Generation of Fluorescent Protein Fusions in <em>Candida</em> Species

Abstract

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis

“…In part, the reason for this is that the number of the molecular tools available that lags behind those existing for C. albicans. However, although several knock-out techniques have been developed to date, no locus has been described as an acceptor sequence for knock-in approaches in C. parapsilosis (Ding and Butler, 2007;Gácser et al, 2007;Holland et al, 2014;Gonia et al, 2017). The goal of this study was to identify and characterize a genomic region for integrating ectopic DNA in C. parapsilosis and develop compatible plasmids.…”

“…Most of the large colonies were confirmed by molecular methods in terms of the correct integration, but we did not know why the small colonies appear. Gonia et al (2017) reported that the transformation procedure represents a serious stress to the cells, that could be an explanation for this phenomenon. This might affect the initial growth rate of the transformants after plating that might result in smaller colonies by the end of the incubation period.…”

“…This effort, however, was complicated due to the absence of a characterized locus for knock-in approaches and a lack of compatible vectors. Recently a protein tagging method was described by Gonia and coworkers, where one allele of the ortholog of C. albicans enolase 1 was fluorescently labeled in C. parapsilosis (Gonia et al, 2017). Nonetheless, the goal of this work was to provide a tool for protein tagging in this species and was presented as a proof of principle rather than introducing a general locus for accepting exogenous sequences.…”

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

“…Fluorescence imaging of this organism has been performed on both a macro scale (2) and on a subcellular scale, though both approaches tend to make use of overexpression of specific targets labeled with a range of fluorescent proteins (3)(4)(5)(6).…”

“…This opportunistic fungal pathogen can cause bloodstream infections, also called candidemia, which has a crude mortality rate of approximately 40% (1). Fluorescence imaging of this organism has been performed on both a macro scale (2) and on a subcellular scale, though both approaches tend to make use of overexpression of specific targets labeled with a range of fluorescent proteins (3-6). Imaging of labeled molecules is often hampered by autofluorescence, which can originate from the presence of many weakly fluorescent molecules such as vitamin B 2 (11, 12).…”