Leiden University Scholarly Publications

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Depelteau, J.S. (2022)

Development and application of cryo-EM tools to study the ultrastructure of microbes in changing environments

Doctoral Thesis
Cryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo-EM techniques to gain insight into the structural changes of the human pathogen, Vibrio cholerae, as it transitions between different environments. A combination of established and novel techniques is used to prepare the individual cells for cryogenic electron tomography (cryo-ET). For example, I designed a manual plunge freezing apparatus to prepare cryo-EM samples off site and subsequently image them with cryo-ET. Furthermore, I used light microscopy and serial block face scanning EM imaging to visualize changes to the cells’ morphology and structure when transitioning from the environment, into the natural host, the zebrafish (Danio rerio), and back into the environment. In addition, this thesis demonstrates how ultraviolet-C radiation of cryo-EM samples of V....Show moreCryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo-EM techniques to gain insight into the structural changes of the human pathogen, Vibrio cholerae, as it transitions between different environments. A combination of established and novel techniques is used to prepare the individual cells for cryogenic electron tomography (cryo-ET). For example, I designed a manual plunge freezing apparatus to prepare cryo-EM samples off site and subsequently image them with cryo-ET. Furthermore, I used light microscopy and serial block face scanning EM imaging to visualize changes to the cells’ morphology and structure when transitioning from the environment, into the natural host, the zebrafish (Danio rerio), and back into the environment. In addition, this thesis demonstrates how ultraviolet-C radiation of cryo-EM samples of V. cholerae and the ICP1 bacteriophage can be used to inactivate the pathogen while retaining their ultrastructural details. Lastly, this thesis outlines current and novel methods for processing of larger, more complex samples for cryo-ET. These techniques, together with new models of host-pathogen interactions, offer new tools for exploring microbial interactions with their environments.Show less
Cryogenic electron microscopy
Cryogenic electron tomography
Single particle analysis
Subtomogram averaging
Vibrio cholerae
Sample preparation
Ultraviolet-C
ICP1 bacteriophage
Host-pathogen interactions
Zebrafish model
All authors
Depelteau, J.S.
Supervisor
Briegel, A.; Meijer, A.H.
Committee
Wezel, G.P. van; Claessen, D.; Blokesch, M.; Koster, A.J.; Spaink, H.P.
Qualification
Doctor (dr.)
Awarding Institution
Institute of Biology Leiden (IBL), Faculty of Science, Leiden University
Date
2022-01-12
ISBN (print)
9789464169751