Cheri M. Hampton
Emory University
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Publication
Featured researches published by Cheri M. Hampton.
Nature Protocols | 2017
Cheri M. Hampton; Joshua D. Strauss; Zunlong Ke; Rebecca S. Dillard; Jason Hammonds; Eric Alonas; Tanay M. Desai; Mariana Marin; Rachel E. Storms; Fredrick Leon; Gregory B. Melikyan; Philip J. Santangelo; Paul Spearman; Elizabeth R. Wright
Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM.
Nature Communications | 2016
Christopher C. Stobart; Christina A. Rostad; Zunlong Ke; Rebecca S. Dillard; Cheri M. Hampton; Joshua D. Strauss; Anne L. Hotard; Jia Meng; Raymond J. Pickles; Kaori Sakamoto; Sujin Lee; Michael G. Currier; Syed M. Moin; Barney S. Graham; Marina S. Boukhvalova; Brian E. Gilbert; Jorge Blanco; Pedro A. Piedra; Elizabeth R. Wright; Martin L. Moore
Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.
Science | 2017
Courtney K. Ellison; Jingbo Kan; Rebecca S. Dillard; David T. Kysela; Adrien Ducret; Cécile Berne; Cheri M. Hampton; Zunlong Ke; Elizabeth R. Wright; Nicolas Biais; Ankur B. Dalia; Yves V. Brun
Elucidating a bacterial sense of touch Bacteria can adhere to surfaces within the host. This leads to tissue colonization, induction of virulence, and eventually the formation of biofilms—multicellular bacterial communities that resist antibiotics and clearance by the immune system (see the Perspective by Hughes and Berg). Hug et al. show that bacteria have a sense of touch that allows them to change their behavior rapidly when encountering surfaces. This tactile sensing makes use of the inner components of the flagellum, a rotary motor powered by proton motif force that facilitates swimming toward surfaces. Thus, the multifunctional flagellar motor is a mechanosensitive device that promotes surface adaptation. In complementary work, Ellison et al. elucidate the role of bacterial pili in a similar surface-sensing role. Science, this issue p. 531, p. 535; see also p. 446 Bacteria sense surfaces via the resistance imparted on retracting surface-bound pili. It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized Caulobacter crescentus pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.
Journal of Histochemistry and Cytochemistry | 2015
Joshua D. Strauss; Zunlong Ke; Eric Alonas; Rebecca S. Dillard; Cheri M. Hampton; Kristen M. Lamb; Jason Hammonds; Philip J. Santangelo; Paul Spearman; Elizabeth R. Wright
Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.
Nature Communications | 2018
Zunlong Ke; Joshua D. Strauss; Cheri M. Hampton; Melinda A. Brindley; Rebecca S. Dillard; Fredrick Leon; Kristen M. Lamb; Richard K. Plemper; Elizabeth R. Wright
Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F–M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.Virus assembly is technically challenging to study. Here the authors use cryo-electron tomography of measles virus-infected human cells to determine native-state virus structure and they locate well-ordered M lattices that organize viral glycoproteins, RNP, and drive assembly.
Viruses | 2017
Adriano Gigante; Cheri M. Hampton; Rebecca S. Dillard; Filipa Gil; Maria João Catalão; José Moniz-Pereira; Elizabeth R. Wright; Madalena Pimentel
All dsDNA phages encode two proteins involved in host lysis, an endolysin and a holin that target the peptidoglycan and cytoplasmic membrane, respectively. Bacteriophages that infect Gram-negative bacteria encode additional proteins, the spanins, involved in disruption of the outer membrane. Recently, a gene located in the lytic cassette was identified in the genomes of mycobacteriophages, which encodes a protein (LysB) with mycolyl-arabinogalactan esterase activity. Taking in consideration the complex mycobacterial cell envelope that mycobacteriophages encounter during their life cycle, it is valuable to evaluate the role of these proteins in lysis. In the present work, we constructed an Ms6 mutant defective on lysB and showed that Ms6 LysB has an important role in lysis. In the absence of LysB, lysis still occurs but the newly synthesized phage particles are deficiently released to the environment. Using cryo-electron microscopy and tomography to register the changes in the lysis phenotype, we show that at 150 min post-adsorption, mycobacteria cells are incompletely lysed and phage particles are retained inside the cell, while cells infected with Ms6wt are completely lysed. Our results confirm that Ms6 LysB is necessary for an efficient lysis of Mycobacterium smegmatis, acting, similarly to spanins, in the third step of the lysis process.
Frontiers in Microbiology | 2017
Cheri M. Hampton; Ricardo C. Guerrero-Ferreira; Rachel E. Storms; Jeannette V. Taylor; Paul A. Gulig; Elizabeth R. Wright
Vibrio vulnificus, a bacterial species that inhabits brackish waters, is an opportunistic pathogen of humans. V. vulnificus infections can cause acute gastroenteritis, invasive septicemia, tissue necrosis, and potentially death. Virulence factors associated with V. vulnificus include the capsular polysaccharide (CPS), lipopolysaccharide, flagellum, pili, and outer membrane vesicles (OMVs). The aims of this study were to characterize the morphology of V. vulnificus cells and the formation and arrangement of OMVs using cryo-electron microscopy (cryo-EM). cryo-EM and cryo-electron tomography imaging of V. vulnificus strains grown in liquid cultures revealed the presence of OMVs (diameters of ∼45 nm for wild-type, ∼30 nm for the unencapsulated mutant, and ∼50 nm for the non-motile mutant) in log-phase growth. Production of OMVs in the stationary growth phase was limited and irregular. The spacing of the OMVs around the wild-type cells was in regular, concentric rings. In wild-type cells and a non-motile mutant, the spacing between the cell envelope and the first ring of OMVs was ∼200 nm; this spacing was maintained between subsequent OMV layers. The size, arrangement, and spacing of OMVs in an unencapsulated mutant was irregular and indicated that the polysaccharide chains of the capsule regulate aspects of OMV production and order. Together, our results revealed the distinctive organization of V. vulnificus OMVs that is affected by expression of the CPS.
Science Advances | 2017
Kathleen F. Mittendorf; Justin T. Marinko; Cheri M. Hampton; Zunlong Ke; Arina Hadziselimovic; Jonathan P. Schlebach; Cheryl L. Law; Jun Li; Elizabeth R. Wright; Charles R. Sanders; Melanie D. Ohi
Reconstitution of the PMP22 protein into lipid bilayers results in membrane assemblies that share common features with myelin. Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin.
Microscopy and Microanalysis | 2016
Rebecca S. Dillard; Rachel E. Storms; Leon De Masi; Cheri M. Hampton; Gaël Panis; Patrick H. Viollier; Elizabeth R. Wright
Many bacterial species express external filamentous structures known as pili that are essential to numerous biological processes . Because of their roles in motility, biofilm formation, and surface colonizaion, pili often serve as important virulence factors for pathogenic bacteria [1]. Caulobacter crescentus is a Gram-negative, oligotrophic bacterium that expresses polar type IVb pili in the swarmer stage of its dimorphic life cycle [2]. These pili are known to be involved in surface attachment at the swarmer to sessile transition [3] and are additionally utilized by the prolate siphophage φCbK in the initial stages of infection by attaching to the pilus portals [4]. Although there is no known homologue to the type IVa retraction ATPase in the C. crescentus genome, it has been hypothesized that pilus retraction aids in both of these functions [2,5]. We investigated the role of pilus retraction in φCbK attachment using bacteriophage adsorption assays, cryo-correlative light and electron microscopy (cryoCLEM), and cryo-electron microscopy and tomography (cryo-EM and cryo-ET).
Microscopy and Microanalysis | 2016
Zunlong Ke; Rebecca S. Dillard; Cheri M. Hampton; Rachel E. Storms; Joshua D. Strauss; Elizabeth R. Wright
Human respiratory syncytial virus (hRSV) is an enveloped RNA virus that belongs to Paramyxoviridae family. HRSV is the most common cause of bronchiolitis and pneumonia in young children in the United States, and severe infections of infants and immuno-compromised adults can lead to death. In order to characterize the molecular mechanisms underlying hRSV assembly and viral and host cellular protein dynamics at high spatiotemporal resolution, we use cryo-electron tomography (cryo-ET) and cryo-correlative light and electron microscopy (cryo-CLEM) to examine hRSV-infected cells.