Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model

Abstract

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

Keywords: Candida albicans; Galleria mellonella; in vivo; luciferase.

Figure 1.

Reliability of the signal emitted by ACT1p-gLUC-PGA59 construct in C. albicans. (A) Evaluation of the luminescence signal emitted by CAF4-2 strain transformed with ACT1p-gLUC-PGA59 construct. Each box corresponds to the Relative Luminescence Unit (RLU) of 10 independent transformants measured in duplicates. Horizontal lines represent mean and 10-90 percentiles. Wiskers reach the minimal and maximal values. (B) Luminescence signals emitted by 2-fold cell suspension dilutions of different strains with the ACT1p-gLUC-PGA59 construct. This analysis was performed with a single transformant for each strain. These strains correspond to 2 different wild-type strains (CAF4-2 and BWP17) and 2 mutants derived from the BWP17 (orf19.719Δ/Δ and zcf13Δ/Δ). Each point corresponds to the average of biological replicates (n = 3) of cell suspensions at defined concentrations. This experiment is representative of 2 independent experiments. Correlation (R²) between RLU and CFU was calculated with a Pearson test (p < 0.0001 for all tested strains) using Graph Pad Prism 6, on this set of data. RLU (−): arbitrary units of luminescent signal.

Figure 2.

Dose response of the luminescence signal of C. albicans luminescent cells detected in the pulp of G. mellonella larvae. Each point corresponds to a single larva. Six animals were injected with a given dose of C. albicans cells. CFU counts and luminescence signals were measured 24 h post-infection. Missing points are due to dead animals at 24 h post-infection. This experiment is representative of 2 independent experiments. Correlation (R²) between RLU and CFU was calculated with a Pearson test (p < 0.0001 for all tested strains) using Graph Pad Prism 6. RLU (−): arbitrary units of luminescent signal.

Figure 3.

Effect of fluconazole on G. mellonella larvae infected with luminescent C. albicans cells. G. mellonella were infected with 3 × 10⁵ C. albicans cells. Each point corresponds to a single larva. The horizontal line corresponds to the mean of the group. Each group contained 8 larvae. Fluconazole was injected at 1 h post-infection. Statistical analysis was performed on processed data to remove outliers using the ROUT (Q = 1%) method. A non-parametric ANOVA analysis was performed with a Dunn post-test comparing each group with the corresponding non-treated group (24 h or 48 h), using Graph Pad Prism 6. *p <0.05, **p < 0.01. These data is representative of 2 independent experiments. RLU (−): arbitrary units of luminescent signal.

Figure 4.

Virulence assay using luminescent C. albicans mutant strains in the G. mellonella infection model. G. mellonella were infected with 3 × 10⁵ C. albicans cells. Results are expressed as percentage of the mean CFU of the wild-type in the same experiment to be able to merge duplicate experiments. (A) Fungal burden assay using 16 C. albicans TF mutants. These data correspond to the merging of 2 independent experiments. Each group of each experiment contained 6 larvae. Central horizontal lines correspond to the mean of the group. Error bars correspond to Standard Deviation (SD). Larvae fungal burden were evaluated at 24h post-infection and expressed for each larva as a percent of the fungal burden mean of larvae infected with the wild-type strain. Calculation was made for each experiment independently. (B) Each point corresponds to a single larva at 24h post-infection. Each group of each experiment contained 6 larvae. Fungal burden prediction using BLI reporter system. CFU prediction was performed using standard curves (see File S2). The two TF mutants were selected previously as displaying larvae low fungal burden as compared to the wild-type strain. These data correspond to 2 independent experiments. The horizontal line corresponds to the mean of the group. In both A and B panels, each larvae fungal burden was expressed as a percent of the fungal burden mean of larvae infected with the wild-type strain. Calculation was made for each experiment independently. Statistical analyses were performed on processed data to remove outliers using the ROUT (Q = 1%) method. Then a non-parametric ANOVA analysis was performed with a Dunn post-test comparing each mutant-infected group with the group infected by the wild type, using Graph Pad Prism 6. **p < 0.01, ***p < 0.001, ****p < 0.0001. RLU (−): arbitrary units of luminescent signal.

Figure 5.

Detection of luminescence signals of C. albicans cells in living animals. Direct detection of the luminescence signal in the living larvae 24 h post-infection was carried out using a ImageQuant LAS 4000 mini CCD camera (GE Healthcare Bio-Sciences). (A) A representative example of recorded signals of larvae infected with EDY2 luminescent strain. Larvae injected with non-luminescent CAF4-2 strain or with PBS were used as controls. (B) Comparison of luminescence signals with enumerated CFUs obtained after sacrifice of the larvae 24 h post-infection. Each point corresponds to a single larva. Results presented here merge data of 2 independent experiments. The first and the second experiments were performed with 3 and 6 animals per group, respectively. RLU (−): arbitrary units of luminescent signal.

Figure 6.

In vivo monitoring of the toxic effect of repeated injection of CTZ on G. mellonella larvae. Twenty animals per group were monitored for survival. At t = 0, larvae were infected with 4 × 10⁵ C. albicans cells in 40 μl. Then larvae were injected twice a day with 40 µl of 250 µM of CTZ or with PBS only. Surviving animals were monitored before every injection. (A) Effect of CTZ diluted in the modified LA buffer. Animals were injected at 12, 24, 48, 72 and 96h post-infection with 40 μl of CTZ 250 μM or PBS. *p < 0.05, **p < 0.01, Log-rank test. (B) Effect of water-soluble CTZ (W-CTZ) diluted in PBS. Animals were injected at 6, 24, 30, 48, 54 and 72h post-infection with 40 μl of W-CTZ 250 μM or PBS. In both experiments, (A) and (B), animal survival was recorded before every CTZ/PBS injection.

Figure 7.

Correlation between luminescence signals of living animal and CFU counts. Each point corresponds to a single larva. Missing points are due to dead animals 24h post-infection. Correlation (R²) between RLU and CFU was calculated with a Pearson test (p < 0.0001 for all tested strains) using Graph Pad Prism 6. Results presented here merged data of 2 independent experiments including each 10 larvae per group. RLU (−): arbitrary units of luminescent signal.

Figure 8.

Kinetic of C. albicans infection in living G. mellonella in the presence of absence of fluconazole. Twenty animals per group were monitored daily for survival and emission of bioluminescence. At t = 0 larvae were infected with 4 × 10⁵ C. albicans cells and placed in 6-wells plate to identify each animal. One hour post-infection animals were injected with 4 mg/kg fluconazole or PBS. At 6, 24, 30, 48, 54 and 72 hours post-infection, animals were monitored for survival and W-CTZ was injected in surviving larvae just before imaging. (A) Survival curve. ****p < 0.0001, Log-rank test. (B) Overall kinetic of infection. At each time-point, each symbol represents an individual larva. *p < 0.05, 2-way ANOVA statistical analysis, using Graph Pad Prism 6 was performed on log normalized data. (C) Kinetic of infection for each untreated (PBS-injected) larva. (D) Kinetic of infection for each fluconazole (4 mg/kg) treated larva. For panel C and D, each line corresponds to an individual larva. Data presented in panel B are the combination of data of panels C and D. RLU (−): arbitrary units of luminescent signal.