Sharp Lindsay, Poole Stephen, Reddi Krisanavane, Fletcher Julie, Nair Sean, Wilson Michael, Curtis Michael, Henderson Brian, Tabonal Peter
Cellular Microbiology Research Group and Microbiology University College London, 256 Gray's Inn Road, London WClX 8LD, UK.
>Division of Endocrinology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK.
Microbiology (Reading). 1998 Nov;144 ( Pt 11):3019-3026. doi: 10.1099/00221287-144-11-3019.
There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivalis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81, 68, 48, 47, 28, 25, 20, 17 and 16 kDa. These LAPs, at concentrations as low as 100 ng ml(-1), were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood to produce the pro-inflammatory cytokine IL-6. The cytokine-inducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous to an internal region of a conserved adhesin domain contained within a family of P. gingivalis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivalis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear to have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influence the cytokine status of the periodontium in patients with the common chronic inflammatory conditions known as the periodontal diseases.
有证据表明，肠道细菌的脂多糖A相关蛋白（LAPs）可诱导白细胞介素1（IL-1）的合成，因此可能是重要的毒力因子。牙龈卟啉单胞菌现已被公认为慢性炎症性牙周疾病的主要病原体，此前有报道称，该菌的粗LAP组分可诱导IL-1和白细胞介素6（IL-6）的合成。在本研究中，对该细菌LAPs的化学和生物学特性进行了进一步表征。SDS-PAGE分析表明，LAPs由9种分子量分别为81、68、48、47、28、25、20、17和16 kDa的蛋白质组成。这些LAPs在低至100 ng ml(-1)的浓度下，可刺激人牙龈成纤维细胞、人外周血单核细胞和全血产生促炎细胞因子IL-6。热灭活和胰蛋白酶处理后，LAPs的细胞因子诱导活性降低，表明该活性并非由污染的脂多糖（LPS）所致。为确定该混合物中哪些蛋白质是活性细胞因子诱导剂，通过聚丙烯酰胺凝胶电泳对LAPs进行了分离。大部分活性与17 kDa的LAP相关。N端序列分析表明，该蛋白质与牙龈卟啉单胞菌细胞外蛋白质家族（包括RI蛋白酶、赖氨酸牙龈蛋白酶、卟啉蛋白酶和血凝素A）中保守黏附素结构域的内部区域同源。除了在黏附中起作用外，这些蛋白质的黏附素结构域也可能具有细胞因子诱导特性。此外，还表明牙龈卟啉单胞菌先前观察到的细胞因子降解可能归因于RI蛋白酶的催化结构域。因此，同一分子内的不同结构域似乎对促炎细胞因子水平具有相反作用，这两种活性之间的平衡可能影响患有常见慢性炎症性疾病即牙周疾病患者牙周组织的细胞因子状态。
