Article

Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants

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Microbial Pathogenesis (Impact Factor: 1.79). 03/2006; 40(2):69-81. DOI: 10.1016/j.micpath.2005.11.002
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ABSTRACT

The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

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    • "The number and variety of molecular tools available for genetic manipulation of C. albicans have increased greatly in the past decade with the development of auxotrophic and drug-resistance markers, epitope tags, fluorescent tags and constitutive or regulatable promoters (Backen et al., 2000; Barelle et al., 2004; Basso et al., 2010; Beckerman et al., 2001; Care et al., 1999; Doyle et al., 2006; Enloe et al., 2000; Finkel et al., 2011; Gerami-Nejad et al., 2001, 2009, 2004, 2012; Gola et al., 2003; Keppler-Ross et al., 2008; Lai et al., 2011; Lebel et al., 2006; Murad et al., 2000; Nobile & Mitchell, 2009; Oh et al., 2010; Park & Choi, 2002; Reijnst et al., 2011; Reuß et al., 2004; Sánchez-Martínez & Pérez- Martín, 2002; Shen et al., 2005; Srikantha et al., 1996; Staab et al., 2003; Stynen et al., 2010; Umeyama et al., 2002; Vieira et al., 2010; Wilson et al., 2000; Xu et al., 2011). Available vectors for the integration of genes into ectopic locations in the C. albicans genome target the HIS1 promoter (orf19.4026), the ENO1 promoter (orf19.935) "
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    ABSTRACT: Candida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, NEUT5L, which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to NEUT5L. These vectors allow rapid, facile cloning through in vivo recombination (gap repair) in Saccharomyces cerevisiae and efficient integration of the construct into the NEUT5L locus. Other useful features of these vectors include a choice of three selectable markers (URA3, the recyclable URA3-dpl200, or NAT1), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into C. albicans, thereby reducing the insert size and preventing integration of non-C. albicans DNA. Importantly, unlike the commonly used RPS1/RP10 locus, integration at NEUT5L has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in C. albicans.
    Full-text · Article · Jan 2013 · Microbiology
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    • "However, when light emission was studied from constitutively active promoters, it was noted that bioluminescence was significantly lower in hyphae than in yeast cells although cell-free extracts from both cell types revealed similar bioluminescence levels. Thus, it was concluded that the uptake of luciferin might be hampered by the reorganised cell wall in C. albicans hyphae [54]. However, the same group also investigated the suitability of the system for studying pathogenesis in mice by BLI [8]. "
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    ABSTRACT: Fungi can cause severe invasive infections especially in the immunocompromised host. Patient populations at risk are increasing due to ongoing developments in cancer treatment and transplantation medicine. Only limited diagnostic tools and few antifungals are available, rendering a significant number of invasive fungal infections life threatening. To reduce mortality rates, a better understanding of the infection processes is urgently required. Bioluminescence imaging (BLI) is a powerful tool for such purposes, since it allows visualisation of temporal and spatial progression of infections in real time. BLI has been successfully used to monitor infections caused by various microorganisms, in particular bacteria. However, first studies have also been performed on the fungi Candida albicans and Aspergillus fumigatus. Although BLI was, in principle, suitable to study the infection process, some limitations remained. Here, different luciferase systems are introduced, and current approaches are summarised. Finally, suggestions for further improvements of BLI to monitor fungal infections are provided.
    Full-text · Article · Jan 2012 · International Journal of Microbiology
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    • "luciferin distribution as a result of the severe clinical symptoms exhibited , as well as the presence of pulmonary lesions that may have been severe enough to restrict oxygen dispersion in the bronchoalveolar tree . In - deed , the researchers found a strong increase in light emis - sion when luciferin was directly administered to lungs ex vivo . Doyle et al . ( 2006b ) developed bioluminescent C . albicans using a codon optimized derivative of the firefly luciferase gene . The researchers found that while luciferase activity in protein extracts taken from C . albicans growing as yeast or hyphae were almost identical , light output from the hyphal stage was massively reduced ( Doyle et al . , 2006a )"
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    ABSTRACT: According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo.
    Full-text · Article · Sep 2010 · FEMS microbiology reviews
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