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

全球播散型志贺毒素(STX)产生的大肠杆菌(STEC)因产生 噬菌体携带的细胞毒素，是致死性食源性疾病的直接介体。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，只有有限的治疗性或预防性武器库 对策是可用的。在人类感染期间进展为危及生命的并发症，如 HUS，与最强的细胞病变毒素亚型Stx2a的产生密切相关。超强毒力作为 表现为Stx2a滴度的增加通过系统发育与循环亚群有关， 流行病学和表型连锁。然而，关于内在基因组的知识还很匮乏。 由高水平的Stx2a生产商组成。这项建议的研究目标是应用系统和 用基因组水平的方法确定与超强毒毒素产生有关的致病原基因座 STEC病毒组。中心假设是分离株个体Stx2a产生的差异 能力与携带的Stx2a-噬菌体中锚定的菌株水平序列变异相关 噬菌体STEC病原体上的序列类型(PST)和外部基因座。这三个具体目标是 对这一假设的检验如下：1)对临床STEC标本的基因组规模变异进行编目 STEC核心和辅助Stx2a噬菌体库存的整体全基因组测序分型。2)至 鉴定一株Stx2a溶原菌的培养库。为了降低基因组的复杂性，我们将创建一个基因组 Stx2a-溶原菌培养的定义变体。通过溶源转换，我们将介绍典型的Stx2a- PST进入非志贺毒素大肠杆菌宿主的基因组背景，其中使用非典型的STX- 从我们的培养收集中检出STEC阴性和残留的胃肠道大肠杆菌。经过改造的Stx2a-溶原菌将 提供一个受控的基因组试验床，系统地确定Stx2a-PST、噬菌体剂量和 染色体定位导致Stx2a产生，独立或与噬菌体外部的基因座结合 在各自的寄主染色体上，以及3)确定与超强毒力Stx2a有关的致病基因组座位 制作。Wt STEC和Wt STEC的系统群和Stx2a-产生致病型的协同测定 工程Stx2a-溶原菌通过以下途径为确定Stx2a生产的调控位点提供了坚实的基础 全基因组联合研究。培养物按Stx2a-噬菌体动员效率表型，全球 转录组变化、Stx2a滴度和体内细胞毒性。每个基因类型变量分别进行测试和 在基因型组中解释多态Stx2a噬菌体和/或宿主的参与和相互作用 精神错乱。这项研究将为改进的生物监测和风险的发展提供有吸引力的目标 抑制性抗Stx2a治疗策略的评估。由于它们之间的进化守恒 Stx2a噬菌体和宿主噬菌体STEC病原体，我们期待开发的翻译应用 原则并确定了临床上重要的优先STEC集体组的Stx2a调节位。

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.