Neurospora Crassa Δccg-8 Compromises Cell Surface Integrity and Antifungal Tolerance: Insights from in Vitro and Galleria Mellonella Studies

A comprehensive reference organism is often lacking when studying the adaptation of filamentous fungi to environmental stressors. This work investigates the mechanism underlying the response of N. crassa to stress conditions by focusing on the deletion mutant for the CCG-8 transcription factor. Our molecular analyses revealed that Δccg-8 severely compromises cell surface structure and metabolic homeostasis. Proteomic profiling demonstrated key dysregulations in ribosome biogenesis (consistent with the clock-controlled nature of CCG-8) and fatty acid β-oxidation. These findings, substantiated by changes in ergosterol and fatty acid composition, confirmed the increased susceptibility of deletion mutant to azoles and echinocandins. Furthermore, glycomic and proteomic data suggested that conidia of N. crassa Δccg-8 exhibit protein and glycan alterations. To validate this structural compromise in vivo, we successfully applied the Galleria mellonella larval model. Despite being non-pathogenic, conidia of N. crassa Δccg-8 were cleared significantly faster by the larval immune system than the wild-type strain, mirroring the in vitro observations. This work provides detailed molecular insights into fungal stress adaptation and establishes N. crassa as a viable non-pathogenic organism for in vivo analysis. This approach substantially broadens application of this filamentous fungus, enabling direct comparative research with pathogenic filamentous fungi in the domain of antifungal resistance and host interaction.