The atypical antipsychotic aripiprazole alters the outcome of disseminated Candida albicans infections.

Invasive fungal infections impose an enormous clinical, social, and economic burden on humankind. One of the most common species responsible for invasive fungal infections is Candida albicans. More than 30% of patients with disseminated candidiasis fail therapy with existing antifungal drugs, including the widely used azole class. We previously identified a collection of 13 medications that antagonize the activity of the azoles on C. albicans. Although gain-of-function mutations responsible for antifungal resistance are often associated with reduced fitness and virulence, it is currently unknown how exposure to azole antagonistic drugs impacts C. albicans physiology, fitness, or virulence. In this study, we examined how exposure to seven azole antagonists affects C. albicans phenotype and capacity to cause disease. Most of the azole antagonists appear to have little impact on fungal growth, morphology, stress tolerance, or gene transcription. However, aripiprazole had a modest impact on C. albicans hyphal growth and increased cell wall chitin content. It also aggravated the disseminated C. albicans infections in mice. This effect was abrogated in immunosuppressed mice, indicating that it is at least in part dependent upon host immune responses. Collectively, these data provide proof of principle that unanticipated drug-fungus interactions have the potential to influence the incidence and outcomes of invasive fungal disease.

Biophysical library screening using a Thermo-FMN assay to identify and characterize Clostridioides difficile FabK inhibitors.

Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism's fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme's FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.

The atypical antipsychotic aripiprazole alters the outcome of disseminated Candida albicans infections.

Invasive fungal infections (IFIs) impose an enormous clinical, social, and economic burden on humankind. For many IFIs, ≥ 30% of patients fail therapy with existing antifungal drugs, including the widely used azole class. We previously identified a collection of 13 approved medications that antagonize azole activity. While gain-of-function mutants resulting in antifungal resistance are often associated with reduced fitness and virulence, it is currently unknown how exposure to azole antagonistic drugs impact C. albicans physiology, fitness, or virulence. In this study, we examined how exposure to azole antagonists affected C. albicans phenotype and capacity to cause disease. We discovered that most of the azole antagonists had little impact on fungal growth, morphology, stress tolerance, or gene transcription. However, aripiprazole had a modest impact on C. albicans hyphal growth and increased cell wall chitin content. It also worsened the outcome of disseminated infections in mice at human equivalent concentrations. This effect was abrogated in immunosuppressed mice, indicating an additional impact of aripiprazole on host immunity. Collectively, these data provide proof-of-principle that unanticipated drug-fungus interactions have the potential to influence the incidence and outcomes of invasive fungal disease.

Glycogen Metabolism in Candida albicans Impacts Fitness and Virulence during Vulvovaginal and Invasive Candidiasis.

The polymorphic fungus Candida albicans remains a leading cause of both invasive and superficial mycoses, including vulvovaginal candidiasis (VVC). Metabolic plasticity, including carbohydrate catabolism, confers fitness advantages at anatomical site-specific host niches. C. albicans possesses the capacity to accumulate and store carbohydrates as glycogen and can consume intracellular glycogen stores when nutrients become limited. In the vaginal environment, estrogen promotes epithelial glycogen accumulation and C. albicans colonization. However, whether these factors are mechanistically linked is unexplored. Here, we characterized the glycogen metabolism pathways in C. albicans and investigated whether these impact the long-term survival of C. albicans, both in vitro and in vivo during murine VVC, or virulence during systemic infection. SC5314 and 6 clinical isolates demonstrated impaired growth when glycogen was used as the sole carbon source, suggesting that environmental glycogen acquisition is limited. The genetic deletion and complementation of key genes involved in glycogen metabolism in Saccharomyces cerevisiae confirmed that GSY1 and GLC3, as well as GPH1 and GDB1, are essential for glycogen synthesis and catabolism in C. albicans, respectively. Potential compensatory roles for a glucoamylase encoded by SGA1 were also explored. Competitive survival assays revealed that gsy1Δ/Δ, gph1Δ/Δ, and gph1Δ/Δ sga1Δ/Δ mutants exhibited long-term survival defects in vitro under starvation conditions and in vivo during vaginal colonization. A complete inability to catabolize glycogen (gph1Δ/Δ sga1Δ/Δ) also rendered C. albicans significantly less virulent during disseminated infections. This is the first study fully validating the glycogen metabolism pathways in C. albicans, and the results further suggest that intracellular glycogen catabolism positively impacts the long-term fitness of C. albicans in nutrient deficient environments and is important for full virulence. IMPORTANCE Glycogen is a highly branched polymer of glucose and is used across the tree of life as an efficient and compact form of energy storage. Whereas glycogen metabolism pathways have been studied in model yeasts, they have not been extensively explored in pathogenic fungi. Using a combination of microbiologic, molecular genetic, and biochemical approaches, we reveal orthologous functions of glycogen metabolism genes in the fungal pathogen Candida albicans. We also provide evidence that extracellular glycogen poorly supports growth across the Candida species and clinical isolates. Competitive fitness assays reveal that the loss of glycogen synthesis or catabolism significantly impacts survival during both in vitro starvation and the colonization of the mouse vagina. Moreover, a global glycogen catabolism mutant is rendered less virulent during murine invasive candidiasis. Therefore, this work demonstrates that glycogen metabolism in C. albicans contributes to survival and virulence in the mammalian host and may be a novel antifungal target.

Single-item depression screening with the Neurobehavioral Symptom Inventory (NSI): Do scores assist clinicians with predicting need for mental health interventions?

The purpose of this study was to examine the relationship between a single item for depression from the Neurobehavioral Symptom Inventory (NSI) and a common depression screening measure to predict need for further mental health consultation for veterans with traumatic brain injury (TBI). Three hundred eighty veterans referred to a Veterans Affairs Health Care System TBI clinic for evaluation were administered the NSI and a common depression screening measure (Beck Depression Inventory-Second Edition; BDI-II). Receiver Operating Characteristic (ROC) curve analyses were conducted to determine best cutoff scores on the BDI-II corresponding with a single item of the NSI item pertaining to depression (i.e., "depressed or sad"). Using multiclass ROC curve analyses, results suggested that a minimum score of 3 (severe) on the specific NSI item indicated need for further mental health referral without warranting additional screening for depression. Analyses further demonstrated that removal of invalid NSI protocols did not significantly change ROC curve findings. Therefore, the NSI item for depression can still be used for making clinical decisions despite the protocol being considered otherwise invalid. Implications for treatment and recommendations for screening are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Identifying Specific Small Molecule-Protein Interactions Using Target Abundance-Based Fitness Screening (TAFiS).

Traditional small molecule antifungal discovery efforts often utilize high-throughput (HTP) biochemical or whole-cell phenotypic screens to identify novel candidates. However, both methods have limitations which hinder the rapid identification of physiologically active compounds that act via a defined mechanism of action. The method described herein is an efficient, sensitive, and HTP compatible approach that utilizes the principles of competitive fitness to rapidly identify small molecules that functionally interact with a specific target protein within whole cells.

Titration of C-5 Sterol Desaturase Activity Reveals Its Relationship to Candida albicans Virulence and Antifungal Susceptibility Is Dependent upon Host Immune Status.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), which depletes cellular ergosterol and promotes synthesis of the dysfunctional lipid 14α-methylergosta-8,24(28)-dien-3ß,6α-diol, ultimately arresting growth. Mutations that inactivate sterol Δ5,6-desaturase (Erg3p), the enzyme that produces the sterol-diol upon S14DM inhibition, enhances Candida albicans growth in the presence of the azoles. However, erg3 null mutants are sensitive to some physiological stresses and can be less virulent than the wild type. These fitness defects may disfavor the selection of null mutants within patients. The objective of this study was to investigate the relationship between Erg3p activity, C. albicans pathogenicity, and the efficacy of azole therapy. An isogenic panel of strains was constructed that produce various levels of the ERG3 transcript. Analysis of the sterol composition confirmed a correspondingly wide range of Erg3p activity. Phenotypic analysis revealed that even moderate reductions in Erg3p activity are sufficient to greatly enhance C. albicans growth in the presence of fluconazole in vitro without impacting fitness. Moreover, even low levels of Erg3p activity are sufficient to support full virulence of C. albicans in the mouse model of disseminated infection. Finally, while the antifungal efficacy of fluconazole was similar for all strains in immunocompetent mice, there was an inverse correlation between Erg3p activity and the capacity of C. albicans to endure treatment in leukopenic mice. Collectively, these results establish that relative levels of Erg3p activity determine the antifungal efficacy of the azoles upon C. albicans and reveal the critical importance of host immunity in determining the clinical impact of this resistance mechanism. IMPORTANCE Mutations that completely inactivate Erg3p enable the prevalent human pathogen C. albicans to endure the azole antifungals in vitro. However, such null mutants are less frequently identified in azole-resistant clinical isolates than other resistance mechanisms, and previous studies have reported conflicting outcomes regarding antifungal resistance of these mutants in animal models of infection. The results of this study clearly establish a direct correlation between the level of Erg3p activity and the antifungal efficacy of fluconazole within a susceptible mammalian host. In addition, low levels of Erg3p activity are apparently more advantageous for C. albicans survival of azole therapy than complete loss of function. These findings suggest a more nuanced but more important role for Erg3p as a determinant of the clinical efficacy of the azole antifungals than previously appreciated. A revised model of the relationship between Erg3p activity, host immunity, and the antifungal susceptibility of C. albicans is proposed.

Anxiety Screening in Polytrauma Patients by Use of Single-Item Reporting With the Neurobehavioral Symptom Inventory: How Brief Is Too Brief?

PURPOSE/OBJECTIVE: This study examined the clinical utility of a single item for anxiety from the Neurobehavioral Symptom Inventory (NSI) in determining the need for mental health referral for veterans with traumatic brain injury (TBI). RESEARCH METHOD/DESIGN: Three hundred eighty veterans referred for TBI evaluation were administered the NSI and a common anxiety screening measure (Beck Anxiety Inventory; BAI). Receiver Operating Characteristic (ROC) curve analyses were conducted to determine ideal BAI total cutoff scores for a single item of the NSI pertaining to anxiety (i.e., "anxious or tense"). RESULTS: Using multiclass ROC curve analyses, NSI scores of 3 and 4 for the sample were comparable to scores of 11 and 22 on the BAI, respectively. Post hoc ROC curve analyses were then conducted on the sample after removal of potentially invalid NSI protocols (i.e., Validity-10 scores greater than 22), and NSI scores 3 and 4 corresponded with scores of 11 and 20, respectively. CONCLUSION/IMPLICATIONS: A minimum score of 3 (severe) on the NSI item was deemed sufficient to indicate the need for further mental health referral without warranting additional screening for anxiety. Further analyses also revealed that removal of positive Validity-10 protocols did not significantly change ROC curve findings, suggesting that the particular NSI item for anxiety can still be used for clinical purposes despite an otherwise invalid protocol. Implications for treatment and recommendations pertaining to when additional screening might be required are discussed.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Identification of Inhibitors of Fungal Fatty Acid Biosynthesis.

Fungal fatty acid (FA) synthase and desaturase enzymes are essential for the growth and virulence of human fungal pathogens. These enzymes are structurally distinct from their mammalian counterparts, making them attractive targets for antifungal development. However, there has been little progress in identifying chemotypes that target fungal FA biosynthesis. To accomplish this, we applied a whole-cell-based method known as Target Abundance-based FItness Screening using Candida albicans. Strains with varying levels of FA synthase or desaturase expression were grown in competition to screen a custom small-molecule library. Hit compounds were defined as preferentially inhibiting the growth of the low target-expressing strains. Dose-response experiments confirmed that 16 hits (11 with an acyl hydrazide core) differentially inhibited the growth of strains with an altered desaturase expression, indicating a specific chemical-target interaction. Exogenous unsaturated FAs restored C. albicans growth in the presence of inhibitory concentrations of the most potent acyl hydrazides, further supporting the primary mechanism being inhibition of FA desaturase. A systematic analysis of the structure-activity relationship confirmed the acyl hydrazide core as essential for inhibitory activity. This collection demonstrated broad-spectrum activity against Candida auris and mucormycetes and retained the activity against azole-resistant candida isolates. Finally, a preliminary analysis of toxicity to mammalian cells identified potential lead compounds with desirable selectivities. Collectively, these results establish a scaffold that targets fungal FA biosynthesis with a potential for development into novel therapeutics.

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Species-Specific Differences in C-5 Sterol Desaturase Function Influence the Outcome of Azole Antifungal Exposure.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol diol that disrupts membrane function. In Candida albicans, sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3. Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensities of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans (CaERG3), Candida glabrata (CgERG3), Candida auris (CaurERG3), Cryptococcus neoformans (CnERG3), Aspergillus fumigatus (AfERG3A-C) and Rhizopus delemar (RdERG3A/B) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored C-5 sterol desaturase activity and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant, confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the MIC and residual growth observed at supra-MICs. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced various levels of 14α-methylergosta-8,24(28)-dien-3ß,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C-5 sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans.

While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase (DHFR), the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1 strain), we were able to demonstrate that Dfr1p is essential for growth in vitro Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when Dfr1p expression is suppressed or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase and, by inference, other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.IMPORTANCE The folate biosynthetic pathway is a promising and understudied source for novel antifungals. Even dihydrofolate reductase (DHFR), a well-characterized and historically important drug target, has not been conclusively validated as an antifungal target. Here, we demonstrate that repression of DHFR inhibits growth of Candida albicans, a major human fungal pathogen. Methotrexate, an antifolate, also inhibits growth but through pH-dependent activity. In addition, we show that C. albicans has a limited ability to take up or utilize exogenous folates as only the addition of high concentrations of folinic acid restored growth in the presence of methotrexate. Finally, we show that repression of DHFR in a mouse model of infection was sufficient to eliminate host mortality. Our work conclusively establishes DHFR as a valid antifungal target in C. albicans.

An Unbiased Drug Screen for Seizure Suppressors in Duplication 15q Syndrome Reveals 5-HT1A and Dopamine Pathway Activation as Potential Therapies.

BACKGROUND: Duplication 15q (Dup15q) syndrome is a rare neurogenetic disorder characterized by autism and pharmacoresistant epilepsy. Most individuals with isodicentric duplications have been on multiple medications to control seizures. We recently developed a model of Dup15q in Drosophila by elevating levels of fly Dube3a in glial cells using repo-GAL4, not neurons. In contrast to other Dup15q models, these flies develop seizures that worsen with age. METHODS: We screened repo>Dube3a flies for approved compounds that can suppress seizures. Flies 3 to 5 days old were exposed to compounds in the fly food during development. Flies were tested using a bang sensitivity assay for seizure recovery time. At least 40 animals were tested per experiment, with separate testing for male and female flies. Studies of K+ content in glial cells of the fly brain were also performed using a fluorescent K+ indicator. RESULTS: We identified 17 of 1280 compounds in the Prestwick Chemical Library that could suppress seizures. Eight compounds were validated in secondary screening. Four of these compounds regulated either serotonergic or dopaminergic signaling, and subsequent experiments confirmed that seizure suppression occurred primarily through stimulation of serotonin receptor 5-HT1A. Additional studies of K+ levels showed that Dube3a regulation of the Na+/K+ exchanger ATPα (adenosine triphosphatase α) in glia may be modulated by serotonin/dopamine signaling, causing seizure suppression. CONCLUSIONS: Based on these pharmacological and genetic studies, we present an argument for the use of 5-HT1A agonists in the treatment of Dup15q epilepsy.

Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris.

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. aurisERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Remasking of Candida albicans ß-Glucan in Response to Environmental pH Is Regulated by Quorum Sensing.

Candida albicans is a commensal yeast of the human gut which is tolerated by the immune system but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host-derived environment cues (pH, hypoxia, and lactate). This cell wall remodeling allows C. albicans to evade or hyperactivate the host's innate immune responses, leading to disease. Previously, we showed that adaptation of C. albicans to acidic environments, conditions encountered during colonization of the female reproductive tract, induces significant cell wall remodeling resulting in the exposure of two key fungal PAMPs (ß-glucan and chitin). Here, we report that this pH-dependent cell wall remodeling is time dependent, with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking of ß-glucan was mediated via the cell density-dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, nonproteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2 This dynamic cell wall remodeling influenced innate immune recognition of C. albicans, suggesting that during infection, C. albicans can manipulate the host innate immune responses.IMPORTANCECandida albicans is part of the microbiota of the skin and gastrointestinal and reproductive tracts of humans and has coevolved with us for millennia. During that period, C. albicans has developed strategies to modulate the host's innate immune responses, by regulating the exposure of key epitopes on the fungal cell surface. Here, we report that exposing C. albicans to an acidic environment, similar to the one of the stomach or vagina, increases the detection of the yeast by macrophages. However, this effect is transitory, as C. albicans is able to remask these epitopes (glucan and chitin). We found that glucan remasking is controlled by the production of farnesol, a molecule secreted by C. albicans in response to high cell densities. However, chitin-remasking mechanisms remain to be identified. By understanding the relationship between environmental sensing and modulation of the host-pathogen interaction, new opportunities for the development of innovative antifungal strategies are possible.

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.

A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein's expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5' end of the ARO1 gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicansIMPORTANCE Investigating a protein's functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach-the insertion of poly-A repeats within the coding sequence of the gene-is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique ß-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the ß-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of ß-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

A Systematic Screen Reveals a Diverse Collection of Medications That Induce Antifungal Resistance in Candida Species.

The increasing incidence of and high mortality rates associated with invasive fungal infections (IFIs) impose an enormous clinical, social, and economic burden on humankind. In addition to microbiological resistance to existing antifungal drugs, the large number of unexplained treatment failures is a serious concern. Due to the extremely limited therapeutic options available, it is critical to identify and understand the various causes of treatment failure if patient outcomes are to improve. In this study, we examined one potential source of treatment failure: antagonistic drug interactions. Using a simple screen, we systematically identified currently approved medications that undermine the antifungal activity of three major antifungal drugs-fluconazole, caspofungin, and amphotericin B-on four prevalent human fungal pathogens-Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis This revealed that a diverse collection of structurally distinct drugs exhibit antagonistic interactions with fluconazole. Several antagonistic agents selected for follow-up studies induce azole resistance through a mechanism that depends on Tac1p/Pdr1p zinc-cluster transcription factors, which activate the expression of drug efflux pumps belonging to the ABC-type transporter family. Few antagonistic interactions were identified with caspofungin or amphotericin B, possibly reflecting their cell surface mode of action that should not be affected by drug efflux mechanisms. Given that patients at greatest risk of IFIs usually receive a multitude of drugs to treat various underlying conditions, these studies suggest that chemically inducible azole resistance may be much more common and important than previously realized.

A Comparison Between the Involuntary and Voluntary Treatment of Patients With Alcohol Use Disorder in a Residential Rehabilitation Treatment Program.

OBJECTIVE: The purpose of the study was to determine if there was a significant difference between veterans who received treatment voluntarily versus involuntarily in regard to length of sobriety. METHOD: A sample of 120 veterans being treated for alcohol use disorder in a residential rehabilitation treatment program was used for this study. Veterans who were admitted under recommendation by court order (n = 60) were matched with veterans who were admitted without recommendation of court order (n = 60). Success of the program was determined by the number of days of sobriety postdischarge. RESULTS: The study revealed that there was no significant difference between types of motivation for residential treatment (i.e., voluntary vs. involuntary treatment) and length of sobriety for veterans with alcohol use disorder posttreatment. CONCLUSIONS: Findings revealed that there was no significant relationship when comparing types of motivation for treatment in a residential treatment program for veterans in regard to length of sobriety posttreatment. Therefore, a veteran's motivation for treatment may not necessarily be an accurate indicator of treatment outcomes (i.e., length of sobriety posttreatment) for residential treatment settings.