guanosine nucleotides by treatment with mycophenolic acid also resulted in increased susceptibility to AmB, with the compounds displaying synergistic interactions in checkerboard MIC assays. A ura2 mutant 
of Candida albicans was likewise hypersensitive to AmB, but uracil as well as uridine ATL-962 site supplementation was able to reverse this effect. Treatment with MPA and 5-FC also reduced AmB MIC for C. albicans, whereas neither drug affected Aspergillus fumigatus AmB susceptibility. Additionally, the C. neoformans ura2 mutant was found to have capsular defects and supplementation with uracil or uridine restored capsule formation suggesting that capsule biosynthesis draws from the pool of salvaged pyrimidine nucleotides. Materials and Methods Strains Studies with Cryptococcus neoformans were performed with wild type Cryptococcus neoformans var grubii strain H99. A C. neoformans ura2 mutant strain was generated by screening for spontaneous ura2 mutant on Yeast Extract Peptone Dextrose agar supplemented with 1 mg/mL 5-Fluoroorotic acid. To confirm a typical mutation of the URA5 gene, PCR primers E-Test of C. neoformans wt on AM3 medium AM3 medium supplemented with 3.25 mg/mL MPA and E-test analysis for AmB susceptibility of C. neoformans wt on AM3 supplemented with 3.25 mg/ mL MPA and 20 mg/mL exogenous guanine. doi:10.1371/journal.pone.0087246.g002 GATACAAGC-39) PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19649254 were designed to amplify the URA5 gene and the purified PCR product was transformed in ura2 mutant by electroporation following which complement strains were screened for the ability to grow in minimal media, in the absence of exogenous uracil, confirming uracil prototrophy. Wild type Candida albicans SC5314 and C. albicans ura2 mutant CAI-4 were kind gifts from Dr. Mira Edgerton, University at Buffalo. Wild type Aspergillus fumigatus strain H237 used in this study was a gift of Dr. Judith Rhodes, University of Cincinnati. All yeast strains were stored in YPD-30% glycerol at 280uC and were subcultured on YPD agar prior to the experiments. The A. fumigatus strain was stored at 4uC on potato dextrose agar slants, subcultured on same media and incubated for 47 days for optimal conidiation prior to experiments. All assays were performed in triplicate. Media and Chemicals Yeast strains were sub-cultured on Yeast Extract Peptone Dextrose agar and A. fumigatus strain was grown on Potato Dextrose Agar. YPD broth was used for growing starter cultures of all yeast strains. Antibiotic medium 3 buffered to pH 7.0 with 10 mM phosphate was used for E-test with all strains. For checkerboard assays with combination of AmB and MPA, AM3 broth supplemented with 2% dextrose was used. RPMI 1640 medium supplemented with 2% dextrose and buffered by 0.165 3propanesulfonic acid and 10 M NaOH to pH 7.0 was used for analysis of AmB and 5-FC combination checkerboard assays. E-test strips containing graded concentration of AmB were supplied by. AmB was 3 Nucleotide Biosynthesis and Amphotericin B obtained from Cellgro, Mediatech Inc. as an aqueous solution. Stock solution of 100 mg/mL was prepared in sterile distilled water; filter sterilized and stored at 220uC. MPA was dissolved in 99% ethanol to make a 12.5 mg/mL stock solution, 100 mL aliquots of the same was placed in microcentrifuge tubes and stored at 220uC. 5-FC was purchased from Sigma Aldrich and dissolved in water to prepare a 2 mg/mL stock solution. Guanine, uracil and uridine bases were obtained as reagent grade powders. 20 mg/mL guanine stock solution wa
