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Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence
Attila Gácser, … , Wilhelm Schäfer, Joshua D. Nosanchuk
Attila Gácser, … , Wilhelm Schäfer, Joshua D. Nosanchuk
Published October 1, 2007
Citation Information: J Clin Invest. 2007;117(10):3049-3058. https://doi.org/10.1172/JCI32294.
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Research Article Microbiology

Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence

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Abstract

Candida parapsilosis is a major cause of human disease, yet little is known about the pathogen’s virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection models that involve inoculation of reconstituted human oral epithelium or murine intraperitoneal challenge. These studies represent what we believe to be the first targeted disruption of a gene in C. parapsilosis and show that C. parapsilosis–secreted lipase is involved in disease pathogenesis. This efficient system for targeted gene deletion holds great promise for rapidly enhancing our knowledge of the biology and virulence of this increasingly common invasive fungal pathogen.

Authors

Attila Gácser, David Trofa, Wilhelm Schäfer, Joshua D. Nosanchuk

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Figure 1

Gene deletion in C. parapsilosis.

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Gene deletion in C. parapsilosis.
               
(A) Design of gene tar...
(A) Design of gene targeting for the C. parapsilosis lipase locus showing the SAT1 flipper cassette with the homologous C. parapsilosis lipase fragments 5′ LIP and 3′ LIP as well as the FLP recombination target sequences (FRT). The probe used to verify correct integration and deletion of the SAT1 flipper by Southern blot hybridization is represented by a black bar. (B) Southern blot hybridization analysis of genomic DNA (PacI, AvrII, double digested) isolated from the WT GA1 (lane 1), the heterozygous mutant CpLIP1-2/Δcplip1-2:SAT1-FLIP (HebFLP) before FLP activation (lane 2), the heterozygous mutant CpLIP1-2/Δcplip1-2:FRT (HE) after excision of SAT1 flipper cassette (lane 3), the homozygous mutant Δcplip1-2/Δcplip1-2:SAT1-FLIP (KobFLP) before FLP activation (lane 4), the homozygous mutant Δcplip1-2/Δcplip1-2:FRT (KO) after excision of SAT1 flipper cassette (lane 5), the reintegration mutant Δcplip1-2/Δcplip1-2/CpLIP2:SAT1-FLIP (RebFLP) before FLP activation (lane 6), and the reconstituted mutant Δcplip1-2/Δcplip1-2/CpLIP2:FRT (RE) after FLP activation (lane 7). Diagrams of the structures and size of the hybridization fragments are shown at right. (C) Nou selection of caSAT1-containing (NouR) transformants (left) and screening for NouS colonies after FLP activation (right). Arrows indicate small NouS colonies.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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