title: PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis.
abstract: Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Gonia
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Britta
authors.initials: B
authors.lastname: Larson
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Gale
mesh_terms: D001426:Bacterial Proteins; D002175:Candida / Q000166:cytology / Q000235:genetics*; D002177:Candidiasis / Q000382:microbiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria
keywords: Candida parapsilosis; NAT1; PCR-mediated gene modification; clinical isolates; fluorescent proteins