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Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells. Author summary: The opportunistic mold pathogen Aspergillus (A.) fumigatus possesses significant health risk, particularly for immunocompromised individuals. This ubiquitous fungus found in soil and decaying organic matter, causes a range of partially life-threatening infections known as aspergillosis. Natural killer (NK) cells, part of the innate immunity, recognize A. fumigatus by pathogen-associated molecular patterns (PAMPs) via their pattern recognition receptors (PRRs), leading to the elimination of infected cells, including direct cytotoxicity and cytokine production. Previously, we identified the NK-cell receptor CD56 as a PRR that recognizes A. fumigatus and triggers potent antifungal activity. However, the A. fumigatus ligand of CD56 has remained unknown. Here, we used different approaches to identify the A. fumigatus ligand of CD56 on NK cells. We found that CD56 directly binds to cell wall galactosaminogalactan. Specifically, deacetylated galactosamine residues of GAG played a role in the interaction with CD56 and triggered strong NK-cell activation, along with potent release of cytotoxic effectors and immune-enhancing chemokines. We also found that GAG-primed NK-cell supernatants activate and enhance effector responses of polymorphonuclear cells. Our study provides new insights on the A. fumigatus-NK cell interplay by identifying GAG as an activating ligand of CD56 on human NK cells, stimulating potent antifungal effector responses. [ABSTRACT FROM AUTHOR]

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation. [ABSTRACT FROM AUTHOR]