P144 Pathogen recognition by the long pentraxin PTX3: Roles of glycosylation and interplay with complement

Abstract

Introduction The long pentraxin PTX3 is a soluble Pattern Recognition Molecule (sPRM) with non-redundant roles in resistance to selected pathogens, such as the opportunistic fungus Aspergillus fumigatus . PTX3 facilitates recognition and phagocytosis of fungal conidia mainly by polymorphonuclear neutrophils (PMNs) via Fc γ receptors (Fc γ Rs) and alternative complement pathway (ACP) mechanisms. The human PTX3 protein contains a single N-glycosylation site with complex type sialylated oligosaccharides that has been involved in a number of PTX3 functions [1] . The present study was designed to characterize the role of this post translational modification in the PTX3-depedent recognition and phagocytosis of A. fumigatus . This microorganism has developed several strategies to evade the immune response, such as attracting onto the conidial wall inhibitors of the ACP, like factor H (FH, a ligand of PTX3). Here we present preliminary evidences on the effect of PTX3 on FH engagement by A. fumigatus . Methods Recombinant as well as natural human PTX3 (i.e., from PMNs) was purified by immunoaffinity chromatography and the attached oligosaccharides were analyzed using glycosidases, lectin blotting and mass spectrometry. A range of PTX3 glycoforms were made in vitro by enzymatic hydrolysis of terminal monosaccharides of the recombinant protein. The opsonic and pro-phagocytic activities of these variants were characterized in buffer solutions and whole human peripheral blood, respectively, by flow cytometry. The influence of PTX3 on FH binding to conidia was studied in competition assays using microtiter plates. Results Complex-type sugars were found on PTX3 of both natural and recombinant origins. These showed heterogeneity with regard to the relative content of terminal sialic acid (SA) and bi-, tri- and tetrantennary glycans, depending on the cellular source. Enzymatic removal of SA caused ∼50% reduction of PTX3 binding to A. fumigatus conidia, with no further change due to additional deglycosylation. Conversely, saccharide hydrolysis had no effect on PTX3 binding to PMNs, where Fc γ Rs have been proposed as putative receptors of this pentraxin [1] . However, consistent with binding data from conidia, desialylated PTX3 showed reduced potentiation of conidia phagocytosis by PMNs as compared to the unmodified protein. Also, we observed that PTX3 inhibits the binding of FH to A. fumigatus conidia, even when these were pre-opsonised with the long pentraxin and FH was applied at physiologically relevant concentrations. Conclusion Different pathogens express neuraminidases on their surface during infection and natural immunity cells, such as PMNs, mobilize intracellular neuraminidases to the plasma membrane upon inflammation. Here, we report that the glycosylation status of the sPRM PTX3 changes, with respect to SA, depending on cellular source and perhaps inducing stimuli. We describe that SA modulates the PTX3-dependent recognition and phagocytosis of A. fumigatus , which points to glycosylation as a strategy to tune PTX3 functions in innate immunity. Furthermore, we provided evidences that PTX3 impairs the interaction of FH with A. fumigatus , where this might link to a new mechanism to counterbalance the complement evasion strategy exploited by this pathogen.