A new type of DNA polymorphism identified in the species-specific DNA region originating from the Candida albicans mitochondrial genome.

Mitochondrial DNA of the pathogenic yeast Candida albicans contains a species-specific DNA region, designated EO3, within the duplicated region in its stem-and-loop structure, as reported in our previous paper. We have revealed that the EO3 region exhibits a DNA-size polymorphism, yielding three fragment length types: L, M, and S. This polymorphism is due to the presence (or absence) of two small nucleotide sequences of ~50 bp (designated a and b) within the EO3 region: type L possesses both a and b, type M possesses a alone, and type S possesses neither a nor b. Here we have identified a new type of EO3, which possesses b but not a, designated type M-II. Restriction fragment analyses with BglII revealed accurate discrimination of EO3 into four types: L, M-I, M-II, and S. Moreover, we show that these small nucleotide segments a and b were encompassed by the characteristic nucleotide sequence with a common inverted repeat structure. In the present report we propose a revised version of EO3 typing, suggesting epidemiological usefulness as an alternative tool for species-specific detection of C. albicans, and discuss the potentiality of EO3 for generating more variations of DNA polymorphism.

Simple method to accurately differentiate Candida albicans isolates concurrently using polymorphic patterns of PCR-amplified, species-specific nuclear and mitochondrial targets.

We describe a simple method to accurately differentiate Candida albicans isolates by concurrent use of the restriction enzyme digestion patterns for PCR products, targeting two species-specific DNA regions originating from genetically different sources, the nuclear and mitochondrial genomes. The target sequence we used as the nuclear gene was derived from the PHO85 gene, a negative regulator of the PHO system, in which we found a restriction size polymorphism within the two alleles of PHO85 in the diploid genome of this fungus. The mitochondrial target was derived from EO3, a species-specific DNA fragment possessing a small size polymorphism among various clinical isolates. Our results should provide a new tool for molecular epidemiological surveys of patients suffering from candidiasis caused by C. albicans.

[Essential genes as potential targets of antifungal agents in pathogenic yeast Candida].

An important point in the development of an antimicrobial agent is whether its target molecules are essential for growth of the microorganism. From this viewpoint, we focused attention on essential genes as potential targets of antifungal agents in the pathogenic yeast Candida. Here we introduce recent attempts for screening, identification, and characterization of essential genes from a haploid yeast Candida glabrata, using temperature-sensitive mutants. Our experimental results suggesting the essentiality of C. albicans PHO85, the homologue of which is known as a negative regulator of the PHO system and as a non-essential gene in Saccharomyces cerevisiae are also described.

[Isolation and molecular characterization of the CaPHO85 gene: a negative regulator of phosphate metabolism (PHO system) in Candida albicans].

The PHO system is an ingenious mechanism by which the yeast Saccharomyces cerevisiae regulates the expression of a set of genes involved in phosphate metabolism in response to the change of phosphate concentrations in the environment. A key factor in this mechanism is the Pho85 kinase, which has been discovered as a negative regulator of the PHO system. One of the genes isolated in our laboratory in screening the protein kinase genes from Candida albicans was identified as a homologue (CaPHO85) of the PHO85 of S. cerevisiae, based on the following results. a) Pho85 is the polypeptide with the highest homology to CaPho85 (62% identity) among the S. cerevisiae genome sequence. b) The position of insertion of the intron is quite similar between CaPHO85 (in the 7th codon of the N-terminal MTGSSSQ) and S. cerevisiae PHO85 (in the 6th codon of the N-terminal MSSSSQ). c) The nucleotide sequences in the intron possess the consensus sequences for yeast intron: the 5-splice-site, internal, and the 3-splice-site sequence. d) CaPHO85 complemented the S. cerevisiae pho85 mutation. e) CaPho85 contains all of the consensus sequences for the ATP-binding domain and for the kinase domain found in S. cerevisiae Pho85.

Effect of amphotericin B dilution with various beverages on the survival of Candida albicans cells.

Amphotericin B (AMPH) has been generally used for prophylaxis or treatment of specific fungal diseases in immunocompromised patients. However, because it is difficult for children to ingest, mainly because of its bitter taste, it is often diluted with soft drinks. We therefore investigated the effect of dilution of AMPH with various beverages on its antifungal activity in vitro. Candida albicans cells were exposed for 30 min to AMPH diluted twofold with each of six commercially available beverages or distilled water, and percent survival was determined. The results showed 60% survival in the dilution with distilled water and higher survival when diluted with Yakult (136%; p < 0.01), orange juice (104%; p < 0.01), and coffee-milk (92%; p < 0.01). By contrast, lower survival was obtained when diluted with gum-syrup (54%), sweet cider (76%), and shaved-ice syrup (52%) with no significant differences from distilled water (60%), suggesting that these three beverages may be useful for diluting AMPH. The results of this study are a warning to medical workers that some methods of making AMPH more palatable considerably decrease its antifungal activity and may have a negative effect on host defenses against infectious diseases.

[Molecular biological approach to screening essential genes as potential targets for antifungal targets in pathogenic yeast Candida].

  We established a system, named ETS system, for screening and identification of essential genes from the pathogenic haploid yeast Candida glabrata by using temperature-sensitive (ts) mutants. Based on the general concepts that ts mutations are generated within essential genes in the genome by virtue of point mutation, the ETS system enabled us to screen and identify a variety of essential genes from the C. glabrata genomic DNA library as the genes that complement ts mutations. The ETS system established in the present study may provide novel potential antifungal targets.