Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism
Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism
复制标题
从噬菌斑衍生的革兰氏阴性微生物中提取白细胞毒素并进行部分表征
作者：
C. Tsai;W. McArthur;P. Baehni;B. F. Hammond;N. Taichman
检测了噬菌斑来源的革兰氏阴性微生物Y 4（鉴定为放线杆菌属成员）的可溶性细胞毒性因子。超声处理或在蒸馏水或正常人血清中孵育活的Y 4微生物导致释放一种可溶性物质，该物质在体外对人多形核白细胞（PMNs）具有细胞毒性。Y 4可溶性声波提取物对人外周血单核细胞也具有细胞毒性。然而，人淋巴细胞、血小板和成纤维细胞，以及兔、大鼠和小鼠白细胞和鸡胚成纤维细胞不会因暴露于Y 4声波浸提液而死亡。在Y 4声波浸提液中未检测到溶血活性。在Y 4声波浸提液中未检测到溶血活性。因此，Y 4声波提取物中的因子被称为Y 4白细胞毒素。Y 4白细胞毒素在4 ℃下无活性，对热敏感（56 ℃，30分钟），并被蛋白酶灭活。Y 4白细胞毒素对中性粒细胞的杀伤作用具有剂量、时间和温度依赖性。白细胞毒素在4 ℃下不与活的PMN结合，但在4 ℃和37 ℃下与死的PMN膜成分结合。此外，牛血清白蛋白（51毫克/毫升）的PMN-Y 4白细胞毒素培养物抑制乳酸脱氢酶的释放从中性粒细胞，但没有阻止细胞的死亡所示的电子显微镜。溶酶体标志物的释放平行于细胞质酶乳酸脱氢酶从Y 4白细胞毒素处理的中性粒细胞。向这些培养物中加入0.02 M乙二胺四乙酸抑制了溶酶体标志物的释放，但增强了乳酸脱氢酶的释放。这些结果表明，可以从活的Y 4中释放出仅对人中性粒细胞和单核细胞具有特异性的可溶性白细胞毒素。这种白细胞毒素在Y 4微生物的致病性中起什么作用尚不清楚。然而，这种白细胞毒素是来自菌斑来源的微生物的第一种物质，在青少年牙周炎的发病机制中具有潜在作用。
The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 4 degrees C, heat sensitive (56 degrees C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 4 degrees C but did bind to dead PMN membrane components at both 4 and 37 degrees C. The addition of bovine serum albumin (51 mg/ml) to PMN-Y4 leukotoxin cultures inhibited the release of lactate dehydrogenase from the PMNs, but did not prevent the death of the cells as indicated by electron microscopy. Lysosomal markers were released in parallel to the cytoplasmic enzyme lactate dehydrogenase from Y4 leukotoxin-treated PMNs. The addition of 0.02 M ethylenedinitrilotetraacetic acid to these cultures inhibited release of lysosomal markers but enhanced the release of lactate dehydrogenase. These results suggested that a soluble leukotoxin with specificity for only human PMNs and monocytes can be liberated from viable Y4. What role this leukotoxin plays in the pathogenicity of the Y4 microorganism is not yet known. However, this leukotoxin is one of the first materials from a plaque-derived microorganism with a potential role in the pathogenesis of juvenile periodontitis.