Structural and mechanistic insights into fungal ß-1,3-glucan synthase FKS1.

The membrane-integrated synthase FKS is involved in the biosynthesis of ß-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal ß-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

Epigenetic gene silencing by heterochromatin primes fungal resistance.

Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent1-3. In the fission yeast Schizosaccharomyces pombe, H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent4,5. Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change6,7. Here we show that heterochromatin-dependent epimutants resistant to caffeine arise in fission yeast grown with threshold levels of caffeine. Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of embedded genes, including some whose mutation confers caffeine resistance. Forced heterochromatin formation at implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, letting wild-type cells adapt to unfavourable environments without genetic alteration. In some isolates, subsequent or coincident gene-amplification events augment resistance. Caffeine affects two anti-silencing factors: Epe1 is downregulated, reducing its chromatin association, and a shortened isoform of Mst2 histone acetyltransferase is expressed. Thus, heterochromatin-dependent epimutation provides a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild type. Isolates with unstable caffeine resistance show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to resistance of plant and human fungal pathogens to such agents.

Naamidine A reveals a promising zinc-binding strategy for topical antifungal therapy.

Fungal disease affects over a billion people worldwide. Naamidine A inhibits the growth of diverse fungal pathogens through an unknown mechanism. Here, we show that the supplementation of medium with excess zinc abolishes the antifungal activity of naamidine A. Furthermore, we highlight that naamidine A has in vitro activity against terbinafine-resistant Trichophyton spp. and in vivo efficacy in a mouse model of dermatomycosis caused by T. mentagrophytes, highlighting its therapeutic potential as a topical treatment.

New arsenals for old armour: Biogenic nanoparticles in the battle against drug-resistant Candidaalbicans.

Candida albicans is a common commensal fungus and fourth most frequent causative agent of nosocomial infections including life-threatening invasive candidiasis in humans. The effectiveness of present antifungal therapies using azoles, polyenes, flucytosine and echinocandins has plateaued in managing fungal infections. The limitations of these antifungal drugs are related to polymorphic morphology, biofilm formation, emergence of drug-resistant strains and production of several virulence factors. Development of new antifungal agents, which can particularly afflict multiple cellular targets and limiting evolving resistant strains are needed. Recently, metal nanoparticles have emerged as a source of new antifungal agents for antifungal formulations. Furthermore, green nanotechnology deals with the use of biosynthetic routes that offer new avenue for synthesizing antifungal nanoparticles coupled with less toxic chemical inventory and environmental sustainability. This article reviews the recent developments on C. albicans pathogenesis, biofilm formation, drug resistance, mode of action of antifungal drugs and antifungal activities of metal nanoparticles. The antifungal efficacy and mode of action of metal nanoparticles are described in the context of prospective therapeutic applications.

Antifungal activity of Cinnamaldehyde derivatives against fluconazole-resistant Candida albicans.

BACKGROUND: Candida albicans is an opportunistic pathogen commonly found in human mucous membranes. In light of the escalating challenge posed by antibiotic resistance of C. albicans strains worldwide, it is an urgently necessary to explore alternative therapeutic options. OBJECTIVE: This study aims to assess the efficacy of two Cinnamaldehyde derivatives, 2-Cl Cinnamaldehyde (2-Cl CA) and 4-Cl Cinnamaldehyde (4-Cl CA), against C. albicans through both in vitro experiments and in vivo murine models and to evaluate their potential as new drug candidates for treating C. albicans. METHODS AND RESULTS: The minimum inhibitory concentrations (MICs) of Cinnamaldehyde 2-Cl and 4-Cl benzene ring derivatives against C. albicans were 25 µg/mL. Time-killing experiments revealed that both Cinnamaldehyde derivatives exhibited fungicidal activity against C. albicans at concentrations of 5 MIC and 10 MIC. In the checkerboard experiment, 4-Cl CA did not show any antagonistic effect when combined with first-line antifungal drugs. Instead, it exhibited additive effects in combination with nystatin. Both 2-Cl and 4-Cl CA demonstrated inhibitory activity against C. albicans biofilm formation, especially at 8 MIC and 16 MIC concentrations. In C. albicans biofilm eradication experiments, although high drug concentrations of 2-Cl and 4-Cl CA were unable to eradicate the biofilm completely, they were still effective in killing C. albicans cells within the biofilm. Moreover, sub-inhibitory concentrations of 4-Cl CA (ranging from 5 to 20 µg/mL) significantly inhibited cell aggregation and hyphal formation. Furthermore, 4-Cl CA effectively inhibited intracellular C. albicans infection in macrophages. Lastly, the effectiveness of 4-Cl CA was evaluated in a mouse model of hematogenous disseminated candidiasis caused by C. albicans, which revealed that 4-Cl CA significantly reduced fungal burden and improved mouse survival compared to the untreated controls. CONCLUSION: The 4-Cl CA exhibited inhibitory effects against C. albicans through both in vivo and in vitro models, demonstrating its therapeutic potential as a promising new drug candidate for treating drug-resistant candidiasis albicans.

Antifungal activity of synthetic xanthenone against fluconazole-resistant Candida auris and its mechanism of action.

Candida auris, an emerging multidrug-resistant fungal pathogen discovered in Japan in 2009, poses a significant global health threat, with infections reported in about 25 countries. The escalation of drug-resistant strains underscores the urgent need for new treatment options. This study aimed to investigate the antifungal potential of 2,3,4,4a-tetrahydro-1H-xanthen-1-one (XA1) against C. auris, as well as its mechanism of action and toxic profile. The antifungal activity of XA1 was first evaluated by determining the minimum inhibitory concentration (MIC), time-kill kinetics and biofilm inhibition. In addition, structural changes, membrane permeability, reactive oxygen species (ROS) production, and in vitro and in vivo toxicity of C. auris after exposure to XA1 were investigated. The results indicated that XA1 exhibited an MIC of 50 µg/mL against C. auris, with time-kill kinetics highlighting its efficacy. Field emission scanning electron microscopy (FE-SEM) showed structural damage in XA1-treated cells, supported by increased membrane permeability leading to cell death. Furthermore, XA1 induced ROS production and significantly inhibited biofilm formation. Importantly, XA1 exhibited low cytotoxicity in human epidermal keratinocytes (HaCaT), with a cell viability of over 90 % at 6.25 µg/mL. In addition, an LD50 of 17.68 µg/mL was determined in zebrafish embryos 24 h post fertilization (hpf), with developmental delay observed at prolonged exposure at 6.25 µg/mL (48-96 hpf). These findings position XA1 as a promising candidate for further research and development of an effective antifungal agent.

Photodynamic Inactivation in agriculture: combating fungal phytopathogens resistant to conventional treatment.

Botrytis cinerea is a severe threat in agriculture, as it can infect over 200 different crop species with gray mold affecting food yields and quality. The conventional treatment using fungicides lead to emerging resistance over the past decades. Here, we introduce Photodynamic Inactivation (PDI) as a strategy to combat B. cinerea infections, independent of fungicide resistance. PDI uses photoactive compounds, which upon illumination create reactive oxygen species toxic for killing target organisms. This study focuses on different formulations of sodium-magnesium-chlorophyllin (Chl, food additive E140) as photoactive compound in combination with EDTA disodium salt dihydrate (Na2EDTA) as cell-wall permeabilizer and a surfactant. In an in vitro experiment, three different photosensitizers (PS) with varying Chl and Na2EDTA concentrations were tested against five B. cinerea strains with different resistance mechanisms. We showed that all B. cinerea mycelial spheres of all tested strains were eradicated with concentrations as low as 224 µM Chl and 3.076 mM Na2EDTA (LED illumination with main wavelength of 395 nm, radiant exposure 106 J cm-2). To further test PDI as a Botrytis treatment strategy in agriculture a greenhouse trial was performed on B. cinerea infected bell pepper plants (Capsicum annum L). Two different rates (560 or 1120 g Ha-1) of PS formulation (0.204 M Chl and 1.279 M Na2EDTA) and a combination of PS formulation with 0.05% of the surfactant BRIJ L4 (560 g Ha-1) were applied weekly for 4 weeks by spray application. Foliar lesions, percentage of leaves affected, percentage of leaf area diseased and AUDPC were significantly reduced, while percentage of marketable plants were increased by all treatments compared to a water treated control, however, did not statistically differ from each other. No phytotoxicity was observed in any treatment. These results add to the proposition of employing PDI with the naturally sourced PS Chl in agricultural settings aimed at controlling B. cinerea disease. This approach seems to be effective regardless of the evolving resistance mechanisms observed in response to conventional antifungal treatments.

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

The progress and future of the treatment of Candida albicans infections based on nanotechnology.

Systemic infection with Candida albicans poses a significant risk for people with weakened immune systems and carries a mortality rate of up to 60%. However, current therapeutic options have several limitations, including increasing drug tolerance, notable off-target effects, and severe adverse reactions. Over the past four decades, the progress in developing drugs to treat Candida albicans infections has been sluggish. This comprehensive review addresses the limitations of existing drugs and summarizes the efforts made toward redesigning and innovating existing or novel drugs through nanotechnology. The discussion explores the potential applications of nanomedicine in Candida albicans infections from four perspectives: nano-preparations for anti-biofilm therapy, innovative formulations of "old drugs" targeting the cell membrane and cell wall, reverse drug resistance therapy targeting subcellular organelles, and virulence deprivation therapy leveraging the unique polymorphism of Candida albicans. These therapeutic approaches are promising to address the above challenges and enhance the efficiency of drug development for Candida albicans infections. By harnessing nano-preparation technology to transform existing and preclinical drugs, novel therapeutic targets will be uncovered, providing effective solutions and broader horizons to improve patient survival rates.

Antifungal Synergy: Mechanistic Insights into the R-1-R Peptide and Bidens pilosa Extract as Potent Therapeutics against Candida spp. through Proteomics.

Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives.

Fluconazole Analogs and Derivatives: An Overview of Synthesis, Chemical Transformations, and Biological Activity.

Fluconazole (2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol), which was patented in 1981 and introduced for commercial use in 1988, is a widely utilized antifungal drug whose mechanism of action involves inhibition of the activity of 14-α lanosterol demethylase. Its safety and effectiveness have established it as one of the most frequently employed antifungal agents. Resistance to azole antifungal drugs is becoming more common. It may be related to a mutation of the gene encoding the enzyme. To address this issue, molecules with modifications in three main regions of fluconazole, namely the hydroxyl group, the aromatic ring, and the 1,2,4-triazole rings, have been synthesized in an attempt to create more potent antifungal drugs. These modifications aim at enhancing the effectiveness against microorganisms and improving pharmacokinetic parameters and safety profiles of the synthesized compounds. The present review explores the synthesis of fluconazole derivatives, accompanied by insights into the results of biological studies evaluating the therapeutic effects of these compounds.

Trichophyton indotineae and other terbinafine-resistant dermatophytes in North America.

Dermatophyte infections (a.k.a. ringworm, tinea) affect an estimated 20%-25% of the world's population. In North America, most dermatophytoses are caused by Trichophyton rubrum or Trichophyton mentagrophytes species complexes. Severe and antifungal-resistant dermatophytoses are a growing global public health problem. A new species of the T. mentagrophytes species complex, Trichophyton indotineae, has recently emerged and is notable for the severe infections it causes, its propensity for antifungal resistance, and its global spread. In this issue of the Journal of Clinical Microbiology, C. F. Cañete-Gibas, J. Mele, H. P. Patterson, et al. (J Clin Microbiol 61:e00562-23, 2023, https://doi.org/10.1128/JCM.00562-23) summarize the results of speciation and AFST performed on North American dermatophyte isolates received at a fungal diagnostic reference laboratory. Within their collection, 18.6% of isolates were resistant to terbinafine (a first-line oral antifungal for dermatophytoses), and similar proportions of T. rubrum and T. indotineae demonstrated terbinafine resistance. The authors also found that T. indotineae has been present in North America since at least 2017. These findings highlight the importance of increased surveillance efforts to monitor trends in severe and antifungal-resistant dermatophytoses and the need for antifungal stewardship efforts, the success of which is contingent upon improving laboratory capacity for dermatophyte speciation and AFST.

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond.

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.

Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor.

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.

Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance.

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.

Candida auris: Epidemiology, biology, antifungal resistance, and virulence.

First described in 2009 in Japan, the emerging multidrug-resistant fungal pathogen Candida auris is becoming a worldwide public health threat that has been attracting considerable attention due to its rapid and widespread emergence over the past decade. The reasons behind the recent emergence of this fungus remain a mystery to date. Genetic analyses indicate that this fungal pathogen emerged simultaneously in several different continents, where 5 genetically distinct clades of C. auris were isolated from distinct geographical locations. Although C. auris belongs to the CTG clade (its constituent species translate the CTG codon as serine instead of leucine, as in the standard code), C. auris is a haploid fungal species that is more closely related to the haploid and often multidrug-resistant species Candida haemulonii and Candida lusitaniae and is distantly related to the diploid and clinically common fungal pathogens Candida albicans and Candida tropicalis. Infections and outbreaks caused by C. auris in hospitals settings have been rising over the past several years. Difficulty in its identification, multidrug resistance properties, evolution of virulence factors, associated high mortality rates in patients, and long-term survival on surfaces in the environment make C. auris particularly problematic in clinical settings. Here, we review progress made over the past decade on the biological and clinical aspects of C. auris. Future efforts should be directed toward understanding the mechanistic details of its biology, epidemiology, antifungal resistance, and pathogenesis with a goal of developing novel tools and methods for the prevention, diagnosis, and treatment of C. auris infections.

Synthesis and antifungal evaluation of phenol-derived bis(indolyl)methanes combined with FLC against Candida albicans.

With the widespread use of azole antifungals in the clinic, the drug resistance has been emerging continuously. In this work, we focus on boron trifluoride etherate catalyzed condensation of indole and salicylaldehydes to form bis(indolyl)methanes (BIMs) in high yields, and in vitro antifungal activity against Candida albicans were evaluated. The results showed that most phenol-derived BIMs combined with fluconazole (FLC) exhibited good antifungal activity against sensitive and drug-resistant C. albicans. Further mechanism study demonstrated that BI-10 combined with FLC could inhibit hyphal growth, result in ROS accumulation, and decrease mitochondrial membrane potential (MMP) as well as altering membrane permeability.

Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

An expanded agar-based screening method for azole-resistant Aspergillus fumigatus.

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A. fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A. fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR53 , TR34 /L98H, TR46 /Y121F/T289A, TR34 /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A. fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A. fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation.

The Catestatin-Derived Peptides Are New Actors to Fight the Development of Oral Candidosis.

Resistance to antifungal therapy of Candida albicans and non-albicans Candida strains, frequently associated with oral candidosis, is on the rise. In this context, host-defense peptides have emerged as new promising candidates to overcome antifungal resistance. Thus, the aim of this study was to assess the effectiveness against Candida species of different Catestatin-derived peptides, as well as the combined effect with serum albumin. Among Catestatin-derived peptides, the most active against sensitive and resistant strains of C. albicans, C. tropicalis and C. glabrata was the D-isomer of Cateslytin (D-bCtl) whereas the efficiency of the L-isomer (L-bCtl) significantly decreases against C. glabrata strains. Images obtained by transmission electron microscopy clearly demonstrated fungal membrane lysis and the leakage of the intracellular material induced by the L-bCtl and D-bCtl peptides. The possible synergistic effect of albumin on Catestatin-derived peptides activity was investigated too. Our finding showed that bovine serum albumin (BSA) when combined with the L- isomer of Catestatin (L-bCts) had a synergistic effect against Candida albicans especially at low concentrations of BSA; however, no synergistic effect was detected when BSA interacted with L-bCtl, suggesting the importance of the C-terminal end of L-bCts (GPGLQL) for the interaction with BSA. In this context in vitro D-bCtl, as well as the combination of BSA with L-bCts are potential candidates for the development of new antifungal drugs for the treatment of oral candidosis due to Candida and non-Candida albicans, without detrimental side effects.