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.

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

“…Extracellular protease secretion was assayed on unbuffered bovine serum albumin (BSA) plates (29), and lipase secretion was assayed on plates containing unbuffered YNB plus 2.5% Tween 80 (30). All plates were prepared with or without DOX.…”

Candida albicans VPS11 Is Required for Vacuole Biogenesis and Germ Tube Formation

“…Secreted aspartyl proteinase (SAP) activity was induced in liquid culture with 0.2% bovine serum albumin (BSA) as a nitrogen source and was assayed on agar containing BSA as described by Crandall and Edwards (12). Secreted lipase activity was detected on egg yolk-containing agar as described by Fu et al (16). In both assays an opaque ring around the colony is indicative of secreted enzymatic activity.…”

Candida parapsilosis Secreted Lipase as an Important Virulence Factor

“…albicans is known to have 10 lipase encoding genes, one (LIP1) characterized by Fu et al [44] and nine (LIP2-LIP10) by Hube et al [31]. Further examinations of C. albicans lipases revealed that individual genes are regulated differently during an infection and gene expression of each ORF was mainly dependent upon the stage of infection [45].…”

“…Fu et al published the first report that suggested the presence of secreted lipases in C. parapsilosis in 1997 [44]. Five years later, Neugnot et al identified two lipase encoding ORFs in this species [51].…”