Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene.

Animal studies with Candida albicans have provided models for understanding fungal virulence and antifungal drug development. To non-invasively monitor long-term Candida murine infections, clinical isolates were stably transformed with a codon-optimized luciferase gene to constitutively express luciferase. Chronic systemic infections were established in mice with engineered strains, and bioluminescent signals were apparent from kidneys by non-invasive imaging using charged-coupled device cameras. These infections were established in immune-competent mice, and bioluminescence was detectable in animals that showed no physiological consequence of infection, as well as those visually succumbing to the disease. Similarly, bioluminescence was measured from the vaginal tissue of mice infected vaginally. Fungal loads determined by plating vaginal lavages showed a similar pattern to the bioluminescent signals measured, and fungal infection could be detected in animals for over 30 days post infection by both modalities. The effect of the antifungal drug miconazole was tested in this model, and clearance in animals was apparent by both direct imaging and fungal load determination. The use of bioluminescence to monitor these and other models of Candida infections will greatly speed up the analysis of drug development studies, both in ease of visualizing infections and decreasing numbers of animals required to run such studies.

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

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.