ROLE OF YAPS IN Y. PESTIS PATHOGENESIS

雅普病在鼠疫杆菌发病机制中的作用

• 批准号: 8081927
• 负责人: VIRGINIA L MILLER
• 金额: $ 35.52万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8081927
• 关键词: Acute; Address; Amino Acids; Animal Model; Antibodies; Bacterial Adhesins; Biological; Biological Assay; Bioterrorism; Blood; Cell surface; Computer Simulation; Country; Cytotoxin; Data; Disease; Disease Outbreaks; Enterobacteriaceae; Escherichia coli; Fever; Fleas; Future; Genome; Goals; Histology; Human; In Vitro; Individual; Infection; Insecta; Laboratories; Life Cycle Stages; Lung; Measures; Membrane; Modeling; Molecular; Molecular Genetics; Mus; Mutation; N-terminal; Organism; Outcome Study; Pathogenesis; Peptide Hydrolases; Peptide Signal Sequences; Phenotype; Plague; Play; Pneumonic Plague; Probability; Production; Proteins; Public Health; Reporter; Rodent; Role; Route; Septicemic plague; Sequence Analysis; Structure; Surface; System; Testing; Trees; Vaccines; Virulence; Virulent; Western Blotting; Work; Yersinia pestis; base; cytokine; genetic manipulation; human morbidity; in vivo; interest; mortality; mouse model; mutant; novel; pathogen; research study; sucking; therapeutic target; tool; transmission process; vaccine development

Yersinia pestis is a Gram-negative bacterial human pathogen that is the causative agent of plague. Historically Y. pestis has been responsible for significant human morbidity and mortality. However, Y. pestis is typically a pathogen of rodents, and is unique among the Enterobacteriaceae in using a blood-sucking insect (flea) for transmission. Y. pestis infection in humans is an acute febrile disease that can have a number of different presentations depending upon the route of inoculation. Although environmental outbreaks of plague in the developed world largely have been controlled, the possible use of this pathogen as an agent of bioterrorism, as well as the re-emergence of plague in several countries, necessitates a more detailed understanding of the pathogenesis of Y. pestis to facilitate the identification of targets for vaccine development and treatment. No vaccine is currently available. A group of proteins that have a high likelihood of contributing to Y. pestis pathogenesis are the autotransporter proteins (ATs). AT proteins consist of three basic domains: a N-terminal signal sequence, a ¿passenger domain¿ and finally a β-domain at the C-terminus which facilitates translocation of the passenger domain across the outer membrane. The passenger domain provides the functional activity and new activities are being attributed to these proteins as more of them are studied in detail. While the β-domains are relatively conserved between ATs the passenger domains can vary significantly. In silico analyses have identified ten AT proteins of Y. pestis (designated yaps/Yaps). The Yaps are of interest to study for a number of reasons. First, while many putative AT proteins have been identified by in silico analyses, relatively few have been studied experimentally and even fewer have been studied in detail. With Y. pestis we have the advantage of being able to pair sophisticated molecular genetic manipulations with an animal model that is actually a natural host for this pathogen. Second, many of the Yaps by sequence analysis are relatively divergent when evolutionary trees of ATs are constructed and thus, the likelihood of identifying new functions by studying this group of ATs is high. Third, analysis of the Yaps may help identify a useful therapeutic target or candidate for inclusion in upcoming Y. pestis vaccine cocktails. We have preliminary data indicating that all ten of the yaps are expressed not only during laboratory conditions but also during infection. We also have constructed deletion mutants in seven yaps in a fully virulent Y. pestis strain and have begun testing the effect of these mutations on virulence. Four yap mutants clearly have phenotypes in a bubonic plague model of infection; these results are consistent with our hypothesis that the yaps play a role in pathogenesis. Our long term goal is to understand at a molecular level the role(s) of the individual Yaps in disease and the Y. pestis life cycle. Thus, we propose the following specific aims: (1) Analysis of the localization and expression of Yaps in Y. pestis; (2) Analysis of the role of Yaps in virulence; (3) Analysis of potential functions of the Yaps.

鼠疫耶尔森氏菌是一种革兰氏阴性细菌，是鼠疫的病原体。从历史上看，鼠疫杆菌是人类发病率和死亡率的主要原因。然而，鼠疫耶尔森氏菌是典型的啮齿动物病原体，在肠杆菌科中使用吸血昆虫(跳蚤)进行传播是独一无二的。人类中的鼠疫杆菌感染是一种急性发热性疾病，根据接种途径的不同，可能会有多种不同的表现。虽然发达国家的鼠疫环境暴发在很大程度上得到了控制，但这种病原体可能被用作生物恐怖主义的媒介，以及鼠疫在几个国家重新出现，这就需要更详细地了解鼠疫杆菌的发病机制，以便于确定疫苗开发和治疗的目标。目前还没有疫苗可用。一组极有可能导致鼠疫杆菌的蛋白质 其发病机制与自身转运蛋白(ATS)有关。AT蛋白由三个基本结构域组成：N-末端信号序列、乘客结构域和C-末端的-结构域，它促进乘客结构域在外膜上的移位。Passenger结构域提供功能活性，随着对更多蛋白质的详细研究，新的活性被归因于这些蛋白质。虽然-结构域在AT之间是相对保守的，但乘客结构域可能会有很大的差异。在计算机分析中已鉴定出鼠疫耶尔森氏菌的10个AT蛋白(命名为YAPS/YAPS)。出于几个原因，YAP引起了人们的研究兴趣。首先，虽然许多假定的AT蛋白已经在计算机分析中被鉴定出来， 进行实验研究的相对较少，详细研究的更少。对于鼠疫杆菌，我们的优势是能够将复杂的分子遗传操作与实际上是这种病原体的自然宿主的动物模型配对。其次，在构建ATS进化树时，通过序列分析得到的许多YAP是相对发散的，因此，通过研究这组AT来识别新功能的可能性很高。第三，对YAP的分析可能有助于确定一个有用的治疗目标或候选对象，以纳入即将到来的鼠疫疫苗鸡尾酒。我们有初步数据表明，所有10个YAP不仅在实验室条件下表达，而且在感染期间也表达。我们还在一株全毒力的鼠疫杆菌菌株中构建了七个年龄段的缺失突变体，并开始测试其效果 在毒力上的突变。四个YAP突变体在腺鼠疫感染模型中明显具有表型；这些结果与我们的假设一致，即YAP在发病机制中发挥作用。我们的长期目标是在分子水平上了解个体YAP在疾病和鼠疫杆菌生命周期中的作用(S)。因此，我们提出了以下具体目标：(1)分析YAPS在鼠疫菌中的定位和表达；(2)分析YAPS在毒力中的作用；(3)分析YAPS的潜在功能。