Zebrafish as a model host for Candida albicans infection

Abstract

In this work, the zebrafish model organism was developed to obtain a minivertebrate host system for a Candida albicans infection study. We demonstrated that C. albicans can colonize and invade zebrafish at multiple anatomical sites and kill the fish in a dose-dependent manner. Inside zebrafish, we monitored the progression of the C. albicans yeast-to-hypha transition by tracking morphogenesis, and we monitored the corresponding gene expression of the pathogen and the early host immune response. We performed a zebrafish survival assay with different C. albicans strains (SC5314, ATCC 10231, an hgc1 mutant, and a cph1/efg1 double mutant) to determine each strain's virulence, and the results were similar to findings reported in previous mouse model studies. Finally, using zebrafish embryos, we monitored C. albicans infection and visualized the interaction between pathogen and host myelomonocytic cells in vivo. Taken together, the results of this work demonstrate that zebrafish can be a useful host model to study C. albicans pathogenesis, and they highlight the advantages of using the zebrafish model in future invasive fungal research.

FIG. 1.

C. albicans kills zebrafish in a dose-dependent manner. The survival rates of adult zebrafish infected with different doses of C. albicans strain SC5314 are shown. Zebrafish were injected with PBS or heat-inactivated C. albicans as controls. Each experimental group contained 20 zebrafish, and each experiment was performed in duplicate.

FIG. 2.

Colonization and invasion of C. albicans in zebrafish. (A) C. albicans burden in infected adult zebrafish. Five C. albicans-injected fish for each dose group were collected at 2, 15, and 23 hpi. Each dot indicates the number of C. albicans CFU in one infected fish. The horizontal lines indicate the mean values of the groups. *, P < 0.05. (B and C) Histological analysis of C. albicans-infected zebrafish. Transverse sections were prepared from zebrafish injected with and killed by 1 × 10⁸ CFU of C. albicans. Abbreviations: L, liver; G, gastrointestinal tract; N, connective tissue; M, muscle.

FIG. 3.

C. albicans hypha-associated gene expression in zebrafish. The hypha-associated ALS3, ECE1, HWP1, and PHR1 genes were highly expressed during the early phase of zebrafish infection. Fish were injected with 1 × 10⁸ CFU of SC5314 and collected at 2, 8, and 15 hpi for the fungal gene expression assay. The relative levels of expression were calculated by comparison with the level of expression of the target gene in the C. albicans culture before injection. The data are the means and standard errors for at least eight samples for each condition. *, P < 0.5; **, P < 0.01.

FIG. 4.

Progression of C. albicans hyphal formation in zebrafish. Time-lapse tissue sections were obtained from fish injected with 1 × 10⁸ CFU of C. albicans cells at (A) 2, (B) 8, and (C) 15 hpi. The arrows indicate C. albicans cells. L, liver; S, swim bladder; I, intestine.

FIG. 5.

C. albicans induces host immune response gene expression. The mRNA levels of IL-1β, TNF-α, IL-10, and iNOS were upregulated in the zebrafish host infected with C. albicans, but the expression of IFN-γ was downregulated. The same group of infected fish was used for the host immune response and fungal gene expression assays. The relative level of expression was calculated by comparing the later time points with the level of expression of the target gene at 2 hpi. The data are the means and standard errors for at least eight samples for each condition. *, P < 0.05; **, P < 0.01.

FIG. 6.

Deficient hyphal formation attenuates C. albicans virulence in zebrafish. Each experimental group of 20 zebrafish was injected with the C. albicans cells indicated. This analysis was performed with strains SC5314, ATCC 10231, HLC84 (cph1/cph1 efg1/efg1 EFG1), HLC54 (cph1/cph1 efg1/efg1), WYZ12.1 (hgc1/hgc1 HGC1), and WYZ12.2 (hgc1/hgc1). Each experiment was performed in duplicate, and the two duplicates showed identical trends. The dashed and solid lines indicate the results for injected doses of 10⁷ and 10⁸ CFU, respectively.

FIG. 7.

The hyphal form of C. albicans was found in dead zebrafish. SC5314, ATCC 10231, HLC84 (cph1/cph1 efg1/efg1 EFG1), and HLC54 (cph1/cph1 efg1/efg1) utilized the dimorphic transition to invade the liver of infected zebrafish. The infected zebrafish were obtained for histological analysis after they died. Scale bar = 50 μm.

FIG. 8.

C. albicans Hgc1 knockout strain formed only pseudohyphae. The Hgc1 mutant of C. albicans was defective in hyphal formation in the liver of infected zebrafish. It only formed pseudohyphae. The arrows indicate the pseudohyphal branches. Zebrafish killed with 10⁸ CFU of WYZ12.1 (hgc1/hgc1 HGC1) or WYZ12.2 (hgc1/hgc1) were obtained for histological analysis.

FIG. 9.

Delayed morphological transition of HLC54. C. albicans SC5314 cells attached to the zebrafish liver became filamentous at 15 hpi, but HLC54 cells were in the yeast form at 15 hpi. The fish were injected with 1 × 10⁸ CFU of C. albicans strain SC5314 (A) or HLC54 (B) and sacrificed at 15 hpi for tissue sectioning. The arrows indicate C. albicans cells.

FIG. 10.

C. albicans virulence gene expression profile in early infection. A lack of CPH1 and EFG1 in C. albicans caused reductions in virulence and in the expression of ALS3, HWP1, ECE1, and SAP4-6. Each zebrafish was injected with 1 × 10⁸ CFU of C. albicans strain SC5314, ATCC 10231, HLC84 (cph1/cph1 efg1/efg1 EFG1), HLC54 (cph1/cph1 efg1/efg1), WYZ12.1 (hgc1/hgc1 HGC1), or WYZ12.2 (hgc1/hgc1) and sacrificed at 15 hpi for analysis by quantitative real-time PCR. The average level of expression of each target gene in SC5314 was defined as the baseline, and the relative level of expression in each strain was compared to this level. The data are the means and standard errors for at least seven samples for each condition. One or two asterisks above a bracket indicate that there were significantly different levels of target gene expression in the two strains connected by the bracket. One or two asterisks above a bar indicate that the level of target gene expression is significantly different from the levels of expression in the other strains. *, P < 0.5; **, P < 0.01.

FIG. 11.

In vivo visualization of C. albicans in an infected zebrafish embryo. (A) Hyphae of C. albicans form in the hindbrain cavity of the zebrafish embryo. A zebrafish embryo was injected with C. albicans strain OG1 in its hindbrain and observed at 18, 26, and 34 hpi using a microscope equipped with a live-imaging apparatus. The arrows indicate the extruding fungal hyphae. (B) Interactions between C. albicans and zebrafish myelomonocytic cells. The yolk of the lyz:DsRed2 zebrafish embryo was injected with GFP-labeled C. albicans strain OG1 and examined at 24 hpi using a Nikon A1R confocal microscope.