2607. Establishment of a Novel High-Throughput Fungal Infection Model in Zebrafish Larvae by Controlled Ablation of Epithelial Cells

BACKGROUND: Efficient animal models are needed in order to investigate fungal pathogenicity and antifungal therapy in the context of epithelial injury, e.g., due to anticancer chemotherapy. Using a Gal4 enhancer trap (GET) zebrafish line facilitating metronidazole (MTZ)-inducible ablation of epithelial (periderm) cells, we aimed to establish a mucositis model predisposing larvae for fungal invasion. METHODS: 4 days post-fertilization Et(Gal4-VP16)(zc1044A);Tg(UAS-1b:nfsB-mCherry)(c264) zebrafish larvae were exposed to 10 mM MTZ in E3 medium for 5 h. After washing, larvae were incubated in Candida albicans or Rhizopus arrhizus spore suspensions (5 × 10(5)–5 × 10(7)/mL) for 16 h. Thereafter, larvae were washed again and survival was monitored until 72 h post-infection. Fungal burden was assessed by 18S qPCR and histopathology. For drug protection studies, 5 µg/mL amphotericin B (AMB) or posaconazole (PCZ) was added to the medium of R. arrhizus-infected larvae at 16 h post-infection. RESULTS: In MTZ-treated GET larvae, inoculum-dependent mortality was found for both R. arrhizus (panel A) and C. albicans (panel B). High inter-experiment reproducibility of survival rates was seen (CV < 0.3). Using a GFP-expressing R. arrhizus strain, fungal invasion of the larval tissue was verified by fluorescence microscopy (panel C). PCZ and AMB improved survival rates of R. arrhizus-infected (5 × 10(6)/mL) larvae from 46% to 85% and 51% to 86%, respectively (P < 0.001). Similarly, significantly reduced fungal burden in AMB and PCZ-treated larvae was documented by qPCR (panel D) and histopathology. In additional validation experiments, the hypo-virulent phenotypes of a CotH-depleted R. arrhizus strain and filamentation-defective C. albicans mutants (Δefg1 and Δcph1) were recapitulated in zebrafish larvae with epithelial cell loss. CONCLUSION: We have established a robust and reliable model of invasive mycoses by controlled ablation of epithelial cells in zebrafish larvae, allowing for rapid immersion-based interrogation of different infection and treatment options. Our proof-of-concept experiments suggest that GET zebrafish larvae are positioned as an appealing high-throughput in vivo system for antifungal drug screening or comparative virulence studies. [Image: see text] DISCLOSURES: All authors: No reported disclosures.