Structural Studies of Host-Microbe Interactions Pubblico
Dillard, Rebecca (Spring 2018)
Published
Abstract
Recent advances in cryo-electron microscopy (cryo-EM) have drastically improved our ability to study the interactions between viruses and their hosts. The development of direct electron detectors has substantially improved image quality, and new techniques such as cryo-correlative light and electron microscopy (cryo-CLEM) are allowing us to understand more intricate systems. We have used cryo-EM to investigate host-microbe interactions at many stages of the viral replication cycle, including entry and assembly, and to provide insights into structural aspects of the host. Identifying regions of interest in the transmission electron microscope can be challenging. We have shown that the use of native immunolabeling is therefore extremely helpful in order to study cellular and viral surface proteins in eukaryotic systems. This technique cannot be used, however, to mark internal components, which require alternative labeling strategies. We developed a workflow for correlated cryo-fluorescence light microscopy and cryo-EM (cryo-CLEM) of fluorescently tagged virus-infected or transfected mammalian cells. Cryo-CLEM combines the spatiotemporal information provided by fluorescence microscopy imaging with structural information from cryo-EM. These studies have provided insights into the assembly and morphology of respiratory syncytial virus (RSV) and entry and fusion events of human immunodeficiency virus type I (HIV-I). We used cryo-electron tomography (cryo-ET) along with several functional assays to study bacteriophage interactions in prokaryotic systems. We found that Vibrio cholerae outer membrane vesicles (OMVs) act as a defense mechanism to protect V. cholerae against bacteriophage infection in a dose- and receptor-dependent manner, suggesting that OMVs are an important consideration for the use of phage therapy. In Caulobacter crescentus, we found that type IVc pilus retraction is required for bacteriophage phiCbK attachment, and that proper assembly of the flagellum, cell motility, and phiCbK adsorption depend on multiple flagellins. In summary, we have used cryo-EM to provide information about both eukarotic and prokaryotic virus interactions with their host cells, allowing us to examine RSV assembly, HIV-1 entry, and bacteriophage interactions with V. cholerae OMVs, C. crescentus pili, and C. crescentus flagella. Our studies illustrate that cryo-EM is a valuable technique for investigating host-microbe interactions.
Table of Contents
TABLE OF CONTENTS Abstract Acknowledgements Table of Contents List of Figures and Tables Chapter 1: Introduction..........1 Chapter 2: Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells..........85 Chapter 3: Vibrio cholerae outer membrane vesicles inhibit bacteriophage infection..........151 Chapter 4: Pilus retraction in Caulobacter crescentus is required for phiCbK phage infection..........179 Chapter 5: Structural specificity of the bacteriophage phiCbK head filament to the Caulobacter crescentus flagellum..........212 Chapter 6: Discussion..........245 Appendix A: Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications..........260 Appendix B: Pleomorphic structures in human blood are red blood cell-derived microparticles, not bacteria..........301 Appendix C: A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation..........324 Appendix D: The Ms6 mycolyl-arabinogalactan esterase LysB is essential for an efficient mycobacteriophage-induced lysis..........385
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