Effects of pig hemoglobin binding on some physical and biological properties of Actinobacillus pleuropneumoniae lipopolysaccharides

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Abstract
Binding of pig hemoglobin (Hb) to Actinobacillus pleuropneumoniae lipopolysaccharide (LPS), either extracted or present at the surface of whole cells, was studied. After a short incubation period, pig Hb seemed to cover the bacterial cell surface and enhanced the cells' contrast when examined by transmission electron microscopy (TEM). Energy-dispersive X-ray spectroscopy analysis showed that the amount of elemental iron detected was increased when cells of A. pleuropneumoniae were incubated with pig Hb. Coating with pig Hb, however, did not interfere with the accessibility of O- and capsular antigens to antibodies on the bacterial cell surface. Binding of pig Hb and polymyxin B to lipid A of A. pleuropneumoniae was confirmed with a fluorescent probe (dansylcadaverine) displacement assay. The binding of pig Hb to extracted LPS resulted in a disaggregation of LPS as observed by TEM after negative staining. Additional evidence for a direct physical interaction between pig Hb and A. pleuropneumoniae LPS was demonstrated by a shift in the sedimentation velocity of LPS-Hb complexes determined by sucrose gradient centrifugation. Pig Hb binding to extracted LPS or to bacterial cells resulted in a significant decrease of chromogenic Limulus amebocyte lysate activation. Finally, the capacity of extracted LPS to induce NO2- in the presence of pig Hb was tested by using cell line J774 and determined by the Greiss' reaction. LPS alone induced, as expected, NO2- production, whereas the presence of pig Hb significantly reduced NO2- production by murine macrophages. Taken together, our results indicate that binding of pig Hb affected some physical and biological properties of A. pleuropneumoniae LPS.