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

Abstract

The Candida albicans PLB1 gene 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 pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a preprotein 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.

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast

“…Sequence-related fungal PLBs include Candida albicans (accession number U59710) [12], and related proteins from S. cerevisiae (accession number L23089) [14], P. notatum (accession number P39457) and T. Delbrueckii (accession number Q11121). Together with the PLB recently identified from the pathogenic fungus, Cryptococcus neoformans [16], these PLBs are secreted enzymes, are heavily glycosylated and are probably involved in virulence in some cases [2].…”

“…In the microorganism Moraxella bovis, the plb gene encodes a protein of 616 amino acids and the enzyme hydrolyses PC/LPC (lysoPC) to produce NEFAs (non-esterified fatty acids, also known as free fatty acids) and glycerophosphorylcholine (GPC) [1]. PLB enzymes have been cloned from different fungi, including Candida albicans [12], Saccharomyces cerevisae [13][14][15], Cryptococcus neoformans [16], Penicillium notatum and Torulaspora delbruekii, and are related by sequence [2], but not to M. bovis PLB. The fungal enzymes when examined indicate that PC is their main substrate.…”

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

“…In C. albicans, LPL, LPTA and PLB activities were identified in crude culture filtrates of the organism, but only enzymes with LPTA activity were purified [18][19][20] ; no PLB activity was detected in purified enzyme preparations [18,21]. Candidal phospholipase activity has been correlated with virulence in mice and may contribute to pathogenicity by damaging host cell membranes [21].…”

High levels of hydrolytic enzymes secreted by Candida albicans isolates involved in respiratory infections

“…Secreted aspartyl proteinases are capable of degrading epithelial and mucosal barrier proteins such as collagen, keratin and mucin, as well as antibodies, complement and cytokines. Cloning and disruption of the genes for these enzymes have shown their involvement in Candida virulence Sanglard et al, 1997;Leidich et al, 1998;Watts et al, 1998;De Bernardis et al, 1999).…”