LIPOPROTEINS OF TREPONEMA-DENTICOLA - THEIR EFFECT ON HUMAN POLYMORPHONUCLEAR NEUTROPHILS

The presence of lipoproteins and lipooligosaccharides in Treponema den ticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 we re metabolically labeled with [H-3]-cis-9-octadecenoic acid and extrac ted with the non-ionic detergent Triton X-114. The extract was phase s eparated, precipitated with acetone and delipidated to remove non-cova lently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradi ography showed [H-3]-cis-9-octadecenoic acid incorporation in bands wi th apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last ban d resolved into a 53 kDa [H-3]-cis-9-octadecenoic acid band upon heati ng for 10 min, at 100 degrees C. The structural relationship of the ou ter sheath major oligomeric polypeptide of strain ATCC 35404 and the 5 3 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa prote in. Proteinase K degraded the [H-3]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [H-3]-fatt y acid labeled compound found in the water phase following phenol-wate r extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophob ic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [H-3]-cis-9-octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8-8 mu g/ml) luminol dependent chemiluminiscence (LDCL) e ffect in human polymorphonuclear neutrophils (PMN) which could be rela ted to protein concentration. The addition of dLPP to PMN together wit h FMLP at submaximal concentration (1 mu M) resulted in a synergistic activation of LDCL. At 21 mu g/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotac tic peptide at 1 mu M. In addition, dLPP (21 mu g/ml) increased additi vely the release of lysozyme caused by 1 mu M FMLP. The release of bet a-glucuronidase was not affected. The modulation of neutrophil activit y was abolished by preincubation of dLPP with proteinase K. The purifi ed 14 kDa had no effect on either LDCL or exocytosis of lysosomal enzy mes of PMN. These data strongly suggest that T. denticola possesses se veral lipoproteins including outer sheath major oligomeric polypeptide s (113-234 kDa) and a lipooligosaccharide of molecular mass oi 14 kDa. In addition, an enriched lipoprotein fraction from this oral spirocha ete modulates oxygen dependent and independent mechanisms for controll ing microorganisms by human PMN.