A 3D <i>Fusarium</i> keratitis model reveals isolate-specific adhesion and invasion properties in the <i>Fusarium solani</i> species complex

Members of the <i>Fusarium solani</i> species complex (FSSC) are major causes of keratitis in humans. The underlying mechanisms leading to this disease are not well understood, partially due to the absence of more complex <i>in vitro</i> infection models. Here, we compared the pathogenicity of keratitis-causing FSSC members <i>F. falciforme</i>, <i>F. keratoplasticum</i>, and <i>F. petroliphilum</i> in a 2D monolayer infection model using a human corneal epithelial cell line and a newly established 3D human cornea infection model which comprises the multilayer epithelium and the stroma. In both models, <i>F. keratoplasticum</i> emerged as the most virulent species, showing extensive invasion and host cell damage and, in rare cases, even inducing the formation of transcellular tunnels. While <i>F. falciforme</i> exhibited strong adhesion to corneal epithelial cells, its capacity for invasion and damage was limited. <i>F. petroliphilum</i> was the least virulent among the FSSC species with low adhesion and invasion. The use of the 3D model allowed the investigation of fungal infection in a more physiologically relevant context and revealed that all three FSSC species disseminated deeper into the cornea than <i>C. albicans</i> under identical conditions. This may partly explain the unfavorable clinical outcome of <i>Fusarium</i> keratitis in patients, as the deep penetration of <i>Fusarium</i> hyphae complicates the accessibility of antifungal drugs to the pathogen. Our data indicate species-specific infection routes which might avoid recognition by the host cell defense system and could contribute to the overall high virulence of the species.IMPORTANCE<i>Fusarium</i> keratitis is a rare fungal infection of the human eye. The outcome for affected patients is often poor, with loss of eyesight or even the entire eye being common. Investigation of this disease is challenging due to the absence of established <i>in vitro</i> complex infection models that go beyond a simple 2D monolayer of a single cell type. Here, we performed a comparative analysis of three <i>Fusarium</i> species in a classic 2D infection model and a newly established 3D human cornea model which comprised the epithelium and the stroma. Our experiments revealed that <i>F. keratoplasticum</i> shows a higher potential for invasion and host cell damage when compared to related species. The 3D human cornea model could be a helpful tool for future investigations of fungal pathogenicity and antifungal drug susceptibility during cornea infections.