DictyMyc: Using Dictyostelium to study the genetic basis of Mycobacterium bovis intracellular infection.

由牛分枝杆菌引起的牛结核病是英国最重要的兽医健康问题之一。在没有改进控制的情况下，预计未来十年英国的经济负担将达到10亿英镑。控制可能需要一种综合方法，牛的疫苗接种是一个关键组成部分。目前还没有获得许可的牛结核病疫苗。然而，活的牛分枝杆菌卡介苗，目前用于人类结核病，是一个令人鼓舞的疫苗接种选择，但研究表明，它只提供保护约70%的动物，形成一个合理的平台，从设计一个改进的疫苗，刺激保护性免疫的所有个人，我们建议有必要更好地了解在分枝杆菌感染的宿主-病原体相互作用的基本机制。有证据表明，操纵分枝杆菌与吞噬细胞的相互作用可以增加免疫原性。分枝杆菌感染的中心特征是在哺乳动物免疫细胞（称为吞噬细胞）内复制的能力。我们和其他人以前研究过参与这些过程的细菌基因。然而，分析参与相互作用的HOST基因一直很困难，因为很难操纵哺乳动物细胞的遗传学。一种解决方法是使用越来越广泛应用于研究的转基因（GM-）小鼠的吞噬细胞。本项目的目的是鉴定和表征非脊椎动物的分枝杆菌感染模型，以替代使用GM-小鼠的吞噬细胞。在这个项目中，我们建议使用自由生活的变形虫Dictyostebellum discoideum作为模型吞噬细胞，因为它很容易操纵它的遗传学，分枝杆菌能够在Dictyostebellum中以类似的方式生存，他们如何在吞噬细胞中生存。因此，在实验中，dictyostelium感染分枝杆菌提供了一种方法，很容易研究在分枝杆菌感染过程中的宿主和病原体基因，同时避免使用有知觉的动物。为了验证网骨藻模型系统并验证它们对牛感染的影响，我们将比较牛巨噬细胞感染中使用的细菌基因与网骨藻感染所需的细菌基因。我们还将使用一种新开发的技术，该技术将允许在分枝杆菌感染期间同时分析40，000个dictyosteroid（宿主）突变体的文库。我们的实验将全面确定哪些宿主和病原体基因参与了牛分枝杆菌在网骨藻中的存活，从而为分枝杆菌如何控制其宿主细胞提供信息，并提供信息以帮助设计更好的抗牛和人类结核病疫苗。该项目将提供技术和证据，以证明其他研究结核病和其他传染病的研究人员利用网骨藻作为模式生物的合理性。

Bovine tuberculosis (TB) caused by Mycobacterium bovis is one of the most important veterinary health problems in the UK. In the absence of improved control, the projected economic burden to the UK over the next decade is predicted to be £1 billion. Control is likely to require an integrated approach with vaccination of cattle representing a key component. Presently there is no licensed vaccine against bovine TB. However, the live M.bovis BCG vaccine, presently used against human TB, represents an encouraging vaccination option, yet studies suggest that it only provides protection to ~70% of animals.To form a rational platform from which to design an improved vaccine that stimulates protective immunity in all individuals, we propose it is necessary to better understand the basic mechanisms of the host-pathogen interaction during mycobacterial infection. Evidence suggests that manipulating the mycobacterial interaction with phagocytic cells can increase immunogenicity.The central feature of mycobacterial infection is an ability to replicate inside mammalian immune cells called phagocytes. We and others have previously studied the bacterial genes involved in these processes. However, analysis of the HOST genes involved in the interactions has been difficult because it is hard to manipulate the genetics of mammalian cells. One solution is to use phagocytes from genetically modified- (GM-) mice which are becoming more widely used in research.THE AIM OF THIS PROJECT IS TO DEVELOP AND CHARACTERISE NON-VERTEBRATE MODELS OF MYCOBACTERIAL INFECTION AS A REPLACEMENT FOR USING PHAGOCYTES FROM GM-MICE. In this project we propose to use the free living amoebae Dictyostelium discoideum as a model phagocyte because it is easy to manipulate its genetics, and mycobacteria are able to survive in dictyostelium in a similar way to how they survive in phagocytes. Thus experiments in which dictyostelium is infected with mycobacteria provide a way to easily study both the host and pathogen genes during mycobacterial infection whilst avoiding the use of sentient animals. To characterise the Dictyostelium model systems and validate them against bovine infection, we will compare the bacterial genes used in infection in bovine macrophages with those required for infection in dictyostelium. We will also use a newly developed technology that will allow the simultaneous analysis of a library of 40,000 dictyostelium (host) mutants during mycobacterial infection. Our experiments will comprehensively identify which host and pathogen genes are involved in the M.bovis survival in dictyostelium informing how the mycobacterium controls its host cell and providing information to help design a better vaccine against bovine and human TB. This project will provide the technology and evidence to justify other researchers working on tuberculosis and other infectious diseases to utilise dictyostelium as a model organism.