Lipoproteins of <i>Treponema denticola</i>: their effect on human polymorphonuclear neutrophils

Abstract

The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 were metabolically labeled with [3H]-cis-9-octadecenoic acid and extracted with the non-ionic detergent Triton X-114. The extract was phase separated, precipitated with acetone and delipidated to remove non-covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]-cis-9-octadecenoic acid incorporation in bands with 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 band resolved into a 53 kDa [3H]-cis-9-octadecenoic acid band upon heating for 10 min, at 100 degrees C. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa protein. Proteinase K degraded the [3H]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]-fatty acid labeled compound found in the water phase following phenol-water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [3H]-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 micrograms/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1 microM) resulted in a synergistic activation of LDCL. At 21 micrograms/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotactic peptide at 1 microM. In addition, dLPP (21 micrograms/ml) increased additively the release of lysozyme caused by 1 microM FMLP. The release of beta-glucuronidase was not affected. The modulation of neutrophil activity was abolished by preincubation of dLPP with proteinase K. The purified 14 kDa had no effect on either LDCL or exocytosis of lysosomal enzymes of PMN. These data strongly suggest that T. denticola possesses several lipoproteins including outer sheath major oligomeric polypeptides (113-234 kDa) and a lipooligosaccharide of molecular mass of 14 kDa. In addition, an enriched lipoprotein fraction from this oral spirochaete modulates oxygen dependent and independent mechanisms for controlling microorganisms by human PMN.