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

1982年，第一次有记录的新型但致命的E。大肠杆菌血清群O157：H7在北美有记录，随后这种情况在世界各地重复发生。虽然这种病原体的食源性暴发数量有限，但疾病的严重程度很高，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。疾病严重程度的主要效应物是滋贺毒素。迄今为止，已有500多个E.已经报道大肠杆菌产生滋贺毒素以及一些其它相关的、甚至不相关的细菌物种，证明了在多种细菌物种中不断出现的致志贺毒素的潜力。使滋贺毒素产生的基因由称为噬菌体的病毒携带，更具体地说是Stx噬菌体。这些病毒直接导致了产志贺毒素的传播，尽管对产志贺毒素的大肠杆菌知之甚少。大肠杆菌（STEC）中，我们对志贺毒素（Stx）知之甚少，它作为滋贺毒素基因的可传播的“生存胶囊”。我们最近已经证明，Stx可以多次感染单个细菌细胞。这增加了细菌细胞内滋贺毒素基因的数量，驱动滋贺毒素的表达增加，使得多重感染的宿主能够潜在地引起更严重的疾病。拟议的项目解决了我们小组发现的Stx噬菌体生物学的几个方面。在鉴定Stx Escherichia coli如何能够多重感染单个宿主细胞时，鉴定了两个未知功能的缺失注释基因。初步的突变体分析表明，这些基因是必要的有效的病毒生产，更重要的是滋贺毒素生产。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，我们已经鉴定出一种新的酶，其驱动我们的模型Stx噬菌体繁殖感染其宿主细胞的能力，是一种非常混杂的整合酶。它在E.大肠杆菌染色体，驱动单向重组反应，没有近亲。由于这些特性，它有可能成为标准实验室技术以及基因治疗和其他第二代分子医学技术中非常有价值的分子工具。最后两个目标集中在了解细菌病毒和其宿主细胞如何相互作用。为了这些目的，使用我们在以前的工作中获得的工具，我们打算检查细菌细胞如何响应它携带的病毒，以及病毒如何通过检查所有基因表达来操纵其宿主细胞，以响应有和没有病毒诱导的单病毒和双病毒携带。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，但它也将告知随后的分析，我们已经开始检查宿主细胞表达的病毒基因的功能。使用qPCR，我们已经能够鉴定与病毒复制和稳定感染状态（其中病毒基因组被认为是沉默携带的）相关的病毒基因表达。这种沉默状态与对各种环境扰动的抵抗力增加有关。我们已经开始研究这些表达的基因在细菌宿主细胞生活方式中的作用。这些数据将使我们能够对病毒携带的益处有所了解，并确定为宿主细胞提供选择性优势的因素，从而推动志贺毒素的进一步传播和出现。这将支持我们在限制这些产生滋贺毒素的人畜共患病病原体未来扩张和传播的战略方面取得进展。

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.