Rebecca S. Dillard

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

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.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation

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.

Obstruction of pilus retraction stimulates bacterial surface sensing

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.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonal packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. The resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

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.

Promotion of virus assembly and organization by the measles virus matrix protein

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.

The Ms6 Mycolyl-Arabinogalactan Esterase LysB is Essential for an Efficient Mycobacteriophage-Induced Lysis

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.

The DREAMS Team: Creating community partnerships through research advocacy training for diverse older adults

ABSTRACT The DREAMS Team research advocacy training program helps clinical faculty and health students introduce basic clinical research concepts to diverse older adults to galvanize their active involvement in the research process. Older adults are frequently underrepresented in clinical research, due to barriers to participation including distrust, historical mistreatment, and their lack of health literacy. The DREAMS Team program aims to involve diverse older adults throughout all phases of research and increase research participation, thereby contributing to the growth of quality patient-centered, evidence-based health care. This course was developed for clinical faculty to deliver to diverse adults aged 55+ in eight 50-minute lectures, followed by half-hour small group discussions moderated by health students. A pilot cohort of 24 individuals was assessed for satisfaction post-program, and self-efficacy before and after the program. Older adult participants improved on a survey measure of self-efficacy, and indicated satisfaction on a post-program questionnaire. All agreed or strongly agreed that they enjoyed participating, and that classes enhanced knowledge/skills about the topics, were high quality, and provided useful information. Twenty-two out of 24 individuals who completed the program indicated they planned to get involved as research advocates. The DREAMS Team program can be offered either on its own, or as a follow-up program to a general health education course led by health students and/or professional researchers or clinicians. Educating older adults about the research process and advocacy through interactive seminars led by congenial and respectful researchers and health students may remove some barriers to research participation and involvement among diverse older adults.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

Background Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. Results Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. Conclusions These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

Analysis of Phage-Pilus Interactions in Caulobacter crescentus

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).