Cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans

Abstract

Extracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes. The opportunistic pathogen Candida albicans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encoding secreted phospholipases, Saccharomyces cerevisiae was transformed with a C. albicans genomic library and screened for lipolytic activity on egg-yolk agar plates, a traditional screen for phospholipase activity. Two identical clones were obtained which exhibited lipolytic activity. Nucleotide sequence analysis identified an ORF encoding a protein of 351 amino acid residues. Although no extensive homologies were identified, the sequence contained the Gly-X-Ser-X-Gly motif found in prokaryotic and eukaryotic lipases, suggesting a similar activity for the encoded protein. Indeed, culture supernatants from complemented yeast cells contained abundant hydrolytic activity against a triglyceride substrate and had no phospholipase activity. The data suggest that C. albicans, in addition to phospholipases, also has lipases. Southern blot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis, Candida glabrata or S. cerevisiae. Northern blot analyses showed that expression of the LIP1 transcript, the cloned gene which encodes a lipase, was detected only when C. albicans was grown in media containing Tween 80, other Tweens or triglycerides as the sole carbon source, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose media. Additionally, carbohydrate supplementation inhibited LIP1 expression. Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this gene in candidal virulence.