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The genotypic and phenotypic impacts of Shiga toxin encoding bacteriophage interactions with their host cells: consequences for food borne zoonoses

志贺毒素编码噬菌体与其宿主细胞相互作用的基因型和表型影响：食源性人畜共患疾病的后果

基本信息

批准号：
BB/I013431/1
负责人：
Heather Allison
金额：
$ 46.2万
依托单位：
University of Liverpool
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2011
资助国家：
英国
起止时间：
2011 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FI013431%2F1
关键词：
genotypic phenotypic impacts Shiga toxin

项目摘要

In 1982, the first recorded outbreak of a novel, but deadly, E. coli serogroup, O157:H7 was recorded in North America, and such episodes were subsequently repeated across the world. Though foodborne outbreaks of this pathogen are limited in number, the severity of the disease is high and results in death or life-long debilitation in a significant proportion of infected individuals, particularly in young children. The major effector for the severity of disease is Shiga toxin. To date, more than 500 serogroup variants of E. coli have been reported to produce Shiga toxin as well as a few other related, and even unrelated, bacterial species, demonstrating the ongoing emergence of Shigatoxigenic potential across a variety of bacterial species. The genes enabling Shiga toxin production are carried by viruses known as bacteriophages, more specifically Stx phages. These viruses are directly responsible for the spread of shigatoxigenic potential, and though mush is known about shigatoxigenic E. coli (STEC), we know little about the Stx phages, which act as transmissible 'survival capsules' for Shiga toxin genes. We have recently demonstrated that Stx phages can actually infect a single bacterial cell multiple times. This increases the number of Shiga toxin genes within a bacterial cell, driving increased expression of Shiga toxin, enabling a multiply infected host to potentially cause more severe disease. The proposed project addresses several aspects of Stx phage biology that have been uncovered by our group. In identifying how Stx phages can multiply infect a single host cell, two miss-annotated genes of unknown function were identified. Preliminary mutant analyses demonstrate that these genes are essential to efficient virus production, and more importantly to Shiga toxin production. We intend to further understand how these genes ae controlled and function as they are crucial to virulence of these foodborne pathogens. Secondly, we have identified that a novel enzyme, which drives the ability of our model Stx phage to multiply infect its host cell, is a very promiscuous integrase. It has multiple recognitions sites in the E. coli chromosome, drives a unidirectional recombination reaction and has no close characterised relatives. Because of the these properties it has the potential to be a very valuable molecular tool for both standard laboratory techniques but also for use in gene therapy and other second generation molecular medicine techniques. The last two objectives focus on understanding how the bacterial virus and its host cell interact. To these ends, using tools we have obtained in previous work, we intend to examine how the bacterial cell responds to the virus it carries and how the viruses manipulates its host cell by examining all gene expression in response to single and double virus carriage with and without viral induction. We can now easily do this through second generation sequencing technologies (SOLiD), which will provide identification of all transcripts and their relative densities. This information will be directly informative, but it will also inform subsequent analyses that we have begun to examine the function of viral genes expressed by the host cell. Using qPCR we have been able to identify viral gene expression that is linked to viral replication and to the stable infection state where the viral genome is supposedly carried silently. This silent state is associated with increased resistance to a variety of environmental perturbations. We have begun to address the role these expressed genes play in the lifestyle of the bacterial host cell. These data will allow us to gain some understanding of the benefits of viral carriage and identify factors that provie a selective advantage to the host cell, driving the further spread and emergence of Shigatoxigenic potential. This will underpin our progress towards strategies to limit the future expansion and spread of these Shiga toxin producing zoonotic pathogens.

1982年，北美记录了第一次有记录的新型但致命的大肠杆菌血清群O157：H7的暴发，随后此类事件在世界各地重复发生。虽然食源性这种病原体的暴发数量有限，但该病的严重性很高，并导致相当大比例的感染者死亡或终身衰弱，特别是在幼儿中。影响病情严重程度的主要因素是志贺毒素。到目前为止，已有500多种大肠杆菌的血清组变体被报道产生志贺毒素以及其他一些相关的、甚至不相关的细菌物种，这表明在各种细菌物种中持续出现产志贺毒素的潜力。能够产生志贺毒素的基因是由被称为噬菌体的病毒携带的，更具体地说是STX噬菌体。这些病毒直接导致志贺氏菌毒素潜力的传播，尽管MUU对产志贺菌毒素大肠杆菌(STEC)已知，但我们对STX噬菌体知之甚少，STX噬菌体充当志贺氏菌毒素基因的可传播“生存胶囊”。我们最近证明，STX噬菌体实际上可以多次感染单个细菌细胞。这增加了细菌细胞内志贺毒素基因的数量，推动志贺毒素表达增加，使多重感染的宿主可能导致更严重的疾病。拟议的项目涉及STX噬菌体生物学的几个方面，这些方面是我们团队已经发现的。在鉴定STX噬菌体如何繁殖感染单个宿主细胞的过程中，鉴定出了两个功能未知的缺失注释基因。初步的突变分析表明，这些基因对于高效的病毒生产是必不可少的，更重要的是对志贺毒素的生产至关重要。我们打算进一步了解这些基因是如何控制和发挥作用的，因为它们对这些食源性病原体的毒力至关重要。其次，我们已经确定了一种新的酶，它驱动我们的模型STX噬菌体复制感染其宿主细胞的能力，是一种非常混杂的整合酶。它在大肠杆菌染色体上有多个识别位点，驱动单向重组反应，没有近亲。由于这些特性，它有可能成为一种非常有价值的分子工具，既可以用于标准的实验室技术，也可以用于基因治疗和其他第二代分子医学技术。后两个目标侧重于了解细菌病毒及其宿主细胞如何相互作用。为此，利用我们在以前的工作中获得的工具，我们打算通过检测在有和没有病毒诱导的情况下单病毒和双病毒携带的所有基因的表达，来研究细菌细胞如何对其携带的病毒做出反应，以及病毒如何操纵其宿主细胞。我们现在可以通过第二代测序技术(SOLID)轻松做到这一点，该技术将提供所有转录本及其相对密度的鉴定。这些信息将直接提供信息，但它也将告知后续的分析，我们已经开始检查宿主细胞表达的病毒基因的功能。利用定量聚合酶链式反应，我们已经能够识别与病毒复制和稳定感染状态有关的病毒基因表达，在这种状态下，病毒基因组被认为是静默携带的。这种沉默状态与对各种环境干扰的抵抗力增强有关。我们已经开始解决这些表达的基因在细菌宿主细胞的生活方式中所起的作用。这些数据将使我们对携带病毒的好处有一些了解，并确定为宿主细胞提供选择性优势的因素，推动志贺氏菌毒素潜力的进一步传播和出现。这将巩固我们在限制这些产生人畜共患病原体的志贺毒素未来扩展和传播的战略方面取得的进展。