CLONING AND CHARACTERIZATION OF A GENE (LIP1) WHICH ENCODES A LIPASE FROM THE PATHOGENIC YEAST CANDIDA-ALBICANS

Extracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes, The opportunistic pathogen Candida albi cans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encod ing 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 activ ity. Two identical clones were obtained which exhibited lipolytic acti vity. Nucleotide sequence analysis identified an ORF encoding a protei n of 351 amino acid residues. Although no extensive homologies were id entified, the sequence contained the Gly-X-Ser-X-Gly motif found in pr okaryotic and eukaryotic lipases, suggesting a similar activity for th e encoded protein. Indeed, culture supernatants from complemented yeas t 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 bl ot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilos is, Candida tropicalis and Candida krusei, but not in Candida pseudotr opicalis, Candida glabrata or S. cerevisiae, Northern blot analyses sh owed that expression of the LIP1 transcript, the cloned gene which enc odes a lipase, was detected only when C. albicans was grown in media c ontaining Tween 80, other Tweens or triglycerides as the sole carbon s ource, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose m edia. Additionally, carbohydrate supplementation inhibited LIP1 expres sion, Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this ge ne in candidal virulence.