A stress responsive protein contributes to pyomelanin biosynthesis and cell wall stress adaptation in Aspergillus fumigatus

The biology of the pathogenic fungus Aspergillus fumigatus remains largely unexplored, in part due to the large number of hypothetical and uncharacterized proteins. In this study, we focused on the protein encoded by the Afu4g10610 gene, which is consistently up-regulated across multiple stress-related transcriptomic datasets, including both in vitro and in vivo infection models. Functional characterization through the generation of mutant strains revealed that deletion of Afu4g10610 compromises the response to cell wall stress induced by Congo Red and Calcofluor White, correlating with the downregulation of key cell wall integrity (CWI) pathway sensors ( wsc1 and midA ). In addition, the mutant exhibits enhanced resistance to osmotic stress, consistent with altered expression of the high-osmolarity glycerol (HOG) pathway effectors mpkC and sakA . The deletion mutant also showed a moderate reduction in cytotoxicity toward A549 epithelial cells and altered TNF production in RAW 264.7 macrophages, whereas the overexpression strain exhibited a significant decrease in TNF levels. GRAsp analysis predicted this gene to be associated with the phenylalanine/tyrosine catabolic pathway. Accordingly, pyomelanin production and related metabolites were analyzed. Moreover, double deletion mutants ∆ maiA ∆ 10610 and ∆ hmgA led to a marked reduction in pyomelanin production, accompanied by altered tyrosine consumption and homogentisic acid (HGA) production. Since pyomelanin biosynthesis depends on the conversion of HGA into benzoquinone acetate (BQA), a step traditionally considered spontaneous, we investigated the potential interaction between Afu4g10610 and HGA. Molecular docking analysis supported the binding of HGA at the predicted dimer interface of the protein and suggested potential binding of FADH₂ to the protein, as an electron donor. Together, these findings identify Afu4g10610 as a stress-associated protein that contributes to cell wall and osmotic stress adaptation and suggest a potential contribution to HGA-to-BQA conversion during pyomelanin biosynthesis. More broadly, our results support the possible involvement of an enzymatic component in a step previously considered spontaneous in A. fumigatus .