Live cell imaging for infectious disease research

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.

Using live-cell imaging technology, we aim to investigate the dynamic interactions between viral, prokaryotic and eukaryotic pathogens and their hosts. One model is Herpes virus infection of B cells. Here, we plan to exploit complete interactome maps for viral gene products and host cell targets to visualise predicted interactions during the infection process in vivo (Haas). This will be complemented by modelling, from infection to recovery, of the fate of infected B cells and their interactions with T cells and macrophages in vivo using fluorescently tagged B cell populations (Nash). Interactions of the cells of the immune system (B cells, T cells and dendritic cells) are also being analysed in the context of Salmonella infection (Gray) and autoimmunity (Anderton). The interactions of eukaryotic pathogens with immune effectors will also be investigated. For example, the cell biology of Leishmania infection into phagocytes will be modelled using a combination of eGFP-tagged parasites and transgenic mice expressing fluorescent reporters in different endocytic compartments (Aebischer). Similarly, the interaction of parasites with the immune system, and the modelling of the evolution of sexual strategies, has been enabled by the development of transgenic, eGFP tagged rodent malaria (Thompson, Reece). Finally, using the extracellular protozoan, Trypanosoma brucei, Matthews is investigating key developmental regulators that operate in unusual intracellular signalling and gene expression pathways. These studies have many nodes of interaction (immune effector molecules, the cell biology of host-pathogen interactions, molecular interactions in vivo, modelling of pathogen infections at the whole organism and evolutionary level). This will ensure synergy, and reagent sharing, between the groups involved, and a wider user-set focused on pathogen biology.