secretome to host-related environmental conditions. Three β-1,3-glucan-modifying enzymes, Mp65, Sun41, and Tos1, are consistently found in large amounts in culture supernatants, suggesting that they are needed for construction and expansion of the cell wall β-1,3-glucan layer and thus correlate with growth and might serve as diagnostic biomarkers. The genes ENG1, CHT3, and SCW11, which encode an endoglucanase, the major chitinase, and a β-1,3-glucan-modifying enzyme, respectively, are periodically expressed and peak in M/G₁. The corresponding protein abundances in the medium correlate with the degree of cell separation during single-yeast-cell, pseudohyphal, and hyphal growth. We also discuss the observation that cells treated with fluconazole, or other agents causing cell surface stress, form pseudohyphal aggregates. Fluconazole-treated cells secrete abundant amounts of the transglucosylase Phr1, which is involved in the accumulation of β-1,3-glucan in biofilms, raising the question whether this is a general response to cell surface stress. Other abundant secretome proteins also contribute to biofilm formation, emphasizing the important role of secretome proteins in this mode of growth. Finally, we discuss the relevance of these observations to therapeutic intervention. Together, these data illustrate that C. albicans actively adapts its secretome to environmental conditions, thus promoting its survival in widely divergent niches of the human body.     

The <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> efficacy of fluconazole in combination with farnesol against <Emphasis Type="Italic">Candida albicans</Emphasis> isolates using a murine vulvovaginitis model

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.     

Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A

Background

Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms.

Methods

Combinations of five antifungal drugs- fluconazole (FLC), voriconazole (VOR), caspofungin (CSP), amphotericin B (AmB) and nystatin (NYT) with cyclosporine A (CSA) were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI) of combination effects. Biofilm growth was analyzed using XTT-metabolic assay.

Results

MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively.

Conclusions

The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.     

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.     

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

Inhibition of Candida albicans Biofilm Formation by Farnesol, a Quorum-Sensing Molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Design of a Simple Model of <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms Formed under Conditions of Flow: Development,Architecture, and Drug Resistance

Candida albicans biofilms on most medical devices are exposed to a flow of body fluids that provide water and nutrients to the fungal cells. While C. albicans biofilms grown in vitro under static conditions have been exhaustively studied, the same is not true for biofilms developed under continuous flow of replenishing nutrients. Here, we describe a simple flow biofilm (FB) model that can be built easily with materials commonly available in most microbiological laboratories. We demonstrate that C. albicans biofilms formed using this flow system show increased architectural complexity compared to biofilms grown under static conditions. C. albicans biofilms under continuous medium flow grow rapidly, and by 8 h show characteristics similar to 24 h statically grown biofilms. Biomass measurements and microscopic observations further revealed that after 24 h of incubation, FB was more than twofold thicker than biofilms grown under static conditions. Microscopic analyses revealed that the surface of these biofilms was extremely compact and wrinkled, unlike the open hyphal layer typically seen in 24 h static biofilms. Results of antifungal drug susceptibility tests showed that C. albicans cells in FB exhibited increased resistance to most clinically used antifungal agents.     

Effect of Tetrandrine against Candida albicans Biofilms

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

220D-F2 from Rubus ulmifolius Kills Streptococcus pneumoniae Planktonic Cells and Pneumococcal Biofilms

Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistence allows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that kill nearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilm structures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to most commonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed the antimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniae planktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktonic cultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterial counts and a LIVE/DEAD bacterial viability assay, 100 and 200 µg/ml of 220D-F2 had significant bactericidal activity against pneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC’s, whether quantified by qPCR or dilution and plating, showed that 80 µg/ml of 220D-F2 completely eradicated overnight cultures of planktonic pneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration (MBIC)₅₀ was obtained incubating biofilms with 100 µg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 also significantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstrate potential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcal biofilms.     

Nonsex Genes in the Mating Type Locus of Candida albicans Play Roles in a/α Biofilm Formation,Including Impermeability and Fluconazole Resistance

The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTL a1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated “nonsex” genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.     

Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

Background

Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models.

Methodology/Principal Findings

The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E₂. In mono-microbial and dual biofilms of C.albicans wild type strains PGE₂ levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE₂ significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE₂ production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE₂ but amounts were significantly lower compared to SC5314.

Conclusion/Significance

In conclision, we identified C. albicans PGE₂ as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.     

Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions

Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O₂. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.