Relationship between respiration deficiency and azole resistance in clinical Candida glabrata

Candida glabrata has become a leading cause of invasive infections around the world and is exhibiting growing resistance to azole antifungals. To study the mechanism of its azole resistance, we analyzed the efflux pumps and found well known increased efflux expression and low metabolic state in all azole-resistant strains. The latter finding led us to further investigate the relationship between respiration status and azole antifungal susceptibility in clinical C.?glabrata by growing them on glycerol-containing agar, measuring the cellular ATP, reactive oxygen species (ROS) levels, oxygen consumption and transmission electron microscopy. All azole-resistant isolates were respiratory-deficient, with reduced generation of ATP and ROS and decreased oxygen consumption; two isolates grew as small colonies and exhibited mitochondrial deficiency. Spot assays and agarose disc diffusion tests were performed to evaluate the effects of respiratory chain inhibitors, sodium azide and salicylhydroxamic acid, on antifungal susceptibility. The results of antifungal susceptibility showed that inhibition of alternative respiration with salicylhydroxamic acid enhanced azole susceptibility of C.?glabrata. In conclusion, clinical azole-resistant C.?glabrata isolates harbor respiratory deficiency exhibiting petite mutant or normal phenotype. The alternative respiratory pathway plays an important role in the decreased susceptibility to azole antifungals.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

Synthesis of new isoxazoles and dihydroisoxazoles and in vitro evaluation of their antifungal activity

New 2-(2,4-dihalogenophenyl)-1-(1H-imidazol-1-yl)-3-(isoxazol-5-yl)propan-2-ols and 2-(2,4-dihalogenophenyl)-1-(1H-imidazol-1-yl)-3-(4,5-dihydroisoxazol-5-yl)propan-2-ols were synthesized by 1,3-dipolar cycloaddition between homopropargylic or homoallylic alcohols and in-situ generated nitrile oxide. Their antifungal activity was evaluated against Candida albicans, C. glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

Membrane fluidity and lipid composition in clinical isolates of Candida albicans isolated from AIDS/HIV patients

In this study, we describe the membrane lipid composition of eight clinical isolates (azole resistant and sensitive strains) of Candida albicans isolated from AIDS/ HIV patients. Interestingly, fluorescence polarization measurements of the clinical isolates displayed enhanced membrane fluidity in fluconazole resistant strains as compared to the sensitive ones. The increase in fluidity was reflected in the change of membrane order, which was considerably decreased (decrease in fluorescence polarization "p" value denotes higher membrane fluidity) in the resistant strains. The ergosterol content in azole susceptible isolates was greater, almost twice as compared to the resistant isolates. However, no significant alteration was observed in phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of phosphatidylethanolamine exposed to the membrane's outer leaflet was higher in the resistant strains as compared to the sensitive strains, indicating increased floppase activity of the two major ABC drug efflux pumps, CDR1 and CDR2 possibly due to their overexpression in resistant strains. The results of the present study suggest that changes in the status of membrane lipid phase especially the ergosterol content and increased activity of drug efflux pumps by overexpression ofABC transporters, CDR1 and CDR2 might contribute to fluconazole resistance in C. albicans isolated from AIDS/HIV patients.     

真菌对唑类抗真菌药物的耐受机制

唑类抗真菌药物广泛用于临床和农业。唑类药物通过与羊毛甾醇14α-去甲基化酶（Erg11p/Cyp51）结合,抑制麦角甾醇合成,同时导致有毒甾醇积累。真菌可快速在转录水平上对唑类药物胁迫作出响应而导致耐药性,尤其是唑类药物外排泵基因和麦角甾醇合成相关基因表达的上调。农业和临床上绝大多数唑类药物耐药菌株的形成都是由麦角甾醇合成基因和唑类药物外排泵表达的变化或是突变所致。一些转录因子（如Pdr1p、Pdr3p、Upc2p、Yap1p、Tac1p、Mrr1p、CCG-8）和信号通路（如cAMP途径、PKC-MAPK途径、HOG MAPK途径、钙调磷酸酶途径）均参与对药物外排泵基因和麦角甾醇合成基因等的调控,影响唑类药物耐药性。针对于这些调控因子设计的抑制剂将有助于提高唑类药物的治疗效果。本文概述了唑类药物的抑菌机制、真菌对唑类药物耐药性形成的原因、真菌对唑类药物适应性响应机理,并对未来此领域的热点和方向进行了展望。     

Quinazolinone fungal efflux pump inhibitors. Part 3: (N-methyl)piperazine variants and pharmacokinetic optimization

Further structure-activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of C. albicans and C. glabrata over-expressing ABC-type efflux pumps are systematically explored. Rat protein binding and pharmacokinetics of selected analogues are reported.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

The activity of echinocandins, amphotericin B and voriconazole against fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates

The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.     

Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole

Candida glabrata is an increasingly important cause of opportunistic fungal infection of humans and appears to be intrinsically resistant to the triazole antifungal fluconazole. However, the mechanisms responsible for reduced susceptibility to azole drugs are not understood. Fluconazole exposure rapidly induced expression of a 169-kDa protein band in plasma membrane fractions of C. glabrata cells. Mass spectrometry of trypsin-digested peptide fragments showed that the induced protein band comprised the ATP binding cassette-type drug efflux transporter CgCdr1p. CgCdr1p was also functionally overexpressed in S. cerevisiae and similarly identified by mass spectrometry. A 61-kDa protein band in the plasma membrane fraction from C. glabrata was also induced by fluconazole exposure. Mass spectrometric peptide fingerprinting identified this band as lanosterol 14alpha-demethylase, the enzyme in the ergosterol biosynthesis pathway targeted by fluconazole. The rapid induction of a multidrug efflux pump and/or overproduction of lanosterol 14alpha-demethylase are mechanisms that could make C. glabrata appear intrinsically resistant to fluconazole. Mass spectrometric fingerprint analysis of SDS-PAGE separated plasma membrane fractions combined with heterologous hyper-expression provides a convenient method for protein identification and functional evaluation of induced proteins, even in an organism where the genome sequence database is incomplete.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts

A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii. KT4561 was precipitated by ethanol and purified by ion-exchange chromatography. The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa. Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h. The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C. glabrata, 0.03 and 0.23 microg/ml for I. orientalis and 1.50 and 2.25 microg/ml for P. guilliermondii. Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics. The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.     

Quinazolinone fungal efflux pump inhibitors. Part 2: In vitro structure-activity relationships of (N-methyl-piperazinyl)-containing derivatives

Structure-activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of Candida albicans and Candida glabrata over-expressing ABC-type efflux pumps are systematically explored.     

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.     

Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA in Candida glabrata

Statins are widely used for lowering cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Yeasts use HMG-CoA reductase for the same enzymatic step as humans, but in yeasts the main end-product of the pathway is ergosterol rather than cholesterol. We considered that insights into the effects of statins in humans could be gained by examination of the effects of simvastatin on the petite-positive yeast Candida glabrata. Simvastatin was found to inhibit growth, and this was associated with lower ergosterol levels. As simvastatin-treated cultures of yeast were passaged, the frequencies of petite cells (respiratory-deficient yeast mutants with deletions in the mitochondrial genome) increased with time and with simvastatin concentration. DNA staining of the petite mutants showed that they were devoid of mtDNA, suggesting a defect in the maintenance of mtDNA. These observations in C. glabrata may provide further insights into the molecular effects of statins in humans undergoing treatment for hypercholesterolemia. In addition, if C. glabrata is a valid model for studying statin treatments, it would be very useful for the preliminary screening of agents to reduce statin side-effects.     

Pdr1蛋白N端携带Flag标签的光滑假丝酵母的构建

近年来,光滑假丝酵母已成为第二位引起侵袭性真菌感染的病原体。光滑假丝酵母对唑类药物(临床一线抗真菌药物)的敏感性低且易发生耐药,一直是研究的热点。介导光滑假丝酵母对唑类药物耐药的关键基因是转录因子pdr1,其功能性突变会使Pdr1蛋白功能过度活跃,导致下游唑类药物外排泵基因高表达,从而对唑类药物耐药。本研究利用同源重组技术,构建在基因pdr1的5′端定点插入3×Flag标签的重组菌株2a2和2b2,为后续利用免疫染色质共沉淀技术寻找Pdr1直接调控基因奠定基础。结果表明,3×Flag标签添加到Pdr1蛋白N端可成功表达Flag-Pdr1蛋白;与野生型菌株相比,表达Flag-Pdr1的菌株对氟康唑的耐药性增强。此外,与野生型菌株相比,表达Flag-Pdr1的菌株中cdr1和pup1基因表达水平显著上升,提示在Pdr1蛋白N端加Flag标签能使其功能活跃,表明N端对Pdr1蛋白功能具有重要意义。     

Synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci

The alarming growth of the number of antibiotic resistant bacteria and in the same time limited possibilities to develop new antimicrobial compounds, lead to an urgent need to keep the sensitivity of bacteria against currently used antibiotics. Bacterial efflux pumps are an important mechanism of antibiotic resistance as the bacteria use efflux pumps for the extrusion of different types of antibiotics and chemicals. The knowledge about inhibitors of efflux pumps from natural sources suggests that this mechanism may be a good target for new drugs based on synergistic interactions of antibiotics with plant extracts, essential oils, or their constituents with efflux pump inhibitory activity. This review summarizes the current knowledge of staphylococcal efflux pumps and potential strategies to overcome them. Natural inhibitors of efflux pumps and their synergistic interactions with antibiotics are summarized.     

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.