Binding of the Staphylococcus aureus leucotoxin LukM to its leucocyte targets

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anglais. The range of leucocytes susceptible to the leucotoxin LukM/F', a two-component pore-forming toxin of Staphylococcus aureus causing mastitis in ruminants, had not been defined. We used fluorescent-labeled LukM to investigate the binding of this toxin to bovine cells and to identify its cellular targets among bovine, human and murine leucocytes. LukM bound to bovine blood neutrophils from all the individuals tested with similar affinity, with an apparent dissociation constant (Kd) of 1.81 +/- 0.14 nM and 13 3100 +/- 506 binding sites. The amount of LukM bound to bovine neutrophils did not depend on the presence of the complementary component LukF, suggesting that the binding of LukM to its ligand does not depend on the formation of pore-forming oligomers, and that the number of bound LukM molecules corresponds to the number of available cell membrane ligands. Other staphylococcal class S components of bipartite leucotoxins (HIgA, HIgC, LukE, LukS-PV) were inefficient competitors of LukM for the binding to bovine neutrophils, indicating that LukM has a distinct ligand on target cells. Bovine blood neutrophils bound slightly more LukM than did milk neutrophils, and much more than did ovine and caprine blood neutrophils. Bovine monocytes and milk macrophages readily bound LukM, whereas blood lymphocytes did not. Human neutrophils bound little LukM and were resistant to LukM/F' at the highest tested concentration (40 nM). Murine neutrophils bound LukM and were susceptible to the toxicity of LukM/F', exhibiting flattening and nucleus alteration beginning at 0.3 nM concentration. Among murine peritoneal exudate cells, T lymphocytes (CD3+) and monocytes/macrophages (F4/80+) bound LukM, whereas binding to B lymphocytes (CD19+) was not detected. These results indicate that cells of the myeloid lineage are the main targets of LukM/F' in dairy ruminants, and that resident or inflammatory migrated phagocytes are susceptible to this toxin. (C) 2010 Elsevier Ltd. All rights reserved.