Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Liu, Jun

Abstract

From the moment Anton van Leeuwenhoek saw bacteria for the first time to the modern era of the ‘resolution revolution’ by the Nobel prize-winning method single particle cryo-electron microscopy (EM), advances in microscopy have led to visual observations that enhance our understanding of life at a wide range of scales, from millimeters to angstroms. The increasingly popular imaging technique cryo-electron tomography (cryo-ET) is uniquely suited for visualizing protein complexes and cellular features at high resolution in their native environment. This technology presents great potential for visualizing host-pathogen interactions. The work within this dissertation utilizes cutting-edge cryo-ET technologies to visualize different stages of bacterial infection and host defenses with an unprecedented level of detail. The first part of this dissertation focuses on determining the in situ structure of bacterial secretion machine. Legionella pneumophila (L. pneumophila) uses a sophisticated nanomachine called the type IV secretion system (T4SS) to inject bacterial substrates into target cells to achieve infection and survival inside hosts. Subtomogram averaging and 3D classification analysis of >9000 nanomachines revealed structural intermediates, suggesting that the T4SS machine is built from the outer membrane towards the cytoplasm. We propose an ‘outside-inside’ model of T4SS machine assembly. Furthermore, focused refinement of the cytoplasmic ATPase complex, composed on DotO and DotB ATPases, revealed that binding of DotB to DotO creates conformational changes leading to opening of a secretion channel that crosses the inner membrane. The second part of this dissertation focuses on how a bacterial pathogen uses the secretion machine to mediate interactions with the host membrane and initiate direct secretion of substrates across the host membrane. Salmonella uses its type III secretion (T3SS) machine, the injectisome, to create intimate contact with the host membrane for substrate translocation to initiate infection. Subtomogram averaging of the contact site between the injectisome needle tip and membrane allowed the first direct visualization of the T3SS translocon embedded in the host membrane. This finding provides critical evidence supporting the long-postulated direct substrate secretion model. The final part of this dissertation focuses on the use of cryo-ET and cutting-edge auxiliary tools, such as cryo-correlative light and electron microscopy (CLEM) and cryo-focused ion beam (FIB) milling, to visualize bacterial pathogens inside host cells. Chapter 4 contains studies characterizing the developmental transitions of Coxiella burnetii (C. burnetii) inside host cells and describes how these transitions impact assembly of T4SS machines. Moreover, high-resolution tomograms of C. burnetii at different stages of developmental transitions revealed that rapid changes in cell size can be facilitated by preserving the inner membrane.

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