Identification of Regulatory Loci of Stx2a Cytotoxin Production in Shiga toxin producing Escherichia coli (STEC)

产志贺毒素大肠杆菌 (STEC) 中 Stx2a 细胞毒素产生调控位点的鉴定

基本信息

批准号：
10612881
负责人：
Mark Eppinger
金额：
$ 37.5万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612881
关键词：
Archives Attenuated Bacteriophages Biotechnology Catalogs Chromosomes Clinical Collection Correlation Studies Cytotoxin Data Development Diagnostic Disease Engineering Epidemiology Equipment and supply inventories Escherichia coli Escherichia coli EHEC Escherichia coli Infections Escherichia coli O157:H7 Food Foundations Frequencies Future Genetic Polymorphism Genome Genomics Genotype Heterogeneity Human Hybrids Individual Infection Knowledge Life Link Location Mediator Methodology Morbidity - disease rate Phenotype Phylogenetic Analysis Preventive Production Prophages Public Health Publishing Research Research Design Resources Risk Assessment Severities Shiga Toxin Specimen System Testing Therapeutic Toxin Vaccines Variant Virulence Work chromosomal location cytotoxicity dosage foodborne gastrointestinal genome annotation genome sequencing genome wide association study genome-wide human disease human morbidity human mortality improved in vivo individual variation mortality novel therapeutics pathogen pathogen genome pathogenic microbe response segregation trait transcriptome translational applications whole genome

Archives Attenuated Bacteriophages Biotechnology Catalogs Chromosomes Clinical Collection Correlation Studies Cytotoxin Data Development Diagnostic Disease Engineering Epidemiology Equipment and supply inventories Escherichia coli Escherichia coli EHEC Escherichia coli Infections Escherichia coli O157:H7 Food Foundations Frequencies Future Genetic Polymorphism Genome Genomics Genotype Heterogeneity Human Hybrids Individual Infection Knowledge Life Link Location Mediator Methodology Morbidity - disease rate Phenotype Phylogenetic Analysis Preventive Production Prophages Public Health Publishing Research Research Design Resources Risk Assessment Severities Shiga Toxin Specimen System Testing Therapeutic Toxin Vaccines Variant Virulence Work chromosomal location cytotoxicity dosage foodborne gastrointestinal genome annotation genome sequencing genome wide association study genome-wide human disease human morbidity human mortality improved in vivo individual variation mortality novel therapeutics pathogen pathogen genome pathogenic microbe response segregation trait transcriptome translational applications whole genome

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项目摘要

Globally disseminated Shiga toxin (Stx)-producing Escherichia coli (STEC) are notorious for producing a phage-borne cytotoxin, that is direct mediator of lethal food borne disease. Human morbidity and mortality remain unacceptably high, as no vaccines and only a limited arsenal of therapeutic or preventive countermeasures are available. Progression to life-threatening complications during human infection, such as HUS, is inexorably linked to the production of the most potent cytopathic toxin subtype Stx2a. Hypervirulence as manifested by increased Stx2a titers has been associated with circulating subpopulations through phylogenetic, epidemiological and phenotypic linkage. However, there is a dearth of knowledge of the intrinsic genomic make-up of high-level Stx2a producers. The research objective of this proposal is to apply a systematic and genome-scale approach to identify causal pathogenome loci responsible for hypervirulent toxin production in the STEC pathogenome. The central hypothesis is that differences in the isolates' individual Stx2a production capability are correlated with strain-level sequence variation anchored in both the carried Stx2a-Phage Sequence Type (PST) and external loci on the phage-hosting STEC pathogenome. The three specific aims to test this hypothesis are as follows: 1) To catalogue genome-scale variation in clinical STEC specimen through holistic Whole Genome Sequencing Typing of the STEC core and accessory Stx2a-phage inventory. 2) To characterize a culture bank of Stx2a lysogen. To reduce the genomic complexity we will create a genomically defined variant of Stx2a-lysogen cultures. Through lysogenic conversion we will introduce archetypical Stx2a- PST into the genome background of non-shigatoxigenic E. coli hosts, using among others, atypical stx- negative STEC and resident gastrointestinal E. coli from our culture collection. Engineered Stx2a-lysogens will provide a controlled genomic testbed to systematically determine how the Stx2a-PST, phage dosage and chromosomal location impart Stx2a production, independently or in combination with loci external to the phage on the respective host chromosomes, and 3) To identify causal genome loci responsible for hypervirulent Stx2a production. The synergistic determination of phylogroup and Stx2a-production pathotype in wt STEC and engineered Stx2a-lysogens provides a robust foundation to identify modulatory loci of Stx2a production through Genome Wide Association Studies. Cultures are phenotyped for Stx2a-phage mobilization efficiency, global transcriptome changes, Stx2a titers, and in vivo cytotoxicity. Each genotypic variable is tested individually and in groups of genotypes to account for the participation and interplay of polymorphic Stx2a phage and/or host loci. This research will provide attractive targets for the development of improved biosurveillance, risk assessment of suppressive therapeutic anti-Stx2a strategies. Due to the evolutionary conservation among Stx2a-phages and phage-hosting STEC pathogenomes, we anticipate translational application of developed principles and identified Stx2a regulatory loci for the collective group of clinically important priority STEC.

全球传播的志贺毒素（STX）产生的大肠杆菌（STEC）臭名昭著，以产生A 噬菌体传播的细胞毒素，是致命食品传播疾病的直接介体。人的发病率和死亡率保持不可接受的高度，因为没有疫苗，只有有限的治疗或预防武器可以使用对策。人类感染期间威胁生命的并发症的发展，例如 HUS与最有效的细胞病毒素亚型STX2A的产生无可避免地联系在一起。过度投资为通过系统发育，流行病学和表型连接。但是，缺乏对固有基因组的知识高级STX2A生产商的构成。该提案的研究目标是应用系统和基因组规模的方法来识别负责过度毒性毒素产生的因果病原体位点 STEC病原体。中心假设是分离株的单个STX2A产生的差异能力与锚定在携带的STX2A-PAGE中的应变级序列变化相关噬菌体托管STEC病原体上的序列类型（PST）和外部基因座。三个特定的目标检验该假设如下：1）通过 STEC核心和配件STX2A-PAGE库存的整体整体基因组测序键入。 2）到特征是STX2A溶酶原培养库。为了降低基因组复杂性，我们将在基因组上创建 STX2A-溶质原培养的定义变体。通过溶菌发生转化，我们将引入原型的STX2A- PST进入非硫酸大肠杆菌宿主的基因组背景，使用了非典型STX- 来自我们的培养物收藏中的负STEC和常驻胃肠道大肠杆菌。设计的STX2A赖糖果会提供受控的基因组测试床，以系统地确定STX2A-PST，噬菌体剂量和染色体位置将STX2A的产生赋予独立或与噬菌体外部的基因座结合在各自的宿主染色体上，3）识别负责STX2A的因果基因组基因群生产。 WT STEC和工程的STX2A赖糖元素为通过基因组广泛的关联研究。将培养物表型用于STX2A-PAGE-PAGE动员效率，全球转录组变化，STX2A滴度和体内细胞毒性。每个基因型变量单独测试，并在基因型组中，以说明多态性STX2A噬菌体和/或宿主的参与和相互作用基因座。这项研究将为开发改善的生物监视，风险提供有吸引力的目标评估抑制性治疗性抗STX2A策略。由于中的进化保护 STX2A-PHAGS和噬菌体托管STEC病原体，我们预计开发的翻译应用原则并确定了集体临床重要优先级STEC组的STX2A调节基因座。