Synergy of nitric oxide and azoles against Candida species in vitro.

The candidacidal activity of nitric oxide (NO) as delivered by a class of compounds termed diazeniumdiolates has been investigated. Diazeniumdiolates are stable agents capable of releasing NO in a biologically usable form at a predicted rate, and three such compounds were examined for activity. One compound, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA-NO), proved to be most suitable for examining NO activity due to its relatively long half-life (20 h) and because of limited candidacidal activity of the uncomplexed DETA nucleophile. DETA-NO was active against six species of Candida for which the MICs necessary to inhibit 50% growth (MIC50s) ranged from 0.25 to 1.0 mg/ml. C. parapsilosis and C. krusei were the most susceptible to the compound. In addition to a determination of NO effects alone, the complex was utilized to investigate the synergistic potential of released NO in combination with ketoconazole, fluconazole, and miconazole. Activity was investigated in vitro against representative strains of Candida albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, and C. dubliniensis. Determination of MIC50, MIC80 and MICs indicated that DETA-NO inhibits all strains tested, with strains of C. parapsilosis and C. krusei being consistently the most sensitive. The combination of DETA-NO with each azole was synergistic against all strains tested as measured by fractional inhibitory concentration indices that ranged from 0.1222 to 0.4583. The data suggest that DETA-NO or compounds with similar properties may be useful in the development of new therapeutic strategies for treatment of Candida infections.

Avirulence of Candida albicans FAS2 mutants in a mouse model of systemic candidiasis.

Disruption of both alleles of the Candida albicans FAS2 gene abolishes the ability of the organism to establish infection in a murine model of systemic candidiasis. Within 72 h all mice inoculated with 10(6) CFU of the parental C. albicans strain had died. In contrast, all animals inoculated with the mutant strain CFD2 survived for the course of the experiment (21 days). Animals infected with either mutant strain CFD1 or CFD3, in which only one FAS2 allele was disrupted, also succumbed to infection, but mortality was not observed until 4 days postinfection and survivors remained for up to 20 days postinfection. The results demonstrate that FAS2 is required for successful C. albicans infection.

Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis.

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.

Purification and characterization of fatty acid synthase from Candida albicans strain 4918 and two derived spontaneous cerulenin-resistant mutants.

Fatty acid synthase from three strains of Candida albicans (parental strain 4918, and two spontaneous cerulenin-resistant mutants, 4918-2 and 4918-10) has been purified and characterized. In all three cases the purification protocol included ammonium sulfate precipitation, fractionation with butyl-Toyopearl, differential centrifugation and sedimentation velocity centrifugation. Inclusion of protease inhibitors, aprotinin, leupeptin and pepstatin was a prerequisite to maximize recoveries. Polyacrylamide gel electrophoresis analysis demonstrated protocol efficacy and showed the apparent molecular mass of the two enzyme sub-units from each strain to be 195 kDa and 210 kDa. The Km (malonyl-CoA) and Vmax of each fatty acid synthase were similar. In contrast, inactivation kinetics of the respective enzymes in the presence of cerulenin showed enzyme activity from both mutants to differ significantly from the parent and from each other. Other experiments suggested in vivo cerulenin resistance of mutant strains is not solely attributable to enzyme alteration.

Phenotypic variation of a virulent Candida albicans strain and two spontaneous, relatively avirulent mutant strain derivatives.

The effect of media and temperature of incubation on colony phenotype of Candida albicans strain 4918, and two relatively avirulent mutant strains, designated 4918-2 and 4918-10, has been investigated. In addition, the strains were characterized on the basis of morphotyping pattern. Colony phenotypes were determined for cultures grown on either Lee's medium supplemented with arginine and zinc, or M63 medium supplemented with casamino acids. Incubation was at either 24 or 37 degrees C for 7 days. The results demonstrated that the predominant colony phenotype observed at 24 degrees C was different from that at 37 degrees C for all three strains, irrespective of the medium. While the growth medium influenced the specific colony phenotypes observed, as well as their categorical distribution, no significant medium effect on switching frequency was apparent. The switching repertoire of strain 4918-10 was consistently more varied than either the parental strain or 4918-2 under the conditions examined. However, categorization of the colony phenotypes shown by the three strains suggested that the pattern exhibited by strain 4918-2 was distinct from that of the other two strains. In addition, individual primary colonies of each phenotype observed were clonally plated in order to examine further the switching frequencies. The results established that all three strains were capable of high frequency switching. Other experiments demonstrated that morphotypes of all three strains were different from one another as expected from the differences in their virulence reported previously.

Mannan composition of the hyphal form of two relatively avirulent mutants of Candida albicans.

We have previously reported the characteristics of mannans isolated from the yeast forms of two relatively avirulent Candida albicans strains, designated 4918-2 and 4918-10. Investigations have been expanded to include an analysis of mannans from the hyphal form of these strains as well as from the hyphal form of the parental strain, 4918. After extraction, mannans were further purified by high-pressure liquid chromatography on a Bio-gel TSK DEAE-5-PW column. Subsequent to either mild acid hydrolysis, alkali hydrolysis, or acetylation followed by acetolysis, the resulting products were fractionated by high-pressure liquid chromatography on an Aminex HPX-42A column. The results of acid hydrolysis showed only minor quantitative differences in the products released from each strain, with mannose constituting the vast majority of product liberated. The profiles of mannooligosaccharides obtained from either alkali hydrolysis or acetolysis for strain 4918-2 showed distinct quantitative differences compared with profiles of the other two strains. Finally, a general characteristic noted is a decrease in the average chain length of mannooligosaccharides in hyphal mannans compared with the yeast counterpart.