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
Source: PubMed


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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    • "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.
    International Journal of Microbiology 01/2012; 2012(1687-918X):956794. DOI:10.1155/2012/956794
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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.
    FEMS microbiology reviews 09/2010; 35(2):360-94. DOI:10.1111/j.1574-6976.2010.00252.x · 13.24 Impact Factor
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    • "Another promising advance has been the isolation and characterization of the luciferase regenerating enzyme from P. pyralis [88]. It has been suggested that co-expression of this enzyme along with Luc could allow for continual bioluminescent production without the need for re-addition of the luciferin substrate [89]. "
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    ABSTRACT: Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed.
    Sensors 11/2009; 9(11):9147-74. DOI:10.3390/s91109147 · 2.25 Impact Factor
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