**Background:** Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), a life-threatening infection in immunocompromised individuals with mortality rates exceeding 50%. Hyphal growth is essential for host colonization, and innate immune cells, particularly neutrophils, are critical for clearing hyphae. The C2H2 zinc finger transcription factor ZfpA was previously identified as a regulator of hyphal branching, septation, and cell wall chitin content in A. fumigatus, but its role during infection and in response to antifungal treatment was unknown.
**Methods:** The authors used a larval zebrafish-Aspergillus infection model with transgenic lines expressing fluorescent neutrophils and macrophages to track fungal burden and immune cell recruitment over 96 hours post infection (hpi). Neutrophil-deficient (Rac2D57N) and ROS-deficient (p22phox-/-) zebrafish lines were used to probe specific immune mechanisms. Ex vivo assays with primary human neutrophils were performed to assess killing, NETosis (SYTOX Green), IL-8 and myeloperoxidase release (ELISA), and hyphal escape. In vitro antifungal susceptibility was measured by colony diameter on solid glucose minimal medium (GMM) with voriconazole (0.1, 0.25 μg/mL), caspofungin (0.25, 0.5, 1, 8 μg/mL), and micafungin (0.25, 0.5, 1, 8 μg/mL). Chitin content was visualized by calcofluor white (CFW) staining and quantified by mean gray value. A CaCl2/CFW pretreatment assay was used to stimulate chitin synthesis prior to caspofungin exposure, with fungal viability measured by PrestoBlue reagent.
**Key Results:** ZfpA deletion (ΔzfpA) attenuated virulence in wild-type zebrafish larvae but not in neutrophil-deficient Rac2D57N larvae, while ZfpA overexpression (OE::zfpA) showed similar virulence to wild-type in both backgrounds. Fungal burden was significantly reduced for ΔzfpA at 96 hpi compared to wild-type and OE::zfpA, but no differences in neutrophil or macrophage recruitment were observed. In human neutrophil co-incubation assays, ΔzfpA germlings were highly susceptible—most died within 1 hour and none survived beyond 5 hours—while OE::zfpA germlings survived significantly longer than wild-type. No differences were detected in NET formation, IL-8 release, or myeloperoxidase release among strains. In p22phox-/- zebrafish (lacking phagocyte ROS), ΔzfpA remained attenuated relative to wild-type and OE::zfpA. In vitro, ΔzfpA showed increased susceptibility to CFW and caspofungin (up to 1 μg/mL), while OE::zfpA was more resistant. At 8 μg/mL caspofungin, all strains exhibited paradoxical growth. Micafungin severely restricted growth of all strains at all concentrations. For voriconazole, OE::zfpA showed reduced susceptibility at 0.1 and 0.25 μg/mL, while ΔzfpA showed a slight decrease in relative colony diameter only at 0.25 μg/mL. In neutrophil-deficient zebrafish, voriconazole (0.1 μg/mL) was least effective against OE::zfpA, while caspofungin (1 μg/mL) significantly improved survival only in ΔzfpA-infected larvae. CFW staining revealed that ΔzfpA had reduced basal chitin and OE::zfpA had increased basal chitin relative to wild-type, but all strains upregulated chitin in response to caspofungin. CaCl2/CFW pretreatment improved wild-type viability by 16% and ΔzfpA viability by 21% following caspofungin exposure, but ΔzfpA viability remained lower than wild-type; OE::zfpA viability was unaffected by pretreatment.
**Clinical Implications:** This study establishes ZfpA as a critical regulator of hyphal stress tolerance during A. fumigatus infection, mediating resistance to both neutrophil killing and echinocandin antifungals. The finding that ZfpA deletion increases susceptibility to caspofungin in vivo suggests that targeting ZfpA or its downstream pathways could enhance echinocandin efficacy. The observation that ZfpA overexpression protects against both immune and antifungal challenges highlights the potential for ZfpA-driven resistance mechanisms in clinical isolates. Understanding the ZfpA regulatory program may identify new therapeutic targets for improving outcomes in invasive aspergillosis, particularly in patients with preserved neutrophil function.