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

Abstract

BackgroundEfficient 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.Methods4 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 × 105–5 × 107/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.ResultsIn 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 × 106/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.ConclusionWe 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. Disclosures All authors: No reported disclosures.