Dr. Iván Conejeros (Gießen / DE), Dr. Zahady Velásquez (Gießen / DE), Gabriel Espinosa (Gießen / DE), Prof. Dr. Carlos Hermosilla (Gießen / DE), Prof. Dr. Anja Taubert (Gießen / DE)
Abstract text
Introduction
Coccidian parasites represent an important threat for animal and human health. Infection-driven innate immune reactions of the hosts depend on the adequate recognition of parasites. One major mechanism relies on the recognition of parasite-own pathogen associated molecular patterns (PAMPs). Among PAMPS, glycan residues on the parasite"s surface are one of the most important drivers of host immune responses. Polymorphonuclear neutrophils (PMN) are one of the first immune cells to arrive at the site of injury or infection. PMN-derived effector mechanisms against coccidian parasites include reactive oxygen species (ROS) production, degranulation and neutrophil extracellular trap (NET) formation. In this study, the glycosylation patterns in Besnoitia besnoiti and Toxoplasma gondii tachyzoites in addition to Eimeria bovis sporozoites and the relevance of different glycans in B. besnoiti- and T. gondii-induced NETosis were studied.
Material and Methods
Glycan pattern in B. besnoiti and T. gondii tachyzoites as well as E. bovis-sporozoites were determined by lectin blots using the following lectins: GNA, SNA, MAA, DSA, PNA. Blockage and modification of B. besnoiti and T. gondii tachyzoite-related glycan motifs was achieved by chemical treatments (NaIO4 oxidation), enzymatic treatments with PNGase F, PNGase A and O-glycosidase or by parasite culture in human foreskin fibroblasts (HFF) treated with the N-glycosylation inhibitor tunicamycin. After treatments, B. besnoiti and T. gondii tachyzoites were confronted with bovine PMN and NET formation was analyzed by confocal microscopy. Percentages of NET formation were calculated by image analysis.
Results and conclusions
Depending on the lectin used, overlapping and differing glycan pattern were revealed in tachyzoite (B. besnoiti, T. gondii) and sporozoite (E. bovis) stages by lectin blots. In contrast to tachyzoites, GNA-detected N-glycans proved absent in E. bovis sporozoites. Glycan oxidation via NaIO4 and PNGase F treatments led to a decrease in NET formation, but also affected tachyzoite viability. In the case of tunicamycin-treated HFF, T. gondii but not B. besnoiti tachyzoites were able to develop F2 tachyzoite generation. F2-tachyzoites proved impaired in their glycosylation repertoire and induced less NETs than those originating from untreated control HFF layers, thereby indicating a potential role of N-glycans in coccidia-induced NETosis.