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：
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
项目类别：
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

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.

全球传播的产志贺毒素 (Stx) 大肠杆菌 (STEC) 因产生噬菌体传播的细胞毒素，是致命的食源性疾病的直接介质。人类发病率和死亡率仍然高得令人无法接受，因为没有疫苗，而且只有有限的治疗或预防手段可以采取对策。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 Stx2a 的产生密切相关。超毒力如通过系统发育，Stx2a 滴度增加与循环亚群相关，流行病学和表型的联系。然而，人们对内在基因组的了解还很缺乏。由高水平 Stx2a 制作人组成。本提案的研究目标是应用系统的、基因组规模的方法来识别负责高毒力毒素产生的致病基因位点 STEC 病原体组。中心假设是分离株个体 Stx2a 产量的差异能力与锚定在所携带的 Stx2a-噬菌体中的菌株水平序列变异相关噬菌体宿主 STEC 病原体组的序列类型 (PST) 和外部基因座。这三个具体目标检验该假设的步骤如下： 1) 通过以下方式对临床 STEC 样本中的基因组规模变异进行编目： STEC 核心和附件 Stx2a 噬菌体库存的整体全基因组测序分型。 2) 至表征 Stx2a 溶原菌培养库。为了降低基因组的复杂性，我们将创建一个基因组 Stx2a-溶原培养物的确定变体。通过溶原转化，我们将引入典型的 Stx2a- PST 进入非产志贺毒素大肠杆菌宿主的基因组背景，其中使用非典型 stx- 来自我们的培养物保藏的阴性 STEC 和常驻胃肠道大肠杆菌。工程化的 Stx2a-溶原菌将提供受控基因组测试平台来系统地确定 Stx2a-PST、噬菌体剂量和染色体位置独立地或与噬菌体外部的位点组合赋予 Stx2a 产生在各自的宿主染色体上，以及 3) 识别导致高毒力 Stx2a 的因果基因组位点生产。 wt STEC 和 STEC 系统发育群和 Stx2a 产生致病型的协同测定工程化的 Stx2a-lysogens 为识别 Stx2a 产生的调节位点提供了坚实的基础全基因组关联研究。对培养物进行全球 Stx2a 噬菌体动员效率表型分析转录组变化、Stx2a 滴度和体内细胞毒性。每个基因型变量都经过单独测试在基因型组中解释多态性 Stx2a 噬菌体和/或宿主的参与和相互作用基因座。这项研究将为改进生物监测、风险的发展提供有吸引力的目标评估抑制性治疗抗 Stx2a 策略。由于进化保守性 Stx2a 噬菌体和噬菌体宿主 STEC 病原体组，我们预计已开发的转化应用原则并确定了临床上重要的优先 STEC 集体组的 Stx2a 调控位点。