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

• 批准号: 10392401
• 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
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392401
• 关键词: Archives; Attenuated; Bacteriophages; Biotechnology; Catalogs; Chromosomes; Clinical; Collection; Cytotoxin; Data; Development; Diagnostic; Engineering; Epidemiology; Equipment and supply inventories; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; Escherichia coli O157:H7; Foundations; Frequencies; Future; Genetic Polymorphism; Genome; Genomics; Genotype; Heterogeneity; Human; Hybrids; Individual; Infection; Knowledge; Lead; Life; Link; Location; Mediator of activation protein; 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; foodborne illness; gastrointestinal; genome sequencing; genome wide association study; genome-wide; human disease; human morbidity; human mortality; improved; in vivo; mortality; novel therapeutics; pathogen; pathogen genome; pathogenic microbe; response; trait; transcriptome; translational applications; whole genome

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.

全球传播的产滋贺毒素（Stx）的大肠杆菌（STEC）因产生 噬菌体携带的细胞毒素，是致命性食源性疾病的直接介质。人类发病率和死亡率 由于没有疫苗，而且只有有限的治疗或预防药物， 反制措施是可行的。在人类感染期间进展为危及生命的并发症，例如 HUS与最有效的细胞病变毒素亚型Stx 2a的产生有着不可分割的联系。毒力亢进， 通过系统发育， 流行病学和表型联系。然而，缺乏内在基因组的知识， 高水平Stx 2a生产商的组成。本建议的研究目的是应用一个系统和 基因组规模的方法，以确定致病病原体基因座负责超毒力毒素的生产， STEC病原体。中心假设是分离株的单个Stx 2a产生的差异 能力与锚定在所携带的Stx 2a-噬菌体 序列类型（PST）和噬菌体宿主STEC病原体组上的外部基因座。三个具体目标是 检验这一假设如下：1）通过 STEC核心和辅助Stx 2a噬菌体库存的整体全基因组测序分型。2)到 表征Stx 2a溶原细胞培养库。为了降低基因组的复杂性，我们将创建一个基因组 Stx 2a-溶原菌培养物的定义变体。通过溶原性转化，我们将引入典型的Stx 2a- PST插入非产志贺毒素E.大肠杆菌宿主，其中使用非典型stx- STEC阴性和胃肠道E.大肠杆菌的样本工程化的Stx 2a-溶原菌将 提供受控的基因组试验平台，以系统地确定Stx 2a-PST、噬菌体剂量和 染色体定位独立地或与噬菌体外部的基因座组合地赋予Stx 2a产生 3）鉴定负责高毒力Stx 2a的致病基因组基因座 生产在野生型STEC和野生型STEC中的Stx 2a组和Stx 2a产生致病型的协同测定 工程化的Stx 2a-溶原菌为通过以下途径鉴定Stx 2a产生的调节基因座提供了坚实的基础： 全基因组关联研究。对培养物的Stx 2a-噬菌体动员效率进行表型分析，全球 转录组变化、Stx 2a滴度和体内细胞毒性。每个基因型变量单独测试， 以解释多态性Stx 2a噬菌体和/或宿主的参与和相互作用 的位点这项研究将提供有吸引力的目标，为发展改进的生物监测，风险 评估抑制性治疗性抗Stx 2a策略。由于进化的保守性， Stx 2a-B1和噬菌体宿主STEC病原体，我们预期已开发的STX 2a-B1的翻译应用。 原则，并确定了临床重要的优先STEC的集体组的Stx 2a调控基因座。