Research

Viral Infection

Visualization of Bacterial Secretion Systems

Molecular Mechanisms of Bacterial Motility

Molecular Machines in Cells

Proteolysis

Bacteriophages are the most abundant biological entity in the biosphere and are responsible for much of bacterial evolution. Most phages utilize elaborate tail machines to eject their genome into a host cell to cause infection. Our studies provide molecular insights into the mechanisms by which tailed phages overcome the multiple barriers of the bacterial cell envelope to deliver their viral DNA and proteins into the host cell cytoplasm. We combine structural biology in situ with genetics and physiology to advance fundamental knowledge of how these highly sophisticated molecular machines infect their bacterial hosts. These studies not only provide insight into the evolution of bacterial pathogenicity but also illuminate basic biological problems, such as molecular host-cell recognition; protein-protein, protein-DNA, and protein-membrane interactions; and penetration of the cell membrane by large macromolecules. Our current research aims to fully elucidate the molecular mechanisms by which the bacteriophage T7 genome ejection machine undergoes massive conformational changes to initiate infection and effect DNA translocation.

This project is supported by NIH/NIGMS.

Gram-negative bacteria use secretion systems for nutrient acquisition, virulence, and the transport of proteins, drugs, and toxins out of the cell or into host cells. We exploit a cutting-edge direct electron detector and high-throughput cryo-electron tomography (cryo-ET) to visualize high-resolution 3D structures of these complex nanomachines - specifically Type III (T3SS or injectisome) and Type IV (T4SS) secretion systems - which inject effector proteins from the bacterium into eukaryotic host cells and mediate the transport of macromolecules across cell membranes. In Shigella flexneri, we revealed the intact injectisome, including cytoplasmic sorting platform, and its interaction with host cells (Hu et al, PNAS 2015). And currently, we are investigating T4SS in the disease-causing pathogens Legionella pneumophila and Coxiella burnetii. By combining advanced cryo-ET with genetic and biochemical approaches, we aim to determine the structure and assembly of the Dot/Icm machine, an essential virulence determinant. Our work stands to elucidate how the Dot and Icm proteins contribute to machine assembly and function at the molecular level, enabling this incredibly versatile apparatus to translocate into host cells a repertoire of over 300 different proteins with distinct biochemical functions and diverse structural properties.

3D visualization of Salmonella-host interaction

In situ structure of the Type III secretion machine in Salmonella

Spirochetes are a phylogenetically distinct group of bacteria of significant importance to human health as they cause major diseases, such as syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi), leptospirosis (Leptospira interrogans), and periodontitis (Treponema spp.). To infect and disseminate in mammalian hosts, spirochetes have evolved a unique morphology and motility that is highly effective at translocating through viscous media and tissue barriers. The organelles essential for spirochetal motility are periplasmic flagella, which reside in the bacterial periplasmic space and are distinct from external flagella in the model systems Escherichia coli and Salmonella enterica. Given that flagella-driven motility is crucial for virulence of pathogenic spirochetes and many other bacteria, our long-term goal is to reveal the molecular mechanisms underlying flagellar assembly and function. We demonstrated at unprecedented resolution that the Lyme disease spirochete B. burgdorferi (Bb) is a great model system for characterizing periplasmic flagella in situ. In collaboration with Drs. Md Motaleb and Chunhao Li, we generated and characterized a large Bb library, including 60 different flagellar and chemotaxis mutants. This work has significantly advanced understanding of the periplasmic flagella and their remarkable capacity in driving the unique spirochetal motility and morphology. We aim to illuminate three fundamental aspects of periplasmic flagella critical to virulence: 1) the structure and function of the flagellar type III secretion apparatus; 2) the mechanism underlying flagellar rotation driven by proton motive force across the membrane; and 3) the mechanisms by which flagella switch rotational directions to control motility and chemotaxis. Together with genetic and biochemical approaches, we use cryo-ET to determine the structure/function relationship of the spirochetal flagellar motor in its native cellular environment at nanometer resolution.

Proteins perform the most important functions in all living cells. In pathogenic bacteria, which must successfully adapt to environmental changes within hosts to achieve infection, proteins must be produced at the correct locales, for the right duration, and in the appropriate quantities. While protein synthesis in pathogenic bacteria is well understood, the regulation of protein degradation remains largely unexplored. Proteolysis is an irreversible process that must be tightly controlled. My research focuses on the human gastroenteritis- and murine typhoid-causing Salmonella enterica serovar Typhimurium to understand how pathogens control proteolysis and virulence via proteases and adaptor molecules. This work will reveal novel strategies used by Enterobacteriaceae to cause infection as well as enhance our understanding of proteolytic systems common in all organisms.