The nucleotide sequence determination of catalases of three medically important yeasts using newly designed degenerated primers.

We have developed degenerated primers for the isolation of several fungal species catalases, based on the known catalase genes of several yeast species. Using a combination of degenerated primers and the nested polymerase chain reaction (PCR) method, we were able to obtain PCR products from Candida dubliniensis, C. tropicalis, and C. glabrata. The nucleotide sequence of the PCR products amplified showed that those fragments contained sequences homologous with the known Candida catalases, indicating the usefulness of the designed primers. We determined the nucleotide sequences of the open reading frames and respective 5' untranscribed regions of these yeasts and compared each sequence with that of the respective related species. The difference between the deduced amino acid sequence of catalase of C. dubliniensis and C. albicans was 5 in 485 amino acids. The nucleotide sequence of C. glabrata catalase was identical to the sequence results from the genome sequence project which was recently released, whereas that of the catalase of the C. tropicalis clinical isolate was not the same as the strain Pk233, n-alkane-utilizing C. tropicalis. The catalase activities of all the strains tested so far were activated by short-term hydrogen peroxide treatment, suggesting that common mechanisms were involved in the induction of catalase activity, although the nucleotide sequences of the 5' untranscribed region of these yeasts were diversified.

Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents.

Catalase-deficient strains of the human pathogenic yeast Candida albicans were constructed using the URA-blaster method. The disruptant was viable and grew normally in an ordinary culture condition, but became extremely sensitive to treatment with hydrogen peroxide. No catalase activity was observed in a catalase (CCT)-gene-disrupted strain, 1F5-4-1, suggesting that there were no other catalase or catalase-like enzymes in this yeast. The disruptant was shown to be sensitive to higher temperature and to low concentrations of SDS, NP-40, or Triton X-100. After a wild-type CCT gene was reintroduced into the disruptant, catalase activity was restored and the strain became moderately sensitive to treatment with hydrogen peroxide. However, neither the temperature sensitivity nor the susceptibility to SDS observed in the disruptant was restored in the CCT-reintroduced strain. A model infection experiment using wild-type and dCCT strains showed that the disruptants disappeared more rapidly than the wild-type strain in mouse liver, lung, and spleen. These results suggest that the catalase plays a significant role in survival in the host immune system and thus leads this organism to establish infection in the host.

Repetitive sequences (RPSs) in the chromosomes of Candida albicans are sandwiched between two novel stretches, HOK and RB2, common to each chromosome.

A novel sequence designated HOK, which is next to the RPS, a repetitive sequence specific to Candida albicans, was cloned and sequenced. HOK hybridized with all of the chromosomes on which the RPSs were located, but did not hybridize with chromosome 3, which does not harbour any RPSs. Sequence determination revealed that a portion of HOK has significant homology with the B and C1 fragments of Ca3, which is used as a molecular epidemiological probe. A homology search of the deduced amino acids of HOK against the protein database showed partial homology with an isocitrate dehydrogenase of Saccharomyces cerevisiae, although an ORF large enough to encode the enzyme was not detected. To verify the existence of other sequences homologous with HOK, a portion of the HOK sequence was amplified using PCR. Sequence determination of the 41 clones from the PCR products resulted in at least six HOK-homologous clones. Another RPS-containing clone, RB2, was isolated from the Pstl-digested chromosome R or 1. It was determined that RB2a, one of the subclones from RB2, hybridized with all of the chromosomes, including chromosome 3, with which neither HOK nor RPS hybridized. The hybridization profile also showed that RPS is located between HOK and RB2a on chromosomes other than chromosome 3.