Bacteria. The flagella and pili of many bacteria are essential for motility, adherence, biofilm formation, and are often crucial virulence factors for pathogenic species. For many species, the two appendages are temporally and spatially regulated and organized to work either in synchrony or alternately in order to coordinate motility and surface colonization. Our long-term goal is to understand 1) the structural implications of the incorporation of multiple flagellins into the flagellar filament and 2) the structural variation between pili at the macromolecular level. Currently, we use two model systems to study bacterial pathogenesis and virulence, bacterial appendage structure and function, and bacteria-bacteriophage interactions. They are highlighted below.

Caulobacter crescentus. Caulobacter is an important model system for molecular developmental biology because of its genetic tractability and well-defined, asymmetric life cycle that features two morphologically and functionally distinct cells. The dimorphic life cycle of Caulobacter is prominently characterized by the presence of the polar stalk or flagellum and pili. We study viral infection in Caulobacter because it is an elegant model system to determine the relationship between a cell’s regulatory pathways and viral infection. φCb13 and φCbK, two Caudovirales, or tailed dsDNA phages, are ideal candidates for this analysis because their infection is in synchrony with the Caulobacter cell cycle and the presence of the polar flagellum and pili.

        Interaction between Caulobacter pilus and the tail of φCb13. Arrowheads point to the pilus filament in

        all images. (A) Slice through a tomogram of an infected Caulobacter cell. The black rectangle highlights

        the area of pilus-phage interaction. (B) Segmented volume imposed in 3D over the same slice in panel

        (A). Phages are depicted in blue, bacteria surface layer in green and pilus in orange. (C) Rotation of

        panel B to position the site of pilus-phage interaction in the front of the view. (D) Enlarged segmented

        volume.

Vibrio species. The virulence of many Vibrio species is specifically regulated by structural components, such as the flagellum, pili, outer membrane vesicles (OMVs), and capsular polysaccharide (CPS). In order to address how ultrastructure of Vibrios influences pathogenesis, we probe the architecture of the cells, the flagella, the pili, OMVs, and the CPS through cryo-ET. These experiments are complemented with fluorescence microscopy, biochemical, genetic, and microbiological assays to develop a spatial, temporal, and functional map of each organism’s pathogenesis profile.

                                                Slice through a 3D reconstruction of a Vibrio vulnificus cell.

                                                Note the cell body, sheathed flagellum, and dispersion of the

                                                outer membrane vesicles (OMVs).

Technology Development. In addition to the biological projects, we are developing and implementing technologies and methods to push the limits of cryo-EM and its correlation with other imaging modalities.

1.  Zernike phase contrast TEM. We are one of the few labs in the United States to have a 200 kV FEG-TEM

    specifically designed and engineered for Zernike phase contrast TEM. This technology provides an advantage for

    achieving higher resolution and higher contrast cryo-EM/cryo-ET images of low contrast biological specimens,

    especially isolated macromolecules, viruses, and bacteria.

                    In focus (left) and Zernike phase contrast (right) images of graphitized carbon taken on

                    the Emory JEOL JEM-2200FS.

2. 2. Correlative light and electron microscopy. We are developing novel equipment and molecular biology approaches for bridging the information gap between cryo-EM and fluorescence microscopy.  This includes the design, manufacture, and use of cryo-stages for confocal microscopy. By rapidly freezing cells cultured on EM grids, we are able to directly correlate fluorescence microscopy images to images collected in the electron microscope. This technology is being applied to fundamental questions about the assembly and trafficking of viruses, like RSV, within host cells.
   

3. 3.Affinity capture of enveloped viruses. We are developing methods for the selective capture and purification of enveloped viruses directly onto cryo-EM grids. Many enveloped viruses are extremely pleiomorphic, grow to low titers in culture, are cell-associated, and require the use of purification strategies that may alter the native structure of the virus. We have applied affinity technologies to address challenges associated with structural studies of enveloped viruses. The improvements realized by using affinity capture will be of benefit as we address basic questions regarding the structures of several families of enveloped viruses.

Viruses. Paramyxoviruses are pleiomorphic enveloped RNA viruses; which include the human pathogens respiratory syncytial virus (RSV), human metapneumovirus (hMPV), measles virus (MeV), and human parainfluenza viruses (HPIVs). Due to the significant variation in virus morphology, cryo-ET is the ideal technology to use for determining the macromolecular (~2 to 10 nm) structure of Paramyxoviridae family viruses.

Respiratory syncytial virus. Our long-term goal is to understand the structural basis of virus assembly in pleiomorphic viruses, using respiratory syncytial virus (RSV) as a model system. The project will explore how the viral proteins, cellular/viral membrane composition, and cell type define the structure of the budding virus.

Measles virus. In collaboration with Richard Plemper, measles virus (MeV) is being studied as a model system for understanding glycoprotein mediated viral fusion. This project addresses two basic questions: What is the spatial organization of paramyxovirus glycoprotein hetero-oligomer complexes in the native, metastable prefusion conformation? How does receptor binding affect this organization?

                                                Slice through a 3D reconstruction of a measles virus.

                                                Surface glycoproteins (white arrowheads) and the matrix

                                                protein coated RNP (black arrowheads) are noted.