CLONING AND DISRUPTION OF CAPLB1, A PHOSPHOLIPASE-B GENE INVOLVED IN THE PATHOGENICITY OF CANDIDA-ALBICANS

The Candida albicans PLB1 gene was cloned using a polymerase chain rea ction-based approach relying on degenerate oligonucleotide primers des igned according to the amino acid sequences of two peptide fragments o btained from a purified candidal enzyme displaying phospholipase activ ity (Mirbod, F., Banno, Y., Ghannoum, M. A, Ibrahim, A. S., Nakashima, S., Yasuo, It., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181-188). Sequence analysis of a 6.7-kilobase pair EcoRI-Cl aI genomic clone revealed a single open reading frame of 1818 base pai rs that predicts for a preprotein of 605 residues. Comparison of the p utative candidal phospholipase with those of other proteins in data ba se revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspor a delbrueckii (48%), and Schizosaccharomyces pombe (38%). Thus, we hav e cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption exper iments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB 1, as assessed in a murine model for hematogenously disseminated candi diasis, was significantly attenuated compared with the isogenic mild-t ype parental strain. Although deletion of caPLB1 did not produce any d etectable effects on candidal adherence to human endothelial or epithe lial cells, the ability of the caplb1 null mutant to penetrate host ce lls was dramatically reduced. Thus, phospholipase B may well contribut e to the pathogenicity of C. albicans by abetting the fungus in damagi ng and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.