The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/defetion of the FASZ gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (Aura3:: imm434 /AuraS::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-hisC sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFDI 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.

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. kruseiwere 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.

A systematic screen for new natural products that displayed antifungal activity by inhibition of fungal fatty acid synthase (FAS) led to the discovery of two new fungal metabolites, designated CT2108A (1) and CT2108B (2). The metabolites were produced by Penicillium solitum (Westling) strain CT2108 and were classified as azaphilones. The structures of these new metabolites were determined using a variety of 1D and 2D NMR experiments, including COSY, HMQC, and HMBC. The chemical conversion of CT2108A to CT2108B was effected using WCl(6). The related metabolite, patulodin (3), was also isolated from the fermentation culture of this P. solitum isolate. Both new compounds inhibited fungal FAS, and neither was found to significantly inhibit human FAS activity.

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.

Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Simple device removal is often impossible, while in addition, resulting candidal infections are difficult to resolve due to their increased resistance to many antifungal agents. Susceptibility studies of clinical isolates are generally performed according to the CLSI standard, which measures planktonic cell susceptibility, but similar standards have not been designed or applied to testing of cells growing within a biofilm. As consistent biofilms from many strains are more difficult to simultaneously obtain and analyze than are independent planktonic cultures, any standard assay must address these concerns. In the present chapter, optimized conditions that promote biofilm formation within individual wells of microtiter plates are described. In addition, the method has proven useful in preparing C. albicans biofilms for investigation by a variety of microscopic and molecular techniques.