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Article Dans Une Revue Trends in Microbiology Année : 2018

The Pathogen–Host Interface in Three Dimensions: Correlative FIB/SEM Applications

Résumé

Pathogens survive and propagate within host cells through a wide array of complex interactions. Tracking the molecular and cellular events by multidimen-sional fluorescence microscopy has been a widespread tool for research on intracellular pathogens. Through major advancements in 3D electron micros-copy, intracellular pathogens can also be visualized in their cellular environment to an unprecedented level of detail within large volumes. Recently, multidimen-sional fluorescence microscopy has been correlated with volume electron microscopy, combining molecular and functional information with the overall ultrastructure of infection events. In this review, we provide a short introduction to correlative focused ion beam/scanning electron microscopy (c-FIB/SEM) tomog-raphy and illustrate its utility for intracellular pathogen research through a series of studies on Shigella, Salmonella, and Brucella cellular invasion. We conclude by discussing current limitations of and prospects for this approach. Studying Pathogens with Light and Electron Microscopy Infection by pathogens occurs at a complex and dynamic interface between the pathogen and host that has been shaped over millions of years of evolution in a so called 'arms race' [1]. The mechanisms of infection and how they can be exploited against the invading pathogen are subject to intensive biological and medical research. Infection by invasive bacteria consists of host cell invasion, residence within the host cell, pathogen replication, propagation to neighboring cells and evasion of the host immune response, all requiring a finely tuned pathogenic strategy that manifests at the pathogen-host interface [2,3] Many of the molecular players and general mechanisms employed at this interface have been identified and characterized in some detail. However, how these function in the three-dimensional cellular environment at subdif-fraction resolution often remains poorly understood. Microscopy has been instrumental in the study of intracellular pathogens for more than 50 years. Light microscopy approaches were, and remain, extremely powerful for this purpose. Fluorescence microscopy provides localization information on specific fluorescently labeled molecules involved in infection processes [often with autofluorescent proteins like the green fluorescent protein (GFP)], and its combination with multidimensional imaging enables dynamic insights to where and when the host and pathogen factors cross-talk [4]. It also provides functional information through the development of a multitude of cellular reporters that are able to capture an ever-growing number of physical parameters [5]. Fluorescence microscopy, however, is limited in its ability to fully describe the three-dimensional cellular architecture of the pathogen-host interface due to both its resolution limit and the visualization of just a few fluorescent tags (normally two or three) against a 'black' background lacking cellular context. Highlights A current challenge in studying pathogen-host interactions is understanding how molecular players and cellular mechanisms function in the three-dimensional cellular environment at subdiffraction resolution. FIB/SEM is an emerging technique that can be used to investigate expansive three-dimensional cellular interfaces between pathogen and host at ultrastructural resolution. The correlation of FIB/SEM with fluor-escence microscopy (c-FIB/SEM) enables the study of transient or rare infection events while also providing molecular specificity within the ultra-structural landscape. Recently, c-FIB/SEM has revealed fundamental aspects of the mechanisms of cellular invasion by Shigella, Salmonella , and Brucella. c-FIB/SEM is becoming an indispensable tool for pathogen research as it can provide a wealth of biological information not obtainable by other technologies.
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pasteur-02000630 , version 1 (31-01-2019)

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Allon Weiner, Jost Enninga. The Pathogen–Host Interface in Three Dimensions: Correlative FIB/SEM Applications. Trends in Microbiology, 2018, xx, ⟨10.1016/j.tim.2018.11.011⟩. ⟨pasteur-02000630⟩
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