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GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

普瓦泽克立克次体的遗传分析

基本信息

批准号：
8053672
负责人：
DAVID WOOD
金额：
$ 13万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-12 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8053672
关键词：
Address Antibiotic Resistance Arthropod Vectors Arthropods Bacteria Binding Biological Assay Bypass Categories Cell Count Cells Classification Code Cytolysis Cytoplasm DNA DNA biosynthesis Detection Development Developmental Process Disease Epidemic Erythromycin Evolution Fever Gene Targeting Genes Genetic Techniques Genetic Transcription Genome Goals Grant Growth Health Homologous Gene Human In Vitro Knock-out Knowledge Link Metabolic Methods Mutagenesis Mutation Participant Pathogenicity Pattern Phase Plasmids Process Property Proteins Publishing Replication Origin Resistance Ribonucleases Rickettsia Rickettsia prowazekii Site Spottings Structure Stuttering System Techniques Testing Transcript Transcriptional Regulation Typhus Virulence base gene function genetic analysis genetic technology genome sequencing human disease in vivo intracellular parasitism member mutant pathogenic bacteria research study rho transcription termination

项目摘要

PROVIDED. Members of the genus Rickettsia are the etiologic agents of rocky mountainand other spotted fevers and endemic, scrub, and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsiaprowazekii, thz etiologic agent of epidemic typhus is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm of a eucaryotic host cell. The ability of rickettsiae to exploit this intracellularniche in animalsas diverse as arthropods and humans and to subsequently cause serious human disease provides the impetus for this study. This proposal focuses on the development and application of genetic techniques to address questions about the pathogenic bacterium R. prowazekii and its obligate intracytoplasmic existence. It exploits the availability of the R. prowazekii genome sequence and the development of rickettsial genetic technologies to test hypotheses related torickettsial gene function, DNA replication, and pathogenic mechanisms. In Specific Aim 1 our goal is to capitalize on a rickettsial transformation system and the identification of a selectable antibiotic resistance gene that can beexpressed inR.prowazekii to discriminate, via knockouts, essential function at the level of single genes. Specifically targeted genes include those that encode products withhomology to known virulencegenes of other bacteria, genes hypothesized to be expressed only in the arthropod vector, genes hypothesized to be non-functional and part of the process of rickettsial reductiveevolution,and finally, genes with homologs within the R.prowazekii genome. In addition, a transposon-based approach will be used to generate random insertion mutants. In Specific Aim 2 our goal is to isolate the functional origin of replication. One approach will attempt to generate a rickettsial minichromosome by linkingputative origin fragments with the selectable erythromycin-resistant gene, ereB. An alternate method will identify the originby the bindingof rickettsial DnaA. Specific Aim 3 will continue our characterization of transcription termination and our identificationof rickettsialtranscriptional changes that occur just prior to lysis of the host cell. Using ribonuclease protection studies we will determine whether these changes reflect a general property of the rickettsiae by examining additional non-intrinsic termination sites and the effect of cell number on terminationat these sites. Modulation of Rho and its correlation to these changes will be: assessed.

提供了立克次体属的成员是落基山脉和其他斑点热的病原体，灌木丛和流行性斑疹伤寒，这些疾病在世界范围内构成了有害的健康威胁。病原性立克次体流行性斑疹伤寒的病原体是一种专性细胞内寄生细菌，只能在细胞质内生长。真核宿主细胞立克次氏体利用这种细胞内生态位的能力在节肢动物和并随后导致严重的人类疾病为这项研究提供了动力。这项建议的重点是发展和应用遗传技术，以解决有关的问题，病原菌R. prowazekii及其专性胞质内存在。它利用了R. prowazekii基因组序列和立克次体遗传技术的发展，以检验立克次体相关假说基因功能、DNA复制和致病机制。在具体目标1中，我们的目标是利用立克次体转化体系的建立和可在普氏立克次体中表达的选择性抗生素抗性基因的鉴定通过敲除，在单个基因水平上区分基本功能。特异性靶向基因包括那些编码的产物与其他细菌的已知毒力基因具有同源性，这些基因假设只在节肢动物载体，假设为非功能性基因和立克次体还原进化过程的一部分，最后，在R.prowazekii基因组内具有同源物的基因。此外，将使用基于转座子的方法来产生随机插入突变体。在具体目标2中，我们的目标是分离复制的功能起点。一一种方法将试图通过将假定的起源片段与可选择的红霉素耐药基因ereB。另一种方法是通过立克次体DnaA的结合来鉴定来源。具体目的3将继续我们的转录终止的特征和我们的鉴定立克次体转录在宿主细胞裂解之前发生的变化。使用核糖核酸酶保护研究，我们将确定是否这些变化反映了立克次氏体的一般性质，通过检查额外的非固有终止位点，细胞数量对这些位点终止的影响。将评估Rho的调节及其与这些变化的相关性。