Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans,Candida glabrata,and Candida krusei

Azoles target the ergosterol biosynthetic enzyme lanosterol 14alpha-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.     

Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system. Existing therapies include amphotericin B, fluconazole, and flucytosine, which are limited by toxic side effects and the emergence of drug resistance. We recently demonstrated that the protein phosphatase calcineurin is required for growth at 37 degrees C and virulence of C. neoformans. Because calcineurin is the target of potent inhibitors in widespread clinical use, cyclosporine and FK506 (tacrolimus), it is an attractive drug target for novel antifungal agents. Here we have explored the synergistic potential of combining the calcineurin inhibitor FK506 or its nonimmunosuppressive analog, L-685,818, with other antifungal agents and examined the molecular basis of FK506 action by using genetically engineered fungal strains that lack the FK506 target proteins FKBP12 and calcineurin. We demonstrate that FK506 exhibits marked synergistic activity with the H(+)ATPase inhibitor bafilomycin A(1) via a novel action distinct from calcineurin loss of function. FK506 also exhibits synergistic activity with the pneumocandin MK-0991/caspofungin acetate (formerly L-743,873), which targets the essential beta-1,3 glucan synthase, and in this case, FK506 action is mediated via FKBP12-dependent inhibition of calcineurin. Finally, we demonstrate that FK506 and fluconazole have synergistic activity that is independent of both FKBP12 and calcineurin and may involve the known ability of FK506 to inhibit multidrug resistance pumps, which are known to export azoles from fungal cells. In summary, our studies illustrate the potential for synergistic activity of a variety of different drug combinations and the power of molecular genetics to define the mechanisms of drug action, as well as identify a novel action of FK506 that could have profound implications for therapeutic or toxic effects in other organisms, including humans.     

Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin

Cyclosporine (CsA) is an immunosuppressive and antimicrobial drug which, in complex with cyclophilin A, inhibits the protein phosphatase calcineurin. We recently found that Cryptococcus neoformans growth is resistant to CsA at 24 degrees C but sensitive at 37 degrees C and that calcineurin is required for growth at 37 degrees C and pathogenicity. Here CsA analogs were screened for toxicity against C. neoformans in vitro. In most cases, antifungal activity was correlated with cyclophilin A binding in vitro and inhibition of the mixed-lymphocyte reaction and interleukin 2 production in cell culture. Two unusual nonimmunosuppressive CsA derivatives, (gamma-OH) MeLeu(4)-Cs (211-810) and D-Sar (alpha-SMe)(3) Val(2)-DH-Cs (209-825), which are also toxic to C. neoformans were identified. These CsA analogs inhibit C. neoformans via fungal cyclophilin A and calcineurin homologs. Our findings identify calcineurin as a novel antifungal drug target and suggest nonimmunosuppressive CsA analogs warrant investigation as antifungal agents.     

The immunosuppressant FK506 and its nonimmunosuppressive analog L-685,818 are toxic to Cryptococcus neoformans by inhibition of a common target protein.

The immunosuppressant FK506 (tacrolimus) is an antifungal natural product macrolide that suppresses the immune system by blocking T-cell activation. In complex with the intracellular protein FKBP12, FK506 inhibits calcineurin, a Ca(2+)-calmodulin-dependent serine-threonine protein phosphatase. We recently reported that growth of the opportunistic fungal pathogen Cryptococcus neoformans is resistant to FK506 at 24 degrees C but sensitive at 37 degrees C and that calcineurin, the target of FKBP12-FK506, is required for growth at 37 degrees C in vitro and pathogenicity in vivo. These findings identify calcineurin as a potential antifungal drug target. In previous studies the calcineurin inhibitor cyclosporin A (CsA) was effective against murine pulmonary infections but exacerbated cryptococcal meningitis in rabbits and mice, likely because CsA does not cross the blood-brain barrier. Although we find that FK506 penetrates the CNS, FK506 also exacerbates cryptococcal meningitis in rabbits. Thus, FK506 immunosuppression outweighs antifungal action in vivo. Like FK506, the nonimmunosuppressive FK506 analog L-685,818 is toxic to C. neoformans in vitro at 37 degrees C but not at 24 degrees C, and FK506-resistant mutants are resistant to L-685,818, indicating a similar mechanism of action. Fluconazole-resistant C. neoformans clinical isolates were also found to be susceptible to both FK506 and L-685,818. Our findings identify calcineurin as a novel antifungal drug target and suggest the nonimmunosuppressive FK506 analog L-685,818 or other congeners warrant further consideration as antifungal drugs for C. neoformans.     

Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms

Calcineurin is a Ca2+-calmodulin-activated serine/threonine-specific protein phosphatase that governs multiple aspects of fungal physiology, including cation homeostasis, morphogenesis, antifungal drug susceptibility, and virulence. Growth of Candida albicans planktonic cells is sensitive to the calcineurin inhibitors FK506 and cyclosporine A (CsA) in combination with the azole antifungal fluconazole. This drug synergism is attributable to two effects: first, calcineurin inhibitors render fluconazole fungicidal rather than simply fungistatic, and second, membrane perturbation by azole inhibition of ergosterol biosynthesis increases intracellular calcineurin inhibitor concentrations. C. albicans cells in biofilms are up to 1,000-fold more resistant to fluconazole than planktonic cells. In both in vitro experiments and in an in vivo rat catheter model, C. albicans cells in biofilms were resistant to individually delivered fluconazole or calcineurin inhibitors but exquisitely sensitive to the combination of FK506-fluconazole or CsA-fluconazole. C. albicans strains lacking FKBP12 or expressing a dominant FK506-resistant calcineurin mutant subunit (Cnb1-1) formed biofilms that were resistant to FK506-fluconazole but susceptible to CsA-fluconazole, demonstrating that drug synergism is mediated via direct calcineurin inhibition. These findings reveal that calcineurin contributes to fluconazole resistance of biofilms and provide evidence that synergistic drug combinations may prove efficacious as novel therapeutic interventions to treat or prevent biofilms.     

国产伏立康唑对临床分离病原真菌体外抗菌活性研究

目的了解国产伏立康唑对北京和我国其他地区临床分离的常见病原真菌体外抗菌活性。方法分别参照CLSIM27-A2和M38-A方案测定伏立康唑对144株酵母和82株产孢丝状真菌的抗菌活性。受试菌株包括念珠菌114株（含氟康唑获得性耐药白念珠菌）、新型隐球菌20株、阿萨希毛孢子菌10株、曲霉62株（含伊曲康唑耐药曲霉及两性霉素B不敏感曲霉）、镰刀菌10株、尖端赛多孢菌10株。结果伏立康唑对念珠菌（不包括氟康唑耐药和剂量依赖敏感白念珠菌）、新型隐球菌、阿萨希毛孢子菌的MIC50≤0．5mg／L、MIC90≤1mg／L；而对氟康唑获得性耐药白念珠菌MIC50和MIC90均〉16mg／L。对曲霉、尖端赛多孢菌的MIC50≤1mg／L、MIC90≤2mg／L，对镰刀菌的MIC50和MIC90分别为4mg／L和〉16mg／L。结论伏立康唑对多数酵母有较强的体外抗菌活性，尤其是对克柔念珠菌和光滑念珠菌等氟康唑天然耐药菌株。该药对多数产孢丝状真菌也有较好的体外抗菌作用，包括伊曲康唑耐药及两性霉素B不敏感的曲霉以及对多种抗真菌药物耐药的尖端赛多孢菌；但其对氟康唑获得性耐药白念珠菌有一定交叉耐药。     

Candida albicans sterol C-14 reductase, encoded by the ERG24 gene, as a potential antifungal target site

The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the ERG24 gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The ERG24 gene of Candida albicans has been isolated by complementation of a Saccharomyces cerevisiae erg24 mutant. Both copies of the C. albicans ERG24 gene have been disrupted by using short homologous regions of the ERG24 gene flanking a selectable marker. Unlike S. cerevisiae, the C. albicans ERG24 gene was not required for growth, but erg24 mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The erg24 mutants were also slightly resistant to the azoles. Most importantly, erg24 mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against C. albicans.     

Antagonism of Azole Activity against Candida albicans following Induction of Multidrug Resistance Genes by Selected Antimicrobial Agents

Antifungal azoles (e.g., fluconazole) are widely used for prophylaxis or treatment of Candida albicans infections in immunocompromised individuals, such as those with AIDS. These individuals are frequently treated with a variety of additional antimicrobial agents. Potential interactions between three azoles and 16 unrelated drugs (antiviral, antibacterial, antifungal, and antiprotozoal agents) were examined in vitro. Two compounds, tested at concentrations achievable in serum, demonstrated an antagonistic effect on azole activity against C. albicans. At fluconazole concentrations two to four times the 50% inhibitory concentration, C. albicans growth (relative to treatment with fluconazole alone) increased 3- to 18-fold in the presence of albendazole (2 microg/ml) or sulfadiazine (50 microg/ml). Antagonism (3- to 78-fold) of ketoconazole and itraconazole activity by these compounds was also observed. Since azole resistance has been correlated with overexpression of genes encoding efflux proteins, we hypothesized that antagonism results from drug-induced overexpression of these same genes. Indeed, brief incubation of C. albicans with albendazole or sulfadiazine resulted in a 3-to->10-fold increase in RNAs encoding multidrug transporter Cdr1p or Cdr2p. Zidovudine, trimethoprim, and isoniazid, which were not antagonistic with azoles, did not induce these RNAs. Fluphenazine, a known substrate for Cdr1p and Cdr2p, strongly induced their RNAs and, consistent with our hypothesis, strongly antagonized azole activity. Finally, antagonism was shown to require a functional Cdr1p. The possibility that azole activity against C. albicans is antagonized in vivo as well as in vitro in the presence of albendazole and sulfadiazine warrants investigation. Drug-induced overexpression of efflux proteins represents a new and potentially general mechanism for drug antagonism.     

Immunization with the Candida albicans membrane fraction and in combination with fluconazole protects against systemic fungal infections

We studied the immunogenicity of a membrane fraction prepared from Candida albicans cells called C. albicans membrane antigen (CMA). The present study revealed that CMA immunization has antifungal activity in mouse models of systemic fungal infection. Immunization of mice by subcutaneous injections of CMA with incomplete Freund adjuvant induced resistance to infections caused not only by C. albicans but also by Aspergillus fumigatus. The level of resistance to candidiasis was as high as that induced by whole-cell immunization. The acquired resistance to candidiasis in the mice immunized with CMA was not diminished by immunosuppressive treatment with cyclophosphamide. The level of resistance to fungal infections was superior to that given by fluconazole (FLC) treatment alone and highly enhanced by the combination with FLC. When CD4(+) cells in CMA-immunized mice were depleted by a monoclonal antibody, the antifungal activity induced by the combination of CMA and FLC was significantly reduced. These results indicate that immunization with CMA is useful for preventing systemic fungal infections and in combination with FLC for increasing resistance after infection.     

CaNdt80 is involved in drug resistance in Candida albicans by regulating CDR1

Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the beta-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.     

Comparison of in vitro activities of camptothecin and nitidine derivatives against fungal and cancer cells

The activities of a series of camptothecin and nitidine derivatives that might interact with topoisomerase I were compared against yeast and cancer cell lines. Our findings reveal that structural modifications to camptothecin derivatives have profound effects on the topoisomerase I-drug poison complex in cells. Although the water-soluble anticancer agents topotecan and irinotecan are less active than the original structure, camptothecin, other derivatives or analogs with substitutions that increase compound solubility have also increased antifungal activities. In fact, a water-soluble prodrug appears to penetrate into the cell and release its active form; the resulting effect in complex with Cryptococcus neoformans topoisomerase I is a fungicidal response and also potent antitumor activity. Some of the compounds that are not toxic to wild-type yeast cells are extremely toxic to the yeast cells when the C. neoformans topoisomerase I target is overexpressed. With the known antifungal mechanism of a camptothecin-topoisomerase I complex as a cellular poison, these findings indicate that drug entry may be extremely important for antifungal activity. Nitidine chloride exhibits antifungal activity against yeast cells through a mechanism(s) other than topoisomerase I and appears to be less active than camptothecin analogs against tumor cells. Finally, some camptothecin analogs exhibit synergistic antifungal activity against yeast cells in combination with amphotericin B in vitro. Our results suggest that camptothecin and/or nitidine derivatives can exhibit potent antifungal activity and that the activities of camptothecin derivatives with existing antifungal drugs may be synergistic against pathogenic fungi. These new compounds, which exhibit potent antitumor activities, will likely require further structural changes to find more selective activity against fungal versus mammalian cells to hold promise as a new class of antifungal agents.     

侵袭性深部真菌感染45例临床和病原学分析

目的了解本院深部真菌感染的发病状况、病原菌特性及对常用抗真菌药的敏感度，为临床深部真菌感染的治疗药物选用提供依据。方法回顾性分析我院2004年1月1日12月31日血、无菌体腔液真菌培养阳性患者的病史，并对收集的病原菌进行体外药敏测定。结果根据诊断标准，确诊为深部真菌感染患者45例。其中社区获得性深部真菌感染22例，医院深部真菌感染23例。社区获得性深部真菌感染的部位以中枢神经系统最常见，共21例，占95．5％；其次为下呼吸道感染1例。医院深部真菌感染最常见的部位为血流感染，共15例，占65．2％；其次为中枢神经系统感染5例；下呼吸道感染2例；腹腔感染2例。社区获得性感染最常见的病原菌为新型隐球菌，共17株，占77．3％；其次为白念珠菌4株；烟曲霉1株。医院感染最常见的病原菌为白念珠菌，共8株；其次为其他念珠菌属。医院感染可能与住院天数较长、高龄、大手术、长时间应用广谱抗生素、深静脉置管等因素有关。结论我院2004年深部真菌感染确诊病例中，医院感染以白念珠菌所致的血流感染最常见；社区获得性感染以隐球菌性脑膜炎最多见。氟康唑仍是治疗敏感念珠菌属尤其是白念珠菌感染的有效药物。     

In vitro synergic antifungal effect of MUC7 12-mer with histatin-5 12-mer or miconazole

OBJECTIVES: MUC7 12-mer (RKSYKCLHKRCR), a cationic peptide derived from human salivary MUC7 mucin, exhibits potent in vitro antifungal activity, as determined by killing assays in phosphate buffer. In this study we examined the MUC7 12-mer antifungal activity alone or in combination with other antifungal agents in LYM medium (modified RPMI 1640). METHODS: Antifungal activities of MUC7 12-mer and other compounds against several fungal strains were first measured by MIC and minimum fungicidal concentration (MFC) tests using broth microdilution assay. The viability of Candida albicans and Cryptococcus neoformans were also determined by killing assays and time kinetics of peptide-mediated killing. Antifungal activities of MUC7 12-mer in combination with other compounds [histatin-5 (Hsn5) 12-mer: AKRHHGYKRKFH, amphotericin B or miconazole] against C. albicans and C. neoformans were determined by chequerboard assays and confirmed by killing assays. Toxicities of individual compounds were determined by haemolytic assays. RESULTS: MICs and MFCs of MUC7 12-mer ranged from 3.13 to 6.25 mg/L for most of the strains tested, and were, in most cases, comparable to those of amphotericin B and miconazole (0.78-6.25 mg/L). ED(50) values of MUC7 12-mer and Hsn5 12-mer were 7.1 and 7.4 micro M (or 11.2 and 11.6 mg/L), respectively, for C. albicans; and 1.2 and 1.1 micro M (or 1.9 and 1.7 mg/L), respectively, for C. neoformans. The killing of C. albicans and C. neoformans was achieved after 30 and 10 min exposure to the peptides, respectively. Combinations of MUC7 12-mer and Hsn5 12-mer, and of MUC7 12-mer and miconazole have a synergic antifungal effect on C. neoformans, with a fractional inhibitory concentration index (FICI) of 0.37 and 0.25, respectively; and a slightly lower than synergic effect on C. albicans, with a FICI of 0.63 and 0.56, respectively. In addition, using human erythrocytes, the two salivary peptides showed low levels of haemolytic activity. CONCLUSIONS: This study suggests that MUC7 12-mer and Hsn5 12-mer peptides may be suitable candidates for use in combination antifungal therapy.     

儿科患者中真菌分布及耐药监测

目的了解我院2000-2006年临床深部真菌感染分离的病原真菌种类及对抗真菌药物的耐药性变化。方法分析2000年1月～2006年12月年我院住院患儿所有送检真菌培养标本中分离出的菌株，药敏试验使用ATB-FUNGUS2INT酵母药敏试条，进行5-氟胞嘧啶、氟康唑和两性霉素B3种抗真菌药敏检测，严格按照2006年CLSIM27-A2规则及标准进行。结果分离出635株真菌中，念珠菌属572株（90．1％），曲霉属29株（4．6％），隐球菌属21株（3．3％），青霉属和酵母属各5株（0．8％），毛孢菌属2株（0．3％），毛霉属1株（0．2％）。念珠菌属中，前3位分离菌是白念珠菌418株（73．1％）；光滑念珠菌64株（11．2％）；热带念珠菌52株（9．1％）。白念珠菌对5-氟胞嘧啶、氟康唑和两性霉素B敏感率分别为97．6％，97．6％和98．4％。结论儿科患者中分离的真菌中以念珠菌属最多，并以白念珠菌为主，曲霉比率也在增多。5-氟胞嘧啶、氟康唑和两性霉素B均有较高的抗真菌活性。应加强真菌分离鉴定和耐药性监测，供合理选用抗真菌药物的参考。     

In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans.

There is currently no rapid, reliable, and reproducible in vitro technique to describe the growth-inhibitory interactions of antifungal drug combinations over a wide range of drug concentrations. We have developed a microdilution plate assay that was used to determine optimal drug combinations and concentrations of one-, two-, and three-drug regimens of amphotericin B (AmphB), fluconazole (FLU), and 5-fluorocytosine (5FC) for growth inhibition of three isolates each of Cryptococcus neoformans and Candida albicans. These growth inhibition data were then used in a multifactorial design technique to (i) generate contour and surface response plots to aid visual interpretation and (ii) develop mathematical equations describing the growth responses of the fungi to a wide range of antifungal concentrations and ratios. Our data indicated that (i) antifungal drug-drug interactions affecting yeast growth are complex functions of the drugs used in combination, their absolute concentrations, and also their relative (proportional) concentrations; (ii) AmphB-FLU combinations had additive effects against C. albicans over wide concentration ranges for each agent but were indifferent (i.e., were less than additive) in their inhibitory effect on C. neoformans; (iii) other two-drug combinations (FLU-5FC or AmphB-5FC) had indifferent effects on the growth of both fungi; and (iv) three-drug combinations (AmphB-FLU-5FC) showed an additive inhibitory effect on the growth of both C. albicans and C. neoformans. The finding that no antagonism was observed in combinations employing AmphB and FLU in this in vitro model is of critical importance since it argues against the current theoretical concept, based on the individual drug's mode of action, of antagonism between these two drugs. These microdilution techniques provide a method to determine rational regimens of antifungal agents in multidrug combinations for future testing to correlate in vitro activity with in vivo response. The use of this approach has made the evaluation of complex antifungal drug-drug interactions possible and provided important new information to the evolving field of antifungal drug combination.     

Candida albicans adhesion to and invasion and damage of vaginal epithelial cells: stage-specific inhibition by clotrimazole and bifonazole

Clotrimazole and bifonazole are highly effective antifungal agents against mucosal Candida albicans infections. Here we examined the effects of low levels of clotrimazole and bifonazole on the ability of C. albicans to adhere, invade, and damage vaginal epithelial cells. Although adhesion and invasion were not affected, damage was greatly reduced upon azole treatment. This clearly indicates that low levels of azoles influence specific activities of C. albicans during distinct stages of vaginal epithelium infections.     

386例假丝酵母菌菌种分布及药敏结果分析

目的 分析假丝酵母菌属临床感染的种类分布及药物耐药性.方法 采用VITEK-AMS全自动微生物鉴定系统、酵母样真菌鉴定卡YBC进行鉴定和药敏实验.结果 所分离的386例假丝酵母菌属中,白色假丝酵母菌占第一位,为64.2%;其次为热带假丝酵母菌,占20.7%;白色假丝酵母菌对唑类的耐药率较低(氟康唑0.6%,酮康唑1.2%);热带假丝酵母菌对唑类的耐药率较白色假丝酵母菌高(氟康唑6.2%,酮康唑7.5%);光滑假丝酵母菌对唑类的耐药率较高(氟康唑16.7%,酮康唑13.3%);克柔假丝酵母菌对氟康唑的耐药率达84.2%.结论 假丝酵母菌属的感染种类不断增多,耐药性上升,对易感患者应加强监测,分析发病原因,合理应用抗菌剂,以延缓和防止真菌耐药性的进一步发展.     

Overexpression of Candida albicans CDR1, CDR2, or MDR1 does not produce significant changes in echinocandin susceptibility

The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.     

MUC7 20-Mer: investigation of antimicrobial activity,secondary structure,and possible mechanism of antifungal action

This study was aimed at examining the spectrum of antimicrobial activity of MUC7 20-mer (N-LAHQKPFIRKSYKCLHKRCR-C; residues 32 to 51 of MUC7, the low-molecular-weight human salivary mucin, comprised of 357 residues) and comparing its antifungal properties to those of salivary histatin 5 (Hsn-5). We also examined the secondary structure of the 20-mer and the possible mechanism of its antifungal action. Our results showed that MUC7 20-mer displays potent killing activity against a variety of fungi and both gram-positive and gram-negative bacteria at micromolar concentrations. Time-dependent killing of Candida albicans and Cryptococcus neoformans by MUC7 20-mer and Hsn-5 indicated differences in killing rates between MUC7 20-mer and Hsn-5. The secondary structure prediction showed that MUC7 20-mer adopts an amphiphilic helix with distinguishable hydrophilic and hydrophobic faces (a characteristic that is associated with antimicrobial activity). In comparison to that of Hsn-5, the fungicidal activity of MUC7 20-mer against C. albicans seems to be independent of fungal cellular metabolic activity, as evidenced by its killing potency at a low temperature (4 degrees C) and in the presence of inhibitors of oxidative phosphorylation in the mitochondrial system. Fluorescence microscopy showed the ability of MUC7 20-mer to cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either C. albicans or C. neoformans.