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

ABSTRACT 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.

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Intravital Imaging of Candida albicans Identifies Differential In Vitro and In Vivo Filamentation Phenotypes for Transcription Factor Deletion Mutants

mSphere ◽

10.1128/msphere.00436-21 ◽

2021 ◽

Author(s):

Rohan S. Wakade ◽

Manning Huang ◽

Aaron P. Mitchell ◽

Melanie Wellington ◽

Damian J. Krysan

Keyword(s):

Candida Albicans ◽

Fungal Infections ◽

Deletion Mutants ◽

Filamentous Form ◽

Content Type ◽

Yeast Form ◽

Common Causes ◽

Do So

Candida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection, which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited.

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Validation and Application of a Novel Target-Based Whole-Cell Screen to Identify Antifungal Compounds

10.21007/etd.cghs.2020.0518 ◽

2020 ◽

Author(s):

◽

Christian DeJarnette ◽

Keyword(s):

Candida Albicans ◽

Fatty Acid ◽

Small Molecules ◽

Dihydrofolate Reductase ◽

Biosynthetic Pathway ◽

Fatty Acid Synthase ◽

Fatty Acid Desaturase ◽

Fungal Growth ◽

Whole Cell ◽

Traditional Approaches

Traditional approaches to drug discovery are inefficient and have several key limitations that constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically-active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein were constructed, tagged with spectrally distinct fluorescent proteins (FPs), and mixed together. The pooled strains were then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture was compared by measuring the intensity of the corresponding FP tags. Chemically-induced population shifts indicated that the bioactivity of a small molecule was dependent upon the target protein’s abundance and thus established a functional interaction. Here, we described the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validated this approach using dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to an extensive array of molecular targets and in any genetically tractable microbe. 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, 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), 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 DFR1 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. Both the fungal fatty acid synthase and desaturase have been established as essential for fungal growth, virulence in a systemic infection, and are structurally distinct from their mammalian counterparts. However, there has been little success in developing clinically relevant inhibitors that target the fungal fatty acid biosynthetic pathway, even though there are agents that inhibit the bacterial orthologs. The purpose of this study was to identify physiologically-active compounds that interact with the fungal fatty acid biosynthetic pathway. Given the challenging nature of targeting these enzymes using traditional approaches, we attempted to apply the newly validated TAFiS method, a target-based whole-cell screen. To accomplish this, Candida albicans strains with altered expression of the fungal fatty acid synthase or the desaturase were used to screen a total of 41,261 small molecules. The primary hits were confirmed through dose responses, and 16 compounds had differential activity on the strains with altered desaturase abundance, indicating a functional chemical-target interaction. Additionally, the growth inhibition exerted by the most potent hits was restored in the presence of exogenous fatty acids, further supporting their inhibition of the fatty acid desaturase. Of the 16 confirmed hits, 11 were structurally related, containing a hydrazide core that was essential for interaction with the desaturase. Collectively, these results have identified a novel scaffold that interacts with the fungal fatty acid desaturase and can be further developed into experimental therapeutics. It also confirms that the TAFiS methodology is highly effective in identifying small molecules that functionally interact with targets that are poorly suited to traditional chemical screening approaches.

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Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions

mSphere ◽

10.1128/msphere.00393-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Franziska Gerwien ◽

Christine Dunker ◽

Philipp Brandt ◽

Enrico Garbe ◽

Ilse D. Jacobsen ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Infections ◽

Laboratory Strain ◽

Infection Process ◽

Vulvovaginal Candidiasis ◽

Host Immune System ◽

Host Pathogen Interactions ◽

Content Type ◽

Host Pathogen

ABSTRACT Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced β-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient. IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

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A Candida albicans Strain Expressing Mammalian Interleukin-17A Results in Early Control of Fungal Growth during Disseminated Infection

Infection and Immunity ◽

10.1128/iai.03057-14 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3684-3692 ◽

Cited By ~ 2

Author(s):

Anna R. Huppler ◽

Natasha Whibley ◽

Carol A. Woolford ◽

Erin E. Childs ◽

Jie He ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Growth ◽

Antibiotic Use ◽

Immune Defense ◽

Indwelling Catheters ◽

Content Type ◽

Immune Parameters ◽

Interleukin 17A

Candida albicansis normally a commensal fungus of the human mucosae and skin, but it causes life-threatening systemic infections in hospital settings in the face of predisposing conditions, such as indwelling catheters, abdominal surgery, or antibiotic use. Immunity toC. albicansinvolves various immune parameters, but the cytokine interleukin-17A (IL-17A) (also known as IL-17) has emerged as a centrally important mediator of immune defense against both mucosal and systemic candidiasis. Conversely, IL-17A has been suggested to enhance the virulence ofC. albicans, indicating that it may exert detrimental effects on pathogenesis. In this study, we hypothesized that aC. albicansstrain expressing IL-17A would exhibit reduced virulencein vivo. To that end, we created aCandida-optimized expression cassette encoding murine IL-17A, which was transformed into the DAY286 strain ofC. albicans.Candida-derived IL-17A was indistinguishable from murine IL-17A in terms of biological activity and detection in standard enzyme-linked immunosorbent assays (ELISAs). Expression of IL-17A did not negatively impact the growth of these strainsin vitro. Moreover, the IL-17A-expressingC. albicansstrains showed significantly reduced pathogenicity in a systemic model ofCandidainfection, mainly evident during the early stages of disease. Collectively, these findings suggest that IL-17A mitigates the virulence ofC. albicans.

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Nitric Oxide-Releasing Macromolecule Exhibits Broad-Spectrum Antifungal Activity and Utility as a Topical Treatment for Superficial Fungal Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01026-17 ◽

2018 ◽

Vol 62 (7) ◽

Cited By ~ 6

Author(s):

Nathan Stasko ◽

Kimberly McHale ◽

Stanley J. Hollenbach ◽

Megan Martin ◽

Ryan Doxey

Keyword(s):

Nitric Oxide ◽

Fungal Infection ◽

Topical Application ◽

Fungal Infections ◽

Drug Product ◽

Nail Plate ◽

Fungal Growth ◽

Content Type ◽

Nitric Oxide Releasing

ABSTRACTCutaneous and superficial fungal infections affecting the skin, nails, and hair of humans are caused primarily by dermatophytes of the generaTrichophytonandEpidermophytonor by yeasts of the generaCandidaandMalassezia.Onychomycosis is a common fungal infection of the nail that frequently coexists with tinea pedis, the most prevalent mycotic skin infection. Efficacy rates for current topical onychomycosis therapies are hampered by low drug penetration across the nail plate, which is theoretically obviated with nitric oxide (NO)-based topical therapies. The Nitricil technology platform is comprised of polysiloxane-based macromolecules that stably release therapeutic levels of NO. In the reported studies, NVN1000, the lead candidate of the platform, was assessed for its spectrum ofin vitroactivity against a broad range of filamentous fungi and yeast species commonly associated with cutaneous fungal infections. Time-kill assays demonstrated that NVN1000 exhibited fungicidal activity as early as 4 h. Additionally, the penetration of several unique NVN1000 NO-releasing drug product formulations (gel, cream, and lacquer) was evaluated following a single topical application in anin vitroinfected human nail assay, with all formulations showing similar inhibition of fungal growth. Repeated topical application in this model demonstrated that a lower-strength dose of NO could achieve the same efficacy as a higher-strength dose after 7 days. Together, thesein vitroresults demonstrate that NO-releasing treatments rapidly penetrate the nail plate and eradicate the fungal infection, representing promising novel topical therapies for the treatment of onychomycosis and other cutaneous fungal infections.

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An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00084-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 6

Author(s):

Brian M. Peters ◽

Arturo Luna-Tapia ◽

Hélène Tournu ◽

Jeffrey M. Rybak ◽

P. David Rogers ◽

...

Keyword(s):

Candida Albicans ◽

Mouse Model ◽

Vaginal Candidiasis ◽

Azole Resistance ◽

Endosomal Trafficking ◽

Wild Type ◽

Content Type ◽

Azole Antifungals

ABSTRACT We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

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In Vitro Interactions between Tacrolimus and Azoles against Candida albicans Determined by Different Methods

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01070-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 409-417 ◽

Cited By ~ 77

Author(s):

Shujuan Sun ◽

Yan Li ◽

Qiongjie Guo ◽

Changwen Shi ◽

Jinlong Yu ◽

...

Keyword(s):

Candida Albicans ◽

Combination Therapy ◽

Fungal Infections ◽

Fungal Growth ◽

Detection Methods ◽

Positive Interactions ◽

Data Generation ◽

Nonparametric Models ◽

In Vitro Interactions

ABSTRACT Combination therapy could be of use for the treatment of fungal infections, especially those caused by drug-resistant fungi. However, the methods and approaches used for data generation and result interpretation need further optimizing. The fractional inhibitory concentration index (FICI) is the most commonly used method, but it has several drawbacks in characterizing antifungal drug interaction. Alternatively, some new methods can be used such as the ΔE model (difference between the predicted and measured fungal growth percentages) and the response surface approach, which uses the concentration-effect relationship over the whole concentration range instead of just the MIC. In the present study, in vitro interactions between tacrolimus (FK506) and three azoles—fluconazole (FLC), itraconazole (ITR), and voriconazole (VRC)-against Candida albicans were evaluated by the checkerboard microdilution method and time-killing test. The intensity of the interactions was determined by visual reading and the spectrophotometric method in a checkerboard assay, and the nature of the interactions was assessed by nonparametric models of FICI and ΔE. Colony counting and colorimetric viable detection methods (2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium hydroxide} [XTT] reduction test) were used for evaluating the combination antifungal effects over time. Synergistic and indifferent effects were found for the combination of FK506 and azoles against azole-sensitive strains, while strong synergy was found against azole-resistant strains analyzed by FICI. The ΔE model gave more consistent results with FICI. The positive interactions were also confirmed by the time-killing test. Our findings suggest a potential role for combination therapy with calcineurin pathway inhibitors and azoles to augment activity against resistant C. albicans.

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Iron Chelator Deferasirox Reduces Candida albicans Invasion of Oral Epithelial Cells and Infection Levels in Murine Oropharyngeal Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02152-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 6

Author(s):

Sumant Puri ◽

Rohitashw Kumar ◽

Isolde G. Rojas ◽

Ornella Salvatori ◽

Mira Edgerton

Keyword(s):

Candida Albicans ◽

Treatment Options ◽

Fungal Growth ◽

Bloodstream Infections ◽

Preventive Treatment ◽

Iron Chelator ◽

Oropharyngeal Candidiasis ◽

Content Type ◽

Oral Epithelial

ABSTRACT Candida albicans, the causative agent of mucosal infections, including oropharyngeal candidiasis (OPC), as well as bloodstream infections, is becoming increasingly resistant to existing treatment options. In the absence of novel drug candidates, drug repurposing aimed at using existing drugs to treat off-label diseases is a promising strategy. C. albicans requires environmental iron for survival and virulence, while host nutritional immunity deploys iron-binding proteins to sequester iron and reduce fungal growth. Here we evaluated the role of iron limitation using deferasirox (an FDA-approved iron chelator for the treatment of patients with iron overload) during murine OPC and assessed deferasirox-treated C. albicans for its interaction with human oral epithelial (OE) cells, neutrophils, and antimicrobial peptides. Therapeutic deferasirox treatment significantly reduced salivary iron levels, while a nonsignificant reduction in the fungal burden was observed. Preventive treatment that allowed for two additional days of drug administration in our murine model resulted in a significant reduction in the number of C. albicans CFU per gram of tongue tissue, a significant reduction in salivary iron levels, and significantly reduced neutrophil-mediated inflammation. C. albicans cells harvested from the tongues of animals undergoing preventive treatment had the differential expression of 106 genes, including those involved in iron metabolism, adhesion, and the response to host innate immunity. Moreover, deferasirox-treated C. albicans cells had a 2-fold reduction in survival in neutrophil phagosomes (with greater susceptibility to oxidative stress) and reduced adhesion to and invasion of OE cells in vitro. Thus, deferasirox treatment has the potential to alleviate OPC by affecting C. albicans gene expression and reducing virulence.

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A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02510-17 ◽

2018 ◽

Vol 62 (6) ◽

Cited By ~ 2

Author(s):

Paris Laskaris ◽

Rocío Vicentefranqueira ◽

Olivier Helynck ◽

Grégory Jouvion ◽

José Antonio Calera ◽

...

Keyword(s):

Fungal Infections ◽

Treatment Options ◽

Fungal Growth ◽

Opportunistic Pathogen ◽

Zinc Metabolism ◽

Pulmonary Aspergillosis ◽

Hyphal Length ◽

Content Type

ABSTRACT Aspergillus fumigatus can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an A. fumigatus wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, and two were polyaminocarboxylates. All three groups showed good in vitro activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with in vivo fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy.

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Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

Eukaryotic Cell ◽

10.1128/ec.00067-15 ◽

2015 ◽

Vol 14 (8) ◽

pp. 834-844 ◽

Cited By ~ 32

Author(s):

Ranjith Rajendran ◽

Elisa Borghi ◽

Monica Falleni ◽

Federica Perdoni ◽

Delfina Tosi ◽

...

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Fungal Infections ◽

Infection Model ◽

Content Type

ABSTRACT Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo . In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

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