Projects

In the Liu lab, I am utilizing single particle cryo-EM analysis and cryo-electron tomography to study the conformational changes of ejection proteins during genome ejection in phages T7 and P22. By combining these state-of-the-art techniques with extensive knowledge from biochemical and genetic studies, we aim to reveal the molecular mechanisms of ejection protein remodeling during genome ejection. Our findings provide detailed structural and functional insights into phage infection.

Efficient genome ejection is a critical yet little understood step in phage infection and requires large conformational changes in the mature tail machine. Specifically, podophages possess short tails that cannot span the Gram-negative cell envelope to allow direct genome ejection into the cell cytoplasm. These phages contain ejection proteins present in the mature capsid, possibly in complex with the genomic dsDNA. As the tail machine changes conformation after productive adsorption, these ejection proteins relocate inside the host bacteria, where they spontaneously assemble, forming a conduit for DNA translocation across the entire cell envelope. By contrast, the ejection proteins of podophages T7 and P22 have different conformations inside mature phages, both assembling a trans-envelope channel during genome ejection (Hu et al, 2013; Wang et al, 2019) using distinct mechanisms.

NCBI My Bibliography

Related lab publications:

• Wang, C., Tu, J., Liu, J., and Molineux, I.J. Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography. Nat Microbiol 4, 1049-1056, 2019
• Hu, B., Margolin, W., Molineux, I.J., and Liu, J. The bacteriophage t7 virion undergoes extensive structural remodeling during infection. Science 339, 576-579, 2013

The emergence of antibiotic-resistant bacteria poses a substantial health threat to humans. As conventional antibiotics are losing their potency, alternative solutions are urgently needed to combat bacterial infections. Bacteria use complex membrane-embedded protein assemblies called nanomachines to acquire nutrients, adhere to various surfaces and cells, and expel toxins from and inject effectors into host cells. Thus, these nanomachines are essential for bacterial virulence and survival, enabling infection and colonization of specific niches. In the Liu Lab, I use cryo-electron tomography to investigate the in situ structural biology of bacterial nanomachines involved in protein secretion and adhesion. Combined with knowledge acquired from biochemical and genetic studies, we aim to reveal key mechanistic insights needed to impair virulence by disrupting the functions of these nanomachines, a promising alternative route to treat bacterial infections without provoking resistance.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system.

Shoichi Tachiyama‡, Yunjie Chang§,¶, Meenakumari Muthuramalingam ‖ , Bo Hu**, Michael L. Barta ‖ 1, Wendy L. Picking‡‡, Jun Liu§,¶2 and William D. Picking‡,‡‡3

From Fig 6: Surface rendering images of Shigella T3SS. Left) The overall architecture of T3SS is described by four main components; the cytoplasmic sorting platform (cytoplasmic side), basal body (between bacterial inner membrane, IM, and outer membrane, OM), needle (extended from the basal body), and tip complex (end of the needle). Right) Cryo-ET provided a view from IM side of T3SS. The cytoplasmic sorting platform and basal body form different numbers of symmetrical rings .

The flagellar motor is a powerful biological nanomachine that drives motility, and thus infectivity and survival, in bacteria. It is the only known molecular machine that can rotate bidirectionally – in both clockwise (CW) and counterclockwise (CCW) senses. Motor rotation relies on the passage of ions through inner membrane-embedded stator units to power the cytoplasmic switch complex (C-ring), thus generating torque. Despite decades of intensive research, the detailed mechanisms that underlie torque generation and directional switching are unclear. I joined the Liu lab to use cryo-electron tomography (cryo-ET) to study this sophisticated mechanism in the model system of the Lyme disease-causing spirochete Borrelia burgdorferi. Our high-resolution analysis of dynamic stator-C-ring interactions will reveal molecular mechanisms responsible for torque generation and rotational switching in the bacterial flagellar motor. In addition, I currently oversee the operation of our state-of-the-art cryo-focused ion beam (FIB) microscope, Aquilos2, which enables extraordinary high-throughput imaging to address diverse biological questions.

Figure: Cryo-FIB milling of the Gram-negative bacterium Proteus in swarming conditions reveals the in-situ structure of the flagellar motor. (Left) Top view of scanning electron microscopy (SEM) image of Proteus swarming. (Right top) Side view of scanning electron microscopy (SEM) image of Proteus swarming. (Right bottom) Final lamella with 4^o tilt.

Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography.

Donghyun Park, Maria Lara-Tejero, M Neal Waxham, Wenwei Li, Bo Hu, Jorge E Galán, Jun Liu.

The overall objective of my research in the Liu lab is to utilize cutting-edge cryo-electron microscopy techniques and instruments to study the molecular mechanisms underlying T3SS- and T4SS-mediated host-pathogen interactions. To enable more sophisticated analyses, I use cryo-correlative light and electron microscopy (CLEM) to locate the target of interest based on fluorescence signals and cryo-focused ion beam (FIB) milling to generate cryo-lamella that will be imaged by cryo-ET to visualize intracellular pathogens. These investigations provide a foundation for the development of novel therapeutic strategies.

From Fig. 2: A tomogram and corresponding 3D rendering of a Salmonella Typhimurium minicell (green) interacting with a HeLa cell (membrane in red) via protein secretion machine.

Related lab publications:

• Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography. D Park, M Lara-Tejero, MN Waxham, W Li, B Hu, JE Galán, J Liu Elife 7, e39514, 2018
• Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding. D Park, D Chetrit, B Hu, CR Roy, J Liu Mbio 11 (1), 2020
• A mammalian system for high-resolution imaging of intact cells by cryo-electron tomography. X Li, D Park, Y Chang, A Radhakrishnan, H Wu, P Wang, J Liu Progress in Biophysics and Molecular Biology, 2020