Heterologous Expression of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal PathogensCandida albicansandCandida glabrataProvides Tools for Antifungal Discovery

ABSTRACTTargeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are modest for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed these problems by expressing inSaccharomyces cerevisiaefunctional, hexahistidine-tagged, full-lengthCandida albicansLDM (CaLDM6×His) andCandida glabrataLDM (CgLDM6×His) for drug discovery purposes and determining their X-ray crystal structures. Compared withS. cerevisiaeoverexpressing LDM6×His (ScLDM6×His), the reduced susceptibility of CgLDM6×His to all azole drugs tested correlated with its level of overexpression. In contrast, the reduced susceptibility to short-tailed (fluconazole and voriconazole) but not medium-tailed (VT-1161) or long-tailed azoles (itraconazole and posaconazole) indicates CaLDM6×His works best when coexpressed with its cognate NADPH-cytochrome P450 reductase (CaNcp1A) rather than the host reductase (ScNcp1). Overexpression of LDM or Ncp1 modified the ergosterol content of yeast and affected growth inhibition by the polyene antibiotic amphotericin B. Affinity-purified recombinantCandidaLDMs bind carbon monoxide and show tight type II binding of a range of azole drugs, including itraconazole, posaconazole, fluconazole, and voriconazole. This study provides a practical basis for the phenotype-, biochemistry-, and structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

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Crystal Structures of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01134-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 9

Author(s):

Mikhail V. Keniya ◽

Manya Sabherwal ◽

Rajni K. Wilson ◽

Matthew A. Woods ◽

Alia A. Sagatova ◽

...

Keyword(s):

Candida Albicans ◽

Crystal Structures ◽

Candida Glabrata ◽

Fungal Pathogens ◽

Catalytic Domain ◽

Fungal Infections ◽

Binding Pocket ◽

Full Length ◽

Content Type ◽

Cure Rates

ABSTRACT Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

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Production and characterization of Green Iron Oxide nanoparticles with antifungal properties against fungal pathogens

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11i2.2206 ◽

2020 ◽

Vol 11 (2) ◽

pp. 2308-2314

Author(s):

Momanyi Kerubo Rachael ◽

Rajiv P

Keyword(s):

Iron Oxide ◽

Green Chemistry ◽

Iron Oxide Nanoparticles ◽

Fungal Pathogens ◽

Crystal Surface ◽

Fungal Infections ◽

Alternative Methods ◽

Oxide Nanoparticles ◽

X Ray ◽

Antifungal Properties

In this study, iron oxide nanoparticles were synthesized throughthe green chemistry method. It involved the use of plant extract for synthesis of metal oxide nanomaterials. Aqueous extract of Tridaxprocumbenswas employed for the production of iron oxide nanoparticles. T. procumbens is a weed and it has good medicinal properties.The green chemistry approach provides a simple, cost-effective and eco-innovative method to the synthesis of nanomaterials as compared to the other alternative methods. Phase, crystal, surface chemistry, size, shape and element composition of nanoparticles were assessed and determined by various techniques like Fourier transform infrared spectroscopy (FTIR),X-Ray diffraction (XRD),Scanning Electron Microscopy (SEM) and Energy dispersion X-Ray (EDX). In addition, the antifungal properties of the biosynthesized nanoparticles were investigated against plant fungal pathogens. Spherical-shaped nanoparticles with a size of 26 ± 5 nm were obtained using the extract of T. procumbens. Bio-molecules from T. procumbens served as reducing, cappingand stabilizing agents for the fabrication of nanoparticles. The highest zone of inhibition was obtained in S. rolfsii(16.5 ± 0.5)at 50 µg/ml of concentration of TFeONPs. Biosynthesized TFeONPs were capable of inhibiting the growth of fungal pathogens such as S. rolfsii and F. oxysporumand they might beemployed as antifungal agents in agriculture to control fungal infections.

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A Heat-Killed Cryptococcus Mutant Strain Induces Host Protection against Multiple Invasive Mycoses in a Murine Vaccine Model

mBio ◽

10.1128/mbio.02145-19 ◽

2019 ◽

Vol 10 (6) ◽

Author(s):

Yina Wang ◽

Keyi Wang ◽

Jorge A. Masso-Silva ◽

Amariliz Rivera ◽

Chaoyang Xue

Keyword(s):

Immune Deficiency ◽

T Cells ◽

Fungal Pathogens ◽

Vaccine Candidate ◽

Fungal Infections ◽

Invasive Fungal Infections ◽

Host Immunity ◽

Content Type ◽

A Subunit ◽

F Box Protein

ABSTRACT Cryptococcus neoformans is a fungal pathogen that infects the lungs and then often disseminates to the central nervous system, causing meningitis. How Cryptococcus is able to suppress host immunity and escape the antifungal activity of macrophages remains incompletely understood. We reported that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase, promotes Cryptococcus virulence by regulating host-Cryptococcus interactions. Our recent studies demonstrated that the fbp1Δ mutant elicited superior protective Th1 host immunity in the lungs and that the enhanced immunogenicity of heat-killed fbp1Δ yeast cells can be harnessed to confer protection against a subsequent infection with the virulent parental strain. We therefore examined the use of heat-killed fbp1Δ cells in several vaccination strategies. Interestingly, the vaccine protection remains effective even in mice depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that vaccinating mice with heat-killed fbp1Δ induces significant cross-protection against challenge with diverse invasive fungal pathogens, including C. neoformans, C. gattii, and Aspergillus fumigatus, as well as partial protection against Candida albicans. Thus, our data suggest that the heat-killed fbp1Δ strain has the potential to be a suitable vaccine candidate against cryptococcosis and other invasive fungal infections in both immunocompetent and immunocompromised populations. IMPORTANCE Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans, elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations.

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Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02114-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 6

Author(s):

Brian C. Monk ◽

Mikhail V. Keniya ◽

Manya Sabherwal ◽

Rajni K. Wilson ◽

Danyon O. Graham ◽

...

Keyword(s):

Fungal Pathogens ◽

Efflux Pumps ◽

Drug Binding ◽

Full Length ◽

Major Facilitator Superfamily ◽

Azole Resistance ◽

Drug Efflux ◽

Content Type ◽

Drug Efflux Pumps ◽

The Impact

ABSTRACTTetrazole antifungals designed to target fungal lanosterol 14α-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain ofCandida albicansLDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-lengthSaccharomyces cerevisiaeLDM with a C-terminal hexahistidine tag (ScLDM6×His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates ofC. albicansandCandida glabrata. VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.

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Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis

Scientific Reports ◽

10.1038/s41598-019-56516-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Ana C. Ebrecht ◽

Naadia van der Bergh ◽

Susan T. L. Harrison ◽

Martha S. Smit ◽

B. Trevor Sewell ◽

...

Keyword(s):

Cytochrome P450 ◽

Cytochrome P450 Monooxygenases ◽

Oxygen Species ◽

Type Ii ◽

Cytochrome P450 Reductase ◽

Terminal Part ◽

X Ray ◽

P450 Monooxygenases ◽

Oxygen Complex ◽

Structural Insights

AbstractCytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.

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Toxoflavin Produced byBurkholderia gladiolifromLycoris aureaIs a New Broad-Spectrum Fungicide

Applied and Environmental Microbiology ◽

10.1128/aem.00106-19 ◽

2019 ◽

Vol 85 (9) ◽

Cited By ~ 7

Author(s):

Xiaodan Li ◽

Yikui Li ◽

Ren Wang ◽

Qizhi Wang ◽

Ling Lu

Keyword(s):

Antifungal Activity ◽

Broad Spectrum ◽

Fungal Pathogens ◽

Antifungal Agents ◽

Fungal Infections ◽

Endophytic Bacterium ◽

Content Type ◽

Burkholderia Gladioli ◽

Lycoris Aurea ◽

Resistant Mutants

ABSTRACTFungal infections not only cause extensive agricultural damage but also result in serious diseases in the immunodeficient populations of human beings. Moreover, the increasing emergence of drug resistance has led to a decrease in the efficacy of current antifungals. Thus, screening of new antifungal agents is imperative in the fight against antifungal drug resistance. In this study, we show that an endophytic bacterium,Burkholderia gladioliHDXY-02, isolated from the medicinal plantLycoris aurea, showed broad-spectrum antifungal activity against plant and human fungal pathogens. An antifungal ability assay indicated that the bioactive component was produced from strain HDXY-02 having an extracellular secreted component with a molecular weight lower than 1,000 Da. In addition, we found that this new antifungal could be produced effectively by liquid fermentation of HDXY-02. Furthermore, the purified component contributing to the antifungal activity was identified to be toxoflavin, a yellow compound possessing a pyrimido[5,4-e][1,2,4]triazine ring.In vitrobioactivity studies demonstrated that purified toxoflavin fromB. gladioliHDXY-02 cultures had a significant antifungal activity against the human fungal pathogenAspergillus fumigatus, resulting in abolished germination of conidia. More importantly, the growth inhibition by toxoflavin was observed in both wild-type and drug-resistant mutants (cyp51Aand non-cyp51A) ofA. fumigatus. Finally, an optimized protocol for the large-scale production of toxoflavin (1,533 mg/liter) has been developed. Taken together, our findings provide a promising biosynthetic resource for producing a new antifungal reagent, toxoflavin, from isolates of the endophytic bacteriumB. gladioli.IMPORTANCEHuman fungal infections are a growing problem associated with increased morbidity and mortality. Moreover, a growing number of antifungal-resistant fungal isolates have been reported over the past decade. Thus, the need for novel antifungal agents is imperative. In this study, we show that an endophytic bacterium,Burkholderia gladioli, isolated from the medicinal plantLycoris aurea, is able to abundantly secrete a compound, toxoflavin, which has a strong fungicidal activity not only against plant fungal pathogens but also against human fungal pathogensAspergillus fumigatusandCandida albicans,Cryptococcus neoformans, and the model filamentous fungusAspergillus nidulans. More importantly, toxoflavin also displays an efficacious inhibitory effect against azole antifungal-resistant mutants ofA. fumigatus. Consequently, our findings provide a promising approach to abundantly produce toxoflavin, which has novel broad-spectrum antifungal activity, especially against those currently problematic drug-resistant isolates.

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A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

mSphere ◽

10.1128/msphere.00002-19 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 16

Author(s):

Lauren Wensing ◽

Jehoshua Sharma ◽

Deeva Uthayakumar ◽

Yannic Proteau ◽

Alejandro Chavez ◽

...

Keyword(s):

Candida Albicans ◽

Human Disease ◽

Transcriptional Repression ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Fungal Infections ◽

Content Type ◽

Guide Rna ◽

Crispr Interference ◽

Functional Genomic Analysis

ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.

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Anticapsular and Antifungal Activity of α-Cyperone

Antibiotics ◽

10.3390/antibiotics10010051 ◽

2021 ◽

Vol 10 (1) ◽

pp. 51

Author(s):

Connor Horn ◽

Govindsamy Vediyappan

Keyword(s):

Antifungal Activity ◽

Fungal Pathogens ◽

Fungal Infections ◽

Antifungal Drugs ◽

Cyperus Rotundus ◽

Drug Resistant ◽

Fungal Virulence ◽

Medical Needs ◽

Number Of Patients ◽

Immunosuppressed Patients

Fungal infections affect 300 million people and cause 1.5 million deaths globally per year. With the number of immunosuppressed patients increasing steadily, there is an increasing number of patients infected with opportunistic fungal infections such as infections caused by the species of Candida and Cryptococcus. In fact, the drug-resistant Can. krusei and the emerging pan-antifungal resistant Can. auris pose a serious threat to human health as the existing limited antifungals are futile. To further complicate therapy, fungi produce capsules and spores that are resistant to most antifungal drugs/host defenses. Novel antifungal drugs are urgently needed to fill unmet medical needs. From screening a collection of medicinal plant sources for antifungal activity, we have identified an active fraction from the rhizome of Cyperus rotundus, the nut grass plant. The fraction contained α-Cyperone, an essential oil that showed fungicidal activity against different species of Candida. Interestingly, the minimal inhibitory concentration of α-Cyperone was reduced 8-fold when combined with a clinical antifungal drug, fluconazole, indicating its antifungal synergistic potential and could be useful for combination therapy. Furthermore, α-Cyperone affected the synthesis of the capsule in Cryp. neoformans, a causative agent of fungal meningitis in humans. Further work on mechanistic understanding of α-Cyperone against fungal virulence could help develop a novel antifungal agent for drug-resistant fungal pathogens.

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Visible Lights Combined with Photosensitizing Compounds Are Effective against Candida albicans Biofilms

Microorganisms ◽

10.3390/microorganisms9030500 ◽

2021 ◽

Vol 9 (3) ◽

pp. 500 ◽

Cited By ~ 1

Author(s):

Priyanka Bapat ◽

Gurbinder Singh ◽

Clarissa J. Nobile

Keyword(s):

Candida Albicans ◽

Photodynamic Therapy ◽

Biofilm Formation ◽

Fungal Pathogens ◽

Fungal Infections ◽

Therapeutic Strategies ◽

Antifungal Drugs ◽

Clinical Settings ◽

Drug Resistant ◽

Candida Albicans Biofilms

Fungal infections are increasing in prevalence worldwide, especially in immunocompromised individuals. Given the emergence of drug-resistant fungi and the fact that there are only three major classes of antifungal drugs available to treat invasive fungal infections, there is a need to develop alternative therapeutic strategies effective against fungal infections. Candida albicans is a commensal of the human microbiota that is also one of the most common fungal pathogens isolated from clinical settings. C. albicans possesses several virulence traits that contribute to its pathogenicity, including the ability to form drug-resistant biofilms, which can make C. albicans infections particularly challenging to treat. Here, we explored red, green, and blue visible lights alone and in combination with common photosensitizing compounds for their efficacies at inhibiting and disrupting C. albicans biofilms. We found that blue light inhibited biofilm formation and disrupted mature biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential. Red and green lights, however, inhibited biofilm formation only in combination with photosensitizing compounds but had no effects on disrupting mature biofilms. Taken together, these results suggest that photodynamic therapy may be an effective non-drug treatment for fungal biofilm infections that is worthy of further exploration.

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Alexidine dihydrochloride has broad spectrum a ctivities against diverse fungal pathogens

10.1101/429944 ◽

2018 ◽

Author(s):

Zeinab Mamouei ◽

Abdullah Alqarihi ◽

Shakti Singh ◽

Shuying Xu ◽

Michael K. Mansour ◽

...

Keyword(s):

Mammalian Cell ◽

Fungal Pathogens ◽

Fungal Infections ◽

Antifungal Drugs ◽

Immunocompromised Patients ◽

Drug Resistant ◽

Cell Toxicity ◽

Diverse Range ◽

Mammalian Cell Toxicity

AbstractInvasive fungal infections due to Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, constitute a substantial threat to hospitalized, immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices, and emergence of drug-resistant fungi such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms which increases the chance of infection relapses. Here, we screened a small molecule Prestwick Chemical Library, consisting of 1200 FDA approved off-patent drugs, against C. albicans, C. auris and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens, and their ability to kill preformed biofilms. Our studies identified the bis-biguanide Alexidine dihydrochloride (AXD), as a drug with the highest antifungal and anti-biofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mice central venous catheters in vivo, highlighting its potential as a pan-antifungal drug.ImportanceThe prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multi-drug resistant strains such as C. auris. High mortality related to fungal infections point to major limitations of current antifungal therapy, and an unmet need for new antifungal drugs. We screened a library of repurposed FDA approved inhibitors to identify compounds with activities against a diverse range of fungi, in varied phases of growth. The assays identified Alexidine dihydrochloride (AXD) to have pronounced antifungal activity including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro, and prevented biofilm growth in vivo. Thus AXD has the potential to be developed as a pan-antifungal, anti-biofilm drug.

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