An alternative respiratory pathway on Candida krusei: implications on susceptibility profile and oxidative stress

Our aim was to detect the presence of an alternative oxidase (AOX) in Candida krusei clinical strains and its influence on fluconazole susceptibility and in reactive oxygen species (ROS) production. Candida krusei clinical isolates were tested to evaluate the presence of AOX. Debaromyces hansenii 2968 (AOX positive) and Saccharomyces cerevisiae BY4742 (AOX negative) were used as control strains. Measurements of oxygen consumption were performed in the presence of 1?mM KCN, an inhibitor of the classical respiratory chain, and 5?mM salicylhydroxamic acid (SHAM). AOX expression was monitored by Western blotting using an AOX monoclonal antibody. Interactions between fluconazole and SHAM were performed using checkerboard assay. ROS production was evaluated in the presence of SHAM plus fluconazole, H(2) O(2) , menadione, or plumbagin. AOX was present in all C.?krusei tested. The combination of fluconazole with SHAM resulted in an indifferent effect. In the presence of SHAM, the treatment with ROS inductors or fluconazole increased ROS production, except in the AOX-negative strain. An alternative respiratory pathway resistant to cyanide is described for the first time as a characteristic of C.?krusei species. This AOX is unrelated to fluconazole resistance; however, it protects C.?krusei from oxidative stress.     

Resistance of human fungal pathogens to antifungal drugs

Resistance mechanisms can be engaged in clinically relevant fungal pathogens under different conditions when exposed to antifungal drugs. Over past years, active research was undertaken in the understanding of the molecular basis of antifungal drug resistance in these pathogens, and especially against the class of azole antifungals. The isolation of various alleles of the gene encoding the target of azoles has enabled correlation of the appearance of resistance with distinct mutations. Resistance mechanisms to azoles also converge to the upregulation of multidrug transporter genes, whose products have the capacity to extrude from cells several chemically unrelated antifungal agents and toxic compounds. Genome-wide studies of azole-resistant isolates are now permitting a more comprehensive analysis of the impact of resistance on gene expression, and may deliver new clues to their mechanisms. Several laboratories are also exploring, as well as possible alternative resistance pathways, the role of biofilm formation by several fungal species in the development of resistance to various antifungals, including azoles.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

Synthesis and antifungal activity of new thienyl and aryl conazoles

Recent studies reported that an first generation azole (tioconazole) was active against Candida glabrata petite mutants, a fluconazole- and voriconazole- resistant strain of fungi characterized as most azole resistant yeast by an overexpression of the efflux pumps. Therefore, monosubstituted 1-[2-(2,4-dichlorophenyl)ethyl]-1H-imidazoles differing from tioconazole by the nature of the linker and of the aromatic ring in their side-chain were synthesized and evaluated against the mutant and the wild-type strain of C. glabrata. New 2-aryl-1-azolyl-3-thienylbutan-2-ols were then designed and synthesized, and their antifungal activity was evaluated against both strains of C. glabrata and two other major human pathogenic fungi, C. albicans and Aspergillus fumigatus. These new compounds exhibited a broad spectrum activity, as well as good efficiency against the petite mutant, suggesting that they may overcome the increased expression of the efflux pumps usually observed in clinical yeast isolates resistant to current azoles.     

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.     

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.     

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH-ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.     

The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata

Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1 / CgCDR2 , but with upregulation of another ATP-binding cassette transporter, CgSNQ2 , which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N -oxide, which is a ScSNQ2 -specific substrate. A putative gain-of-function mutation, P822L, was identified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas introduction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1 . Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the development of CgPDR1 -dependent azole resistance in C. glabrata . The overlapping in function and regulation between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata .     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.     

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。     

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C.?glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.     

Respiration during seed maturation

The contribution of cytochrome and alternative pathways to respiration during soybean [ Glycine max (L.) Merr. cv. Chippewa 64] seed formation and maturation was measured by oxygen consumption of embryonic axis and cotyledon tissues and of mitochondria isolated from immature seeds. Respiration rates (dry weight basis) declined duing the last month of seed maturation. Cyanide sensitive respiration (cytochrome pathway) accounted for 80 to 90% of total oxygen consumption throughout seed formation and maturation. The proportion of the total respiration resistant to 1 m M cyanide and inhibited by 1 m M salicylhydroxamic acid (alternative pathway) did not exceed 6%. A residual oxygen consumption, accounting for 5 to 15% of total respiration, persisted in the presence of both inhibitors. Likewise, virtually no alternative respiration was found in mitochondria prepared from immature seeds. The alternative respiratory pathway seems absent during soybean seed formation and maturation regardless of whether grown in the greenhouse or field.     

Involvement of alternative oxidase in the regulation of growth, development, and resistance to oxidative stress of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum.     

Drug induced proteome changes in Candida albicans: comparison of the effect of beta(1,3) glucan synthase inhibitors and two triazoles,fluconazole and itraconazole

The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.     

A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.