EFFECTS OF PIG HEMOGLOBIN BINDING ON SOME PHYSICAL AND BIOLOGICAL PROPERTIES OF ACTINOBACILLUS-PLEUROPNEUMONIAE LIPOPOLYSACCHARIDES

Binding of pig hemoglobin (Hb) to Actinobacillus pleuropneumoniae lipo polysaccharide (LPS), either extracted or present at the surface of wh ole 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-disper sive X-ray spectroscopy analysis showed that the amount of elemental i ron detected was increased when cells of A. pleuropneumoniae were incu bated with pig Hb. Coating with pig Hb, however, did not interfere wit h the accessibility of O- and capsular antigens to antibodies on the b acterial cell surface. Binding of pig Hb and polymyxin B to lipid A of A. pleuropneumoniae was confirmed with a fluorescent probe (dansylcad averine) displacement assay. The binding of pig Hb to extracted LPS re sulted in a disaggregation of LPS as observed by TEM after negative st aining. 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 gradi ent centrifugation. Pig Hb binding to extracted LPS or to bacterial ce lls resulted in a significant decrease of chromogenic Limulus amebocyt e 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, NO 2- production, whereas the presence of pig Hb significantly reduced NO 2- production by murine macrophages. Taken together, our results indic ate that binding of pig Hb affected some physical and biological prope rties of A. pleuropneumoniae LPS.