Ergosterol Biosynthesis Inhibitors Research Articles

Antifungal properties and inhibitory effects upon aflatoxin production of Thymus vulgaris L. by Aspergillus flavus Link

The antifungal and antiaflatoxigenic properties of Thymus vulgaris essential oil (TEO) were evaluated upon Aspergillus flavus “in vitro”. Suspension containing 106 of A. flavus were cultivated with TEO in concentrations ranging from 50 to 500μg/mL. TEO reached minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) at 250μg/mL. Inhibition of ergosterol biosynthesis was detected at a concentration of 100μg/mL of TEO. Morphological evaluation performed by both light microscopy and scanning electron microscopy showed that antifungal activity of TEO could be detected starting at a concentration of 50μg/mL and the fungicide effect at a concentration of 250μg/mL. TEO completely inhibited production of both B1 and B2 aflatoxins (AFB1 and AFB2) at a concentration of 150μg/mL. This way, fungal biomass development and aflatoxin production were dependent on TEO concentration. Therefore, TEO was capable of controlling the growth of A. flavus and its production of aflatoxins.

Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.

TOXICITY OF SOME INSECTICIDES AND THEIR MIXTURES WITH ERGISTROL BIOSYNTHESIS INHIBITING FUNGICIDES AGAINST THE 2ND INSTAR LARVAE OF SPODOPTERA LITTORALIS (BOISD)

The toxicity of organophosphorus insecticide (profenofos), pyrethroid insecticide (esfenvalirate), insect growth regulators, (flufenoxuron) and biocide as natural products (spinosad) increased when mixed with the butylated hydroxyanisol (BHA) and ergosterol biosynthesis inhibiting (EBI) fungicides to 2nd instar larvae of Spodoptera littoralis. Present study was carried out by feeding 2nd instar larvae on castor bean leaves treated with different concentrations of profenofos, esfenvalirate, flufenoxuron and spinosad either alone or in mixtures with BHA, EBI fungicide or IGR. Interestingly, these BHA and EBI did not kill the larvae at concentration up to 100 ppm, moreover 20 ppm of each BHA or EBI fungicide with concentrations of the insecticides increased the toxicity of these insecticides. Also, all data indicated that the calculated LC50 of the insecticides alone were higher than the insecticides and BHA, EBI fungicides or IGR. For instance, the LC50 of profenofos alone was 1.90 ppm, decreased to 0.08, 0.20, 0.24 and 0.22 ppm when mixed with BHA, difenoconazol, penconazol and flufenoxuron respectivily. Also, the insecticidal action increased by 23.75, 9.5, 7.91 and 8.63 folds. The LC50 values decreased when esfenvalirate mixed with BHA, difenoconazol, penconazol and flufenoxuron reached 0.32, 0.21, 0.28 and 0.33 ppm compared with 1.58 ppm when esfenvalirate used alone. Also, the insecticidal action increased by 4.93, 7.52, 5.64 and 4.78 folds when mixed with the mentioned BHA, EBI fungicides or the IGR respectively. Concerning flufenoxuron LC50 values alone was 1.83 decreased to 0.12, 0.19 and 0.22 ppm when mixed with BHA, difenoconazol, penconazol and flufenoxuron respectively, insecticidal action increased by 15.25, 9.63 and 8.31 folds respectively. LC50 values of spinosad was 1.44 ppm when used alone, decreased to 0.21, 0.21, 0.25 and 0.20 ppm when was mixed with BHA, difenoconazol, penconazol and flufenoxuron respectively, insecticidal action was increased by 6.85, 6.85, 5.76 and 7.2 folds respectively. These results suggested that the antioxidant butylated hydroxyanisol (BHA), ergosterol biosynthesis inhibiting (EBI) fungicides and the IGRs enhanced the insecticidal activity of profenofos, esfenvalirate and spinosad against 2nd instar S.littoralis.

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea.

The high-osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high-osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7-1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7-1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high-osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7-1 are restored by genetic complementation of the mutant with the wild-type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.

The Effects of Combretum zeyheri Leaf Extract on Ergosterol Synthesis in Candida albicans

The in vitro effects of Combretum zeyheri leaf ethanol extract (CZLE) on ergosterol biosynthesis of Candida albicans were investigated by quantifying the ergosterol in C. albicans in the presence and absence of the extract using UV-visible spectrophotometric analyses. Miconazole was used as the positive control. CZLE showed ergosterol biosynthesis inhibition at sub-inhibitory concentration for growth of C. albicans and also at the MIC (0.08 mg.mL−1). The decrease in ergosterol in C. albicans cells was dose-dependant with 67%, 79%, and 100% after growing in 20, 40, and 80 μg.mL−1 of CZLE, respectively. CZLE exerted its fungicidal effects by targeting the ergosterol biosynthesis in C. albicans and disrupting the membrane integrity.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Synthesis of amino acid appended indoles: Appreciable anti-fungal activity and inhibition of ergosterol biosynthesis as their probable mode of action

Rationally designed compounds consisting of mono- and di-peptide appendages on bis-indole template were synthesized in appreciable yield. Some of these compounds exhibited significant antifungal activities against Candida albicans with their MIC80 in μg/ml range. However, when used in combination with azoles, the antifungal activities of the azoles were considerably enhanced. The growth inhibition appeared to be specific to the fungal cells and mammalian cells were not affected by these compounds. It is shown that these compounds lower ergosterol levels in the fungal cells and probably act by targeting lanosterol 14α-demethylase, a key enzyme in the sterol biosynthetic pathway of C. albicans. The compounds do not appear to directly act on the fungal cell wall. Hence, the sensitivity of the fungal cells to these compounds cannot be attributed to cell wall damage and consequent accumulation of the compounds in the cell, though defects in cell wall due to defective sterol biosynthesis cannot be completely ruled out.

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development.

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. In this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors – posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compound- and strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.

Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops

Assessment of the toxicity of individual pesticides to honeybees is routinely assessed. However, few data have been generated for realistic mixtures of neonicotinoid insecticides and fungicides particularly with regard to exposure levels used. Assessment of the effects of exposure of bees to predicted residues following sprayed applications of ergosterol biosynthesis inhibitor fungicides on the contact and oral toxicity of a range of neonicotinoid insecticides (thiamethoxam, clothianidin, imidacloprid and thiacloprid) showed only low levels of synergism (<3-fold maximum). Further studies showed that the scale of increase in toxicity was fungicide dose dependent with greater synergy of oral toxicity of thiamethoxam following contact dosing with propiconazole. This underlines the need for the use of realistic exposure levels and routes in studies.

In Vitro Activity of the Antifungal Azoles Itraconazole and Posaconazole against Leishmania amazonensis

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs could represent novel therapies for the treatment of leishmaniasis, either alone or in combination with other agents.

Neurospora crassa Protein Arginine Methyl Transferases Are Involved in Growth and Development and Interact with the NDR Kinase COT1

The protein arginine methyltransferaseas (PRMTs) family is conserved from yeast to human, and regulates stability, localization and activity of proteins. We have characterized deletion strains corresponding to genes encoding for PRMT1/3/5 (designated amt-1, amt-3 and skb-1, respectively) in Neurospora crassa. Deletion of PRMT-encoding genes conferred altered Arg-methylated protein profiles, as determined immunologically. Δamt-1 exhibited reduced hyphal elongation rates (70% of wild type) and increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole. In ▵amt-3, distances between branches were significantly longer than the wild type, suggesting this gene is required for proper regulation of hyphal branching. Deletion of skb-1 resulted in hyper conidiation (2-fold of the wild type) and increased tolerance to the chitin synthase inhibitor polyoxin D. Inactivation of two Type I PRMTs (amt-1 and amt-3) conferred changes in both asymmetric as well as symmetric protein methylation profiles, suggesting either common substrates and/or cross-regulation of different PRMTs. The PRMTs in N. crassa apparently share cellular pathways which were previously reported to be regulated by the NDR (Nuclear DBF2-related) kinase COT1. Using co-immunprecipitation experiments (with MYC-tagged proteins), we have shown that SKB1 and COT1 physically interacted and the abundance of the 75 kDa MYC::COT1 isoform was increased in a Δskb-1 background. On the basis of immunological detection, we propose the possible involvement of PRMTs in Arg-methylation of COT1.

Effects of azole treatments on the physical properties of Candida albicans plasma membrane: A spin probe EPR study

EPR spectroscopy was applied to investigate the effects of the treatment of Candida albicans cells with fluconazole (FLC) and two newly synthesized azoles (CPA18 and CPA109), in a concentration not altering yeast morphology, on the lipid organization and dynamics of the plasma membrane. Measurements were performed in the temperature range between 0°C and 40°C using 5-doxyl- (5-DSA) and 16-doxyl- (16-DSA) stearic acids as spin probes. 5-DSA spectra were typical of lipids in a highly ordered environment, whereas 16-DSA spectra consisted of two comparable components, one corresponding to a fluid bulk lipid domain in the membrane and the other to highly ordered and motionally restricted lipids interacting with integral membrane proteins. A line shape analysis allowed the relative proportion and the orientational order and dynamic parameters of the spin probes in the different environments to be determined. Smaller order parameters, corresponding to a looser lipid packing, were found for the treated samples with respect to the control one in the region close to the membrane surface probed by 5-DSA. On the other hand, data on 16-DSA indicated that azole treatments hamper the formation of ordered lipid domains hosting integral proteins and/or lead to a decrease in integral protein content in the membrane. The observed effects are mainly ascribable to the inhibition of ergosterol biosynthesis by the antifungal agents, although a direct interaction of the CPA compounds with the membrane bilayer in the region close to the lipid polar head groups cannot be excluded.

Flucytosine Antagonism of Azole Activity versus Candida glabrata: Role of Transcription Factor Pdr1 and Multidrug Transporter Cdr1

Infections with the opportunistic yeast Candida glabrata have increased dramatically in recent years. Antifungal therapy of yeast infections commonly employs azoles, such as fluconazole (FLC), but C. glabrata frequently develops resistance to these inhibitors of ergosterol biosynthesis. The pyrimidine analog flucytosine (5-fluorocytosine [5FC]) is highly active versus C. glabrata but is now rarely used clinically due to similar concerns over resistance and, a related concern, the toxicity associated with high doses used to counter resistance. Azole-5FC combination therapy would potentially address these concerns; however, previous studies suggest that 5FC may antagonize azole activity versus C. glabrata. Here, we report that 5FC at subinhibitory concentrations antagonized the activity of FLC 4- to 16-fold versus 8 of 8 C. glabrata isolates tested. 5FC antagonized the activity of other azoles similarly but had only indifferent effects in combination with unrelated antifungals. Since azole resistance in C. glabrata results from transcription factor Pdr1-dependent upregulation of the multidrug transporter gene CDR1, we reasoned that 5FC antagonism might be similarly mediated. Indeed, 5FC-FLC antagonism was abrogated in pdr1Δ and cdr1Δ strains. In further support of this hypothesis, 5FC exposure induced CDR1 expression 6-fold, and this upregulation was Pdr1 dependent. In contrast to azoles, 5FC is not a Cdr1 substrate and so its activation of Pdr1 was unexpected. We observed, however, that 5FC exposure readily induced petite mutants, which exhibit Pdr1-dependent CDR1 upregulation. Thus, mitochondrial dysfunction resulting in Pdr1 activation is the likely basis for 5FC antagonism of azole activity versus C. glabrata.

Metabolic Differentiation of Saccharomyces cerevisiae by Ketoconazole Treatment

Abstract Azole fungicides are one of the most wide-spreadantifungal compounds in agriculture and pharmaceutical applications.Their major mode of action is the inhibition of ergosterolbiosynthesis, giving depletion of ergosterol, precursors andabnormal steroids. However, metabolic consequences of suchinhibition, other than steroidal metabolitesare not well established.Comprehensive metabolic profiles of Saccharomyces cerevisiaehas been presented in this study. Wild type yeast was treated eitherwith glucose as control or azole fungicide (ketoconazole). Bothpolar metabolites and lipids were analyzed with gas chromatography-mass spectrometry. Approximately over 180 metabolites werecharacterized, among which 18 of them were accumulated ordepleted by fungicide treatment. Steroid profile gives the mostprominent differences, including the accumulation of lanosteroland the depletion of zymosterol and ergosterol. However, thepolar metabolite profile was also highly different in pesticidetreatment. The concentration of proline and its precursors,glutamate and ornithine were markedly reduced by ketoconazole.Lysine and glycine level was also decreased while the concentrationsof serine and homoserine were increased. The overall metabolicprofile indicates that azole fungicide treatment induces thedepletion of many polar metabolites, which are important in stressresponse.Keywords azole · metabolomics · mode of action · pesticide toxicity · proline · Saccharomyces cerevisiaeIntroductionAzole-type fungicides (e.g., ketoconazole) are widely used inmany agricultural and medicinal applications. Their major modeof action is the inhibition of steroid biosynthesis, being one of themost important primary metabolites in fungal cell membrane(Schwinn, 1984; Zarn et al, 2003). The primary target of thesefungicides is C-14 demethylase, which catalyze the consecutivedemethylation at 14

Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae

We have investigated the functions of three endocytosis-related proteins in the filamentous fungus Aspergillus oryzae. Yeast two-hybrid screening using the endocytic marker protein AoAbp1 (A.oryzae homolog of Saccharomyces cerevisiae Abp1p) as a bait identified four interacting proteins named Aip (AoAbp1 interacting proteins). In mature hyphae, EGFP (enhanced green fluorescent protein) fused to Aips colocalized with AoAbp1 at the hyphal tip region and the plasma membrane, suggesting that Aips function in endocytosis. aipA is a putative AAA ATPase and its function has been dissected (Higuchi et al., 2011). aipB, the homolog of A. nidulans myoA, encodes an essential class I myosin and its conditional mutant showed a germination defect. aipC and aipD do not contain any recognizable domains except some proline-rich regions which may interact with two SH3 (Src homology 3) domains of AoAbp1. Neither aipC nor aipD disruptants showed any defects in their growth, but the aipC disruptant formed less conidia compared with the control strain. In addition, the aipC disruptant was resistant to the triazole antifungal drugs that inhibit ergosterol biosynthesis. Although no aip disruptants showed any defects in the uptake of the fluorescent dye FM4-64, the endocytosis of the arginine permease AoCan1, one of the MCC (membrane compartment of Can1p) components, was delayed in both aipC and aipD disruptants. In A. oryzae, AoCan1 localized mainly at the plasma membrane in the basal region of hyphae, suggesting that different endocytic mechanisms exist in apical and basal regions of highly polarized cells.

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.

Vanillin Inhibits Growth, Morphogenesis and Biofilm Formation by Candida albicans

Vanillin, a major component of the vanilla is known to exhibit various antimicrobial properties including inhibition of bacterial biofilms. However, a systematic study on activities of vanillin against the most common fungal pathogen, Candida albicans, is lacking. In this study, broth micro dilution method was used to assess vanillin's activity on the planktonic growth of C. albicans. Minimum inhibitory concentration (MIC) was established at 1 mg ml-1 of vanillin. Prevention of yeast to hyphae morphogenesis was evident at 500 μg ml-1 of vanillin, in standard micro plate based assay. Adhesion, biofilm development and mature biofilms were studied in an in vitro biofilm model using XTTmetabolic assay and light microscopy. Significant (p < 0.05) reduction in biofilm development was observed in presence of 500 μg ml-1 vanillin. Concentration dependent decrease of cellular ergosterol in presence of vanillin indicated that inhibition of ergosterol biosynthesis may be a probable target. Our study, for the first time reveals vanillin as a potential inhibitor of biofilm formation by C. albicans.

Posaconazole Exhibits In Vitro and In Vivo Synergistic Antifungal Activity with Caspofungin or FK506 against Candida albicans

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the β-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen Candida albicans. Although current drug treatments for Candida infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and C. albicans infections. Our studies reported here demonstrate that posaconazole exhibits in vitro synergy with caspofungin or FK506 against drug susceptible or resistant C. albicans strains. Furthermore, these combinations also show in vivo synergy against C. albicans strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 β-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future.

A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects on total ergosterol biosynthesis

Increasing resistance of clinically relevant fungi is causing major problems in anti-mycotic therapy. Particularly for immunosuppressed patients fungal infections are of concern and increasing resistance against clinically used antimycotic drugs is hampering successful treatment. In the search for new antifungals ergosterol biosynthesis still is the most prominent target. However, several pitfalls in the bioactivity testing of such substances remain. Two of the major drawbacks certainly are the membrane association of most enzymes participating in ergosterol biosynthesis, and the difficulty to selectively associate growth inhibitory effects with the target pathway (ergosterol biosynthesis). Here we describe a GC–MS based cellular assay for target identification and selective potency determination of test components. In the qualitative part of the assay GC–MS analysis of cell lysates allows target identification by analysis of the changes in the sterol pattern. The quantitative part of the assay makes use of 13C-acetate feeding combined with GC–MS analysis allowing the selective quantification of a compound’s effect on total ergosterol biosynthesis. The described cellular assay was analytically and biologically validated and used to characterize the novel ergosterol biosynthesis inhibitor JK-250.

Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China

False smut, caused by Ustilaginoidea virens, is an important emerging disease of rice (Oryza sativa L.) in China. Up to now, as most varieties with high yielding and good quality are susceptible or even highly susceptible to false smut in most rice-growing ecological regions, especially in Anhui Province, chemical control with fungicides would be an important measure for the control of this disease. The ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, difenoconazole, propiconazole and tebuconazole, are extensively used in China for the control of rice diseases, such as rice sheath blight and rice blast. In this study, a total of 102 U. virens isolates (from Anhui Province of China) were tested for their sensitivity to these four EBI fungicides during the stage of mycelial growth. The EC50 ranges of values for prochloraz, difenoconazole, propiconazole and tebuconazole inhibiting mycelial growth of the 102 U. virens isolates were 0.04–0.75, 0.04–1.08, 0.04–0.38 and 0.03–0.57 μg ml−1, with the average EC50 values of 0.32 ± 0.08, 0.45 ± 0.08, 0.19 ± 0.03 and 0.21 ± 0.06 μg ml−1, respectively. These values suggested that the tested U. virens isolates were very sensitive to these four EBI fungicides. Results of field trials showed that two sprays of three of the fungicides exhibited greater control efficacy than a single spray for the control of rice false smut. Two sprays of each was better than a single spray for the control of rice sheath blight. Two sprays of 50% propiconazole EC at 300 g a.i. ha−1 gave the best control of rice false smut at both two sites during the two consecutive years, 2010 and 2011, with the control efficacy ranging from 71.5 to 74.3%. Sensitivity of the field U. virens isolates to EBI fungicides should be monitored. Mixtures, as well as alternation with other fungicides with different modes of action, should be tested.