MECHANISM OF BACTERIAL METASTASIS IN PLAGUE

鼠疫的病原体--鼠疫耶尔森氏菌的聚乳酸突变体 特别是在引起传播性感染的能力上有缺陷。不像 通过皮下注射迅速传播的全毒力菌株 这些突变只会造成局部的皮下病变。他们的LD5O由 皮下注射比等基因注射高一百万倍 家长。尽管如此，它们仍然具有很高的传染性，并在 注射部位，显示对抗菌素有很大抗药性 免疫前宿主的防御。在以下情况下，病毒仍保持高度致命性 静脉注射，肝脾均达高滴度。 皮下注射部位进行组织学检查时，主要的 在突变型和野生型皮损之间观察到的区别是 后者含有较少的中性粒细胞(PMN)。这些结果意味着 即(1)即使对天然寄主具有高水平抵抗力的细菌 防御需要一些特殊的功能才能产生传播 在其他健康的宿主中感染，(2)此功能由 该作用的关键作用可能是降低PMN。 积聚在感染灶处，阻止形成微小脓肿 足够的完整性以防止细菌逃逸。 这项提案的主要目标是了解人民解放军是如何行动起来的 系统性疾病。该基因编码一个32kD的外膜蛋白水解酶 能在特定位置切割补体C3和备解素，阻断C3 消耗人血清，还能激活纤溶酶原。因此， 另一种补体途径被破坏，这是 感染早期的化学诱导剂，可能是中性粒细胞有限流入的原因 由Pla细菌引起的损伤。尽管它不能很容易地解释 减少PMN迁移，纤溶酶原激活也可能在 通过破坏组织完整性传播细菌，这是一种功能 已知它在哺乳动物肿瘤的转移中起作用。这个 补体假说将在VIVA和补体中进行检验 在体外，通过分析Pla对小鼠肝细胞生长的影响 补充成分。和系统功能的补充。纤溶酶原 激活假说将通过替代替代来检验 鼠疫杆菌纤溶酶原激活剂及其对纤溶酶的抑制作用 活体内活动。这些方法将通过更彻底的 Pla和Pla-突变体的组织病理学比较及进一步研究 Pla结构和功能的表征，重点是Pla的定义 蛋白水解酶活性部位。因为Pla的同源物出现在其他 革兰氏阴性细菌(包括大肠杆菌)，可能广泛存在，他们的 加强传播性感染发展的潜力将是 通过确定E.ColiPla同源物是否与 临床分离株中的菌血症，并测定大肠杆菌酶 可替代鼠疫杆菌中的Pla。播散性革兰氏阴性菌感染 每年在美国造成的死亡人数超过10万人。这项工作应该是 对剖析分子机制有重大贡献 对它们的起源做出了贡献。

pla mutants of Yersinia pestis, the causative agent of plague, are specifically defective in ability to cause disseminated infections. Unlike fully virulent strains which rapidly spread from subcutaneous injection sites, the mutants produce only a local subcutaneous lesion. Their LD5O by subcutaneous injection is a million-fold higher than that of the isogenic parent. Nonetheless, they remain highly infectious and grow at the injection site, indicating substantial resistance to antibacterial defenses of the pre-immune host. Thee also remain highly virulent when injected intravenously and reach high titers in both liver and spleen. When subcutaneous injection-sites are examined histologically, the major difference observed between mutant and wild-type lesions is that the latter contain many fewer polymorph neutrophils (PMN). These results imply that (1) even bacteria with high levels of resistance to innate host defenses require some specialized function to produce disseminated infections in an otherwise healthy host, (2) this function is provided by pla, and (3) the crucial activity of this function may be reduction of PMN accumulation at infectious foci, blocking formation of a micro-abscess of sufficient integrity to prevent escape of the bacteria. The major goal of this proposal is to understand how pla acts to produce systemic disease. This gene encodes a 32 kD outer membrane protease which can cleave complement C3 and properdin at specific sites, block C3 consumption in human serum, and also activate plasminogen. Thus, disruption of the alternative complement pathway, a major source of chemoattractant early in infection, may account for limited PMN influx to lesions caused by Pla+ bacteria. Although it does not readily explain reduced PMN immigration, plasminogen activation could also play a role in dissemination of the bacteria by disrupting tissue integrity, a function it is known to perform in the metastasis of mammalian tumors. The complement hypothesis will be tested both in viva, with complement deficient mice, and in vitro, by analysis of the effect of Pla on complement components. and complement system function. The plasminogen activation hypothesis will be tested by substitution of an alternative plasminogen actIvator for Pla in Y. pestis, and by inhibition of plasmin activity in vivo. These approaches will be enhanced by a more thorough histo-pathological comparison Pla+ and Pla- mutants and further characterization of Pla structure and function, focusing on definition of the proteolytic active site. Because homologues of Pla occur in other Gram-negative bacteria, (including E. coli,) and may be widespread, their potential to enhance the development of disseminated infections will be assessed by determining if the E. coli Pla homologues correlate with bacteremia among clinical isolates, and determining if the E. coli enzyme can substitute for Pla in Y. pestis. Disseminated Gram-negative infections cause in excess of 100,000 deaths in the U. S. each year. This work should contribute significantly to dissecting the molecular mechanisms contributing to their genesis.