Live cell imaging for infectious disease research

用于传染病研究的活细胞成像

• 批准号: BB/E012442/1
• 负责人: Keith Matthews
• 金额: $ 30.89万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE012442%2F1
• 关键词: Live; cell; imaging; infectious; disease

In the past, the analysis of the cellular events and molecular interactions of pathogens with their hosts was limited by the availability of suitable antibodies to detect particular proteins, or by inference from biochemical studies. These approaches, however, represent experimental 'autopsies' in which dynamic cellular processes must be deduced from a fixed sample. Developments in the imaging of molecular events in live cells, however, now mean that it is possible to follow, in real time, the location, movement and interactions of molecules within cells, and the interactions between cells- for example when a virus or bacterium infects cells of the body. These developments have been made possible by the engineering and expression within cells of proteins fused to naturally fluorescent proteins and by improvements in microscope technology. In particular, current confocal microscope systems enable both conventional high resolution positioning of proteins within fixed samples and the detection of fluorescent proteins in live samples under conditions that limit the cell damage that results from fluorescence illumination. The application of live cell imaging technology is particularly powerful for analysis of the interactions of a pathogen with its host, or host cell. Recently, it has become technically possible to engineer infectious agents such as viruses, bacteria or protozoan parasites to express one or more proteins conjugated to a fluorescent protein. When combined with existing technology for the expression of distinct fluorescent proteins within a mammalian host cell, it is possible to track molecular interactions between the pathogen and host, or track the infection pathway as a pathogen moves through distinct host cellular compartments, for example. These technologies have very wide ranging applications in infectious disease research, with researchers comprising this application being interested in such diverse processes as: - The complete interactions of viral proteins with other viral proteins and the proteins of the host cell. - The use of fluorescently labelled viruses to track the infection process in host cells and tissues. - The presentation of antigens by antibody producing cells to the immune system and the signalling events and protein interactions of immune effector and regulatory cells. - The infection process, developmental biology and evolutionary strategies of protozoan pathogens such as Leishmania, trypanosomes and malaria. The advantage of this technology- the ability to image live pathogens and their interactions with host cells- also presents a limitation- the need for safe containment of the pathogen. In consequence facilities available routinely in University departments are not available for use with infectious agents. This application proposes to establish a live cell imaging facility for infectious agents housed under suitable conditions for effective and safe containment. The equipment requested, a Leica TMC-SP5, has the capability for high resolution imaging of fixed and live samples and of high-speed capture, at excellent resolution, of dynamic cellular events. The equipment will be available to a large group of researchers contained within the Centre for Infectious Diseases at the University of Edinburgh, and provide a high-end facility with the potential to encourage resource, technology and knowledge sharing among a far wider group.

在过去，病原体与其宿主的细胞事件和分子相互作用的分析受到检测特定蛋白质的合适抗体的可用性或生物化学研究的推断的限制。然而，这些方法代表了实验性的“尸检”，其中动态细胞过程必须从固定的样品中推导出来。然而，活细胞中分子事件成像的发展现在意味着可以真实的跟踪细胞内分子的位置、运动和相互作用，以及细胞之间的相互作用--例如，当病毒或细菌感染身体细胞时。这些发展已成为可能的工程和细胞内的蛋白质融合到天然荧光蛋白的表达和显微镜技术的改进。特别地，当前的共聚焦显微镜系统使得能够在限制由荧光照明导致的细胞损伤的条件下，在固定样品内对蛋白质进行常规高分辨率定位以及对活样品中的荧光蛋白进行检测。活细胞成像技术的应用对于分析病原体与其宿主或宿主细胞的相互作用特别强大。最近，在技术上已经可以工程化感染因子，如病毒、细菌或原生动物寄生虫，以表达一种或多种与荧光蛋白缀合的蛋白质。例如，当与用于在哺乳动物宿主细胞内表达不同荧光蛋白的现有技术结合时，可以跟踪病原体和宿主之间的分子相互作用，或者当病原体移动通过不同的宿主细胞区室时跟踪感染途径。这些技术在感染性疾病研究中具有非常广泛的应用，包括该应用的研究人员对以下不同的过程感兴趣：-病毒蛋白与其他病毒蛋白和宿主细胞蛋白的完全相互作用。- 使用荧光标记的病毒来追踪宿主细胞和组织中的感染过程。- 抗体产生细胞向免疫系统呈递抗原，以及免疫效应细胞和调节细胞的信号事件和蛋白质相互作用。- 利什曼原虫、锥虫和疟疾等原生动物病原体的感染过程、发育生物学和进化策略。这项技术的优势--能够对活病原体及其与宿主细胞的相互作用进行成像--也存在一个局限性--需要安全地遏制病原体。因此，大学各系常规提供的设施不能用于感染性病原体。本申请提出建立一种活细胞成像设施，用于在适当条件下容纳的传染性病原体，以实现有效和安全的遏制。所要求的设备是Leica TMC-SP 5，具有对固定和活样本进行高分辨率成像的能力，以及以优异分辨率高速捕获动态细胞事件的能力。这些设备将提供给爱丁堡大学传染病中心的一大批研究人员，并提供一个高端设施，有可能鼓励更广泛的群体分享资源、技术和知识。

项目成果

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system.

• DOI: 10.1371/journal.ppat.1002340
• 发表时间: 2011-10
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Mott GA;Wilson R;Fernando A;Robinson A;MacGregor P;Kennedy D;Schaap D;Matthews JB;Matthews KR
• 通讯作者: Matthews KR

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei.

• DOI: 10.1093/nar/gkr1106
• 发表时间: 2012-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Walrad PB;Capewell P;Fenn K;Matthews KR
• 通讯作者: Matthews KR

A surface transporter family conveys the trypanosome differentiation signal.

• DOI: 10.1038/nature07997
• 发表时间: 2009-05-14
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Dean, Samuel;Marchetti, Rosa;Kirk, Kiaran;Matthews, Keith R.
• 通讯作者: Matthews, Keith R.