A Luciferase Reporter for Gene Expression Studies and Dynamic Imaging of Superficial Candida albicans Infections
Real-time imaging of fungal infections is becoming integral to the study of host–pathogen interactions, as it allows monitoring of the spatial and temporal progression of pathogen growth or of the host response in a single animal as well as reducing the n
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Introduction The study of host–pathogen interactions has recently benefited from the development of real-time imaging in small animals. Realtime imaging approaches take advantage of sensitive charge-coupled device (CCD) cameras to detect low levels of light emitted from luciferase reporters in vivo (1). They allow monitoring of the spatial and temporal progression of pathogen growth or of the host response in a single animal, making it possible to reveal the spread of pathogens to unexpected infection sites as well as significant variations in pathogen/host responses that can be masked by the heterogeneous
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_39, © Springer Science+Business Media, LLC 2012
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behavior of individual animals. Moreover, real-time monitoring allows for a reduction in the number of animals required to generate statistically significant datasets (1). Real-time imaging of Candida albicans infections has been pioneered by Doyle et al. (2, 3) who used the firefly luciferase gene expressed under the control of the C. albicans ENO1 promoter to image different forms of C. albicans infections. This approach was successful in a vulvovaginal candidiasis model and allowed monitoring of the efficacy of an antifungal treatment. However, it suffered drawbacks, such as a limited permeability of hyphal cells to the firefly luciferase substrate, luciferin, and monitoring of disseminated candidiasis was unsuccessful. In this chapter, we present a novel luciferase reporter whereby the naturally secreted Gaussia princeps luciferase (4) is targeted to the cell surface of C. albicans using the targeting signals of the C. albicans GPI-linked cell wall protein Pga59 (5) (Fig. 1a). This approach resulted in a highly sensitive reporter, referred to as gLUC59, localized at the cell surface and allowing detection of luciferase in intact yeast and hyphal cells even when expressed from a weak promoter (6). gLUC59 has been used successfully to monitor different forms of superficial C. albicans infections where the luciferase substrate coelenterazine can be directly delivered at the site of infection (Fig. 2). The efficacy of antifungal treatments, e.g., a β-glucan-conjugate vaccine and anti-β-glucan antibodies, against the development of different superficial C. albicans infections could be efficiently monitored (6, 7). Importantly, these studies showed that imaging superficial candidiasis with gLUC59-expressing C. albicans strains was more reliable than colony-forming unit (CFU) counts in assessing the extent and duration of these infections. However, it should be noted that monitoring systemic candidiasis has so far been unsuccessful when using C. albicans strains expressing the gLUC59 luciferase (6, 8). Here, we present protocols for measuring C. albicans promoter activity in in vitro grown cells and monitoring of superficial, namely cutaneous, subcutaneous, and vaginal,
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