Yoshinori Nozawa

Cloning and Disruption of caPLB1, a Phospholipase B Gene Involved in the Pathogenicity of Candida albicans

The Candida albicans PLB1gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M. A., Ibrahim, A. S., Nakashima, S., Yasuo, K., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181–188). Sequence analysis of a 6.7-kilobase pairEcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues. Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae(45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe(38%). Thus, we have cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain. Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced. Thus, phospholipase B may well contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.

Anti-candidal activity of GBR-14206, a new imidazole derivative: Intravenous administration in lipid emulsion form.

GBR-14206, a new imidazole derivative, has been evaluated against systemic infection with Candida albicans in normal and immunocompromised mice. The therapeutic effect of GBR-14206 (as a lipid emulsion) given intravenously to mice infected systemically with C. albicans was superior to that of miconazole. GBR-14206 also showed a candicidal effect in the blood stream at the therapeutic dose. In addition, GBR-14206 inhibited the hyphal growth (yeast to hyphal transformation) in Eagles minimum essential medium and methionine synthetic medium at the respective concentrations of 0.20 and 0.05μg/ml, which were approximately 3 to 7 times more active than miconazole.These properties suggested that GBR-14206 may be useful for the therapy of systemic candidiasis in human.