title: Fusion of the Fc part of human IgG1 to CD14 enhances its binding to gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils.
abstract: Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.
authors.affiliation: Department of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Vida
authors.forename: Bart
authors.initials: B
authors.lastname: Bardoel
authors.forename: Fin
authors.initials: F
authors.lastname: Milder
authors.forename: László
authors.initials: L
authors.lastname: Majoros
authors.forename: Andrea
authors.initials: A
authors.lastname: Sümegi
authors.forename: Attila
authors.initials: A
authors.lastname: Bácsi
authors.forename: György
authors.initials: G
authors.lastname: Vereb
authors.forename: Kok P M
authors.initials: KP
authors.lastname: van Kessel
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.forename: Péter
authors.initials: P
authors.lastname: Antal-Szalmás
mesh_terms: D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D005434:Flow Cytometry; D015870:Gene Expression / Q000276:immunology; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000254:growth & development / Q000276:immunology*; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000254:growth & development / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology* / Q000382:microbiology; D009895:Opsonin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology; D011485:Protein Binding; D011961:Receptors, Fc / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D009895:Opsonin Proteins; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins