Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth

Abstract

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.

Fig. 1.

Injection of C. albicans into zebrafish larvae causes disseminated infection and significant mortality. Wild-type GFP-expressing (WT-GFP) C. albicans cells (11 ± 4 CFU, as measured in homogenates at 0 hpi) were injected into the hindbrain ventricle of wild-type AB larvae at the prim25 stage. The results in each panel are representative of at least three independent experiments. (A) Confocal imaging of disseminated infection at 24 hpi. Bars, 100 μm in large images and 50 μm in insets. (B) Fungal burdens determined by serial dilution and growth on YPD plates. Error bars represent standard deviations. (C) Kaplan-Meier survival curve from a representative experiment. WT-GFP strain-infected fish had significantly more mortality than PBS-injected controls (P < 0.0001 by log rank test).

Fig. 2.

Noninvasive imaging of innate immunity-fungus interactions. For all panels, prim25-stage embryos were infected with 10 to 15 CFU of CAF2-yCherry in the hindbrain ventricle. Bars = 10 μm. (A) Infected fli1:EGFP larvae with green macrophages were imaged at 0, 6, and 24 hpi by confocal microscopy. Images at 0 and 6 hpi are individual optical sections, and images at 24 hpi are z-stack projections of 15 optical slices. (B) Percentages of filaments in wild-type AB control morphants. Data shown are the averages and standard deviations for three independent experiments. (C) An infected mpx:GFP embryo was imaged from 0.25 to 4.5 hpi by confocal microscopy. Shown is a z-stack projection of 30 optical slices from 4 hpi. A movie of the three-dimensional reconstruction of these slices is viewable as Movie S3 in the supplemental material. (D) An infected fli1:EGFP larva was imaged from 2 to 2.2 hpi by confocal microscopy. Individual optical sections are shown. The entire image series is viewable as Movie S4. (E) An infected mpx:GFP larva was imaged from 0.25 to 4.5 hpi by confocal microscopy. The EGFP-negative macrophage followed in the time-lapse series is indicated with a white arrow. The figure panels are z-stack projections of 19 to 30 optical slices covering the entire z space of the hindbrain ventricle. The entire time-lapse series is viewable as Movie S5. (F and G) An infected mpx:GFP larva was imaged at 5 hpi by confocal microscopy. Shown are single optical slices from a z series encompassing a single EGFP-positive neutrophil with two fungi inside. The entire z series is viewable as Movie S6. (F) Phagosome with live fungus. yCherry is concentrated within the fungal cytoplasm and limited to the fungal cell. Arrows indicate the position of intact fungus. (G) Phagosome with killed fungus. yCherry is no longer concentrated and limited to the fungal cytoplasm and has dispersed throughout the phagosome. Arrowheads indicate the remaining cell wall.

Fig. 3.

Hyphal morphogenesis is required for virulence. Wild-type AB fish at the prim25 stage were infected in the hindbrain ventricle with 20 CFU of CAF2-yCherry or 35 CFU of efg1Δ/Δ cph1Δ/Δ-yCherry C. albicans. (A) A hypofilamentous mutant is less virulent. Wild-type CAF2-yCherry (red) or the filamentation-defective efg1Δ/Δ cph1Δ/Δ-yCherry strain (green) was used to infect 100 prim25-stage fish. The difference in survival is significant (P < 0.0001 by log rank test). (B) Fungal burdens are reduced in hypofilamentous efg1Δ/Δ cph1Δ/Δ-yCherry strain-infected larvae compared to those in CAF2-yCherry-infected larvae (P < 0.001 by Student's t test). Note that at 0 hpi, the infection dose was established, and for this experiment, there was a higher dose for the efg1Δ/Δ cph1Δ/Δ-yCherry strain (35 ± 6) than for CAF2-yCherry (20 ± 0). Error bars represent standard deviations for triplicate measurements. (C) Confocal imaging of infected fish at 18 hpi reveals reduced but not absent filamentation in efg1Δ/Δ cph1Δ/Δ-yCherry strain-infected larvae (right) in comparison to CAF2-yCherry-infected larvae (left). Despite growing as filaments, the efg1Δ/Δ cph1Δ/Δ-yCherry strain grows only as pseudohyphae. Bars = 50 μm. Results for all three panels were derived from the same experiment and are representative of at least three independent experiments in which doses were comparable and the dose of the efg1Δ/Δ cph1Δ/Δ-yCherry strain was greater than or equal to the dose of CAF2-yCherry.

Fig. 4.

Respiratory burst is required for resistance to infection. Fertilized 1-cell eggs were injected with 2.5 nanograms of control or p47^phox morpholino. (A) p47^phox morphants have a dampened respiratory burst compared to control morphants. At 48 h postfertilization, p47^phox or control morphants were assayed for respiratory burst activity by PMA stimulation in the presence of H₂DCF-DA. Monte Carlo analysis with 1,000 replicates was used to find the average ratio of fluorescence from 12 induced versus 12 uninduced fish, and this difference was significant (P < 0.001). Results are representative of three independent experiments. Error bars represent 95% confidence intervals. (B and C) One hundred morphants were infected at the prim25 stage with approximately 20 CFU of CAF2-yCherry C. albicans. (B) p47^phox morphants had reduced survival compared to control morphants (n = 100 for each group; P = 0.003 by log rank test). Note that there were significant differences (P ≤ 0.01; Kaplan-Meier and log rank tests) between PBS- and CAF2-yCherry-injected larvae (both control and p47^phox morphants) but no difference (P = 0.68) between PBS-injected control and p47^phox morphants. The survival curves represent pooled data from three independent experiments with similar results. (C) p47^phox morphants had significantly increased fungal burdens at 12 hpi (P = 0.05), 18 hpi (P = 0.002), and 24 hpi (P = 0.004), as measured by Student's t test. Each bar in panel C shows the average and standard deviation of measurements from three independent experiments. Note that the average and standard deviation of the infectious dose in these three experiments, as measured at 0 hpi, was 17 ± 5 CFU for controls and 21 ± 6 CFU for p47^phox morphants.

Fig. 5.

Knockdown of the p47^phox subunit of the phagocyte NADPH oxidase enhances fungal filamentation and pathogenesis. (A) Control and p47^phox morphants were infected with CAF2-yCherry C. albicans and imaged at 0, 6, and 24 hpi. At 0 hpi, larvae were injected with approximately 20 yeast cells in the hindbrain ventricle. By 6 hpi, C. albicans germinated within control and p47^phox morphants. At 24 hpi, control morphants had only yeast-form fungi present, while p47^phox morphants had large chains and branches of filaments. Bar = 10 μm. (B) p47^phox morphants had a larger number of fungi in filamentous form than did controls at 24 hpi. Individual fish were crushed with glass coverslips onto slides and then imaged, and images were quantified for presence of yeast or filaments as described in Materials and Methods and illustrated in Fig. S6 in the supplemental material. Three independent experiments were performed (n = 11 controls and 15 p47^phox morphants), and data were pooled for analysis. There was a significantly higher level of filamentous growth in p47^phox morphants (P = 0.005 by nonparametric Wilcoxon/Kruskal-Wallis test).

Fig. 6.

C. albicans is subject to NADPH oxidase-dependent oxidative stress in vivo. (A) Schematic of dual-fluorescence oxidative stress reporter WT-OXYellow. C. albicans expressing EGFP from the CTA1 promoter was transformed with the P_ADH1-yCherry plasmid to drive constitutive expression of yCherry. For panels B to D, one-cell fertilized eggs were injected with 2.5 ng of control or p47^phox morpholino and infected with 10 to 15 CFU of WT-OXYellow at the prim25 stage. (B) Fungi infecting p47^phox morphants exhibit little to no EGFP fluorescence, indicating no oxidative stress to the Candida. Infected control and p47^phox morphants were imaged by confocal microscopy at 6 and 24 hpi. Bar = 10 μm. (C) Infected control and p47^phox morphants were imaged by Vivatome microscopy, and line histograms of red and green fluorescence were compared. Yeast cells within controls (top) had correlated green and red fluorescence, indicative of cytoplasmic fluorescence. In contrast, yeast cells within p47^phox morphants (bottom) displayed only red fluorescence and no corresponding green fluorescence. (D) Quantitation of confocal images of individual yeast cells and filament segments taken at 24 hpi was performed using ImageJ software to determine the average fluorescence per pixel as described in Materials and Methods. There was a significantly higher average green/red ratio at 24 hpi for control morphants than that for p47^phox morphants (P < 0.0001; Student's t test) and that for controls at 6 hpi (P < 0.0001; Student's t test). Box plot whiskers represent the 1.5 interquartile range either below or above the lower or upper quartile, respectively. The results are representative of two independent experiments.