MECHANISM OF BACTERIAL METASTASIS IN PLAGUE

鼠疫细菌转移机制

• 批准号: 2061735
• 负责人: Jon D. Goguen
• 金额: $ 25.74万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061735
• 关键词: Escherichia coli; Yersinia pestis; Yersinia pestis disease; bacterial proteins; chemotaxis; complement; complement pathway; enzyme activity; histopathology; laboratory mouse; laboratory rabbit; molecular pathology; mutant; neutrophil; nucleic acid sequence; plasminogen activator; protease inhibitor; tissue /cell culture; virulence

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.

鼠疫的病原体鼠疫耶尔森氏菌的pla突变体， 特别是在引起传播性感染的能力上有缺陷。不像 从皮下注射迅速传播的完全毒力菌株 突变体仅产生局部皮下病变。其LD 5 O由 皮下注射比同基因组高出百万倍 家长尽管如此，它们仍然具有高度传染性， 注射部位，表明对抗菌药物具有实质性耐药性 免疫前宿主的防御。当病毒感染时， 静脉内注射并在肝脏和脾脏中达到高滴度。 当对皮下注射部位进行组织学检查时， 在突变型和野生型病变之间观察到的差异是， 后者含有少得多的多形中性粒细胞（PMN）。这些结果意味 （1）即使是对先天宿主具有高水平抗性的细菌， 防御需要一些专门的功能来产生传播的 感染，否则健康的主机，（2）这一功能是由 pla，（3）该功能的关键活动可能是中性粒细胞的减少 在感染灶处积聚，阻塞微脓肿的形成， 足够的完整性，以防止细菌的逃逸。 本提案的主要目标是了解解放军如何采取行动， 全身性疾病。该基因编码一种32 kD的外膜蛋白酶， 可以在特定位点切割补体C3和备解素， 在人血清中消耗，并且还激活纤溶酶原。因此，在本发明中， 替代补体途径的破坏，这是 感染早期的化学引诱物，可能是有限的PMN流入， 由Pla+细菌引起的病变。虽然这并不能很好地解释 减少PMN迁移，纤溶酶原激活也可能在 通过破坏组织完整性来传播细菌， 已知其在哺乳动物肿瘤的转移中发挥作用。的 补体假说将在体内进行检验，补体 缺陷小鼠，并在体外，通过分析的影响， 互补成分并补充系统功能。纤溶酶原 激活假设将通过替代另一种假设来检验 纤溶酶原激活剂。鼠疫，并通过抑制纤溶酶 体内活性。 这些方法将得到加强， 组织病理学比较Pla+和Pla-突变体， PLA结构和功能的表征，重点是定义 蛋白水解活性位点。因为Pla的同源物存在于其他 革兰氏阴性菌（包括E.大肠杆菌，），并可能是广泛的，他们的 增加传播性感染发展的潜力将是 通过确定E.大肠杆菌Pla同源物与 临床分离株的菌血症，并确定是否E.大肠杆菌酶 可以替代Y.鼠疫播散性革兰阴性菌感染 导致美国超过10万人死亡。S.每年.这项工作应 对剖析生物进化的分子机制 有助于它们的起源。