Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid

Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.     

Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines

Bacteriophage T4 capsid is an elongated icosahedron decorated with 155 copies of Hoc, a nonessential highly antigenic outer capsid protein. One Hoc monomer is present in the center of each major capsid protein (gp23*) hexon. We describe an in vitro assembly system which allows display of HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer, on phage T4 capsid surface through Hoc-capsid interactions. In-frame fusions were constructed by splicing the human immunodeficiency virus (HIV) genes to the 5' or 3' end of the Hoc gene. The Hoc fusion proteins were expressed, purified, and displayed on hoc(-) phage particles in a defined in vitro system. Single or multiple antigens were efficiently displayed, leading to saturation of all available capsid binding sites. The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response. The phage T4 system offers new direction and insights for HIV vaccine development with the potential to increase the breadth of both cellular and humoral immune responses.     

Repeat regions R1 and R2 in the P97 paralogue Mhp271 of Mycoplasma hyopneumoniae bind heparin, fibronectin and porcine cilia

Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the centre of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modelling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)‐like and one non‐Ig domain at the C‐terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent‐exposed module containing domains 2 and 3. This model is consistent with the dumbbell‐shaped cryo‐EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C‐terminal 25 amino acids, which are predicted to form two β‐strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent‐exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus and exposing the tail for more efficient contact with the host cell surface prior to infection.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.     

Structure of the three N-terminal immunoglobulin domains of the highly immunogenic outer capsid protein from a T4-like bacteriophage

The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.     

Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a “glue” between neighboring hexameric capsomers, forming a “cage” that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 Å resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.     

Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles

Chitosan-based nanoparticles (NPs) are widely used in drug delivery, device-based therapy, tissue engineering, and medical imaging. In this aspect, a clear understanding of how physicochemical properties of these NPs affect the cytological response is in high demand. The objective of this study is to evaluate the effect of surface charge on cellular uptake profiles (rate and amount) and intracellular trafficking. We fabricate three kinds of NPs (～ 215 nm) with different surface charge via SPG membrane emulsification technique and deposition method. They possess uniform size as well as identical other physicochemical properties, minimizing any differences between the NPs except for surface charge. Moreover, we extend our research to eight cell lines, which could help to obtain a representative conclusion. Results show that the cellular uptake rate and amount are both positively correlated with the surface charge in all cell line. Subsequent intracellular trafficking indicates that some of positively charged NPs could escape from lysosome after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged NPs prefer to colocalize with lysosome. These results are critical in building the knowledge base required to design chitosan-based NPs to be used efficiently and specifically.     

Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold nanoparticles

Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.     

Synthesizing and staining manganese oxide nanoparticles for cytotoxicity and cellular uptake investigation

Background

For decades, contrast agents have been used to reduce longitudinal (T₁) or transverse (T₂) relaxation times. High toxicity of gadolinium-based contrast agents leads researchers to new T₁ contrast agents. Manganese oxide (MnO) nanoparticle (NP) with the lower peril and good enough signal change ability has been offered as a new possibility for magnetic resonance imaging (MRI).

Methods

The synthesized NPs were investigated for physicochemical and biological properties by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscope, dynamic light scattering (DLS), inductively coupled plasma, enzyme-linked immunosorbent assay, and 3 T magnetic resonance imaging.

Results

Due to physical contact importance of T₁ contrast agents with tissues' protons, extremely thin layer of the surfactant, less than 2 nm, was coated on NPs for aqueous stabilizing. The hydrophilic gentisic acid with low Dalton, around 154, did that role truly. Moreover, decreasing NP size to 5 nm which increases available surface for the proton relaxation is another important parameter to reach an appropriate longitudinal relaxation rate. The NPs didn't reveal any side effects on the cells, and cellular uptake was considerable.

Conclusions

The synthesized NPs represented a promising result in comparison to clinical gadolinium chelates, due to higher r₁ relaxivity and lower toxicity.

General significance

In addition to considerable signal change and cellular uptake, Prussian blue was tried on MnO NPs for the initial time, which can be observed within cells by pale blue color.     

Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, n-CeO(2)) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials.     

Prediction of chlortetracycline adsorption on the Fe3O4 nanoparticle using molecular dynamics simulation

Abstract

Molecular dynamics (MD) simulation was applied to investigate the adsorption mechanism of chlortetracycline (CTC) antibiotic molecule as the aqueous pollutant on the Fe₃O₄ nanoparticle (NP). Two different NP sizes with a diameter of about 1.4?nm and 3.5?nm were selected. Initially, the stability of both NPs in water was investigated by calculating radial distribution function curves of NP atoms. Simulation results confirmed the stable crystallographic structures of both NPs. However, small NP induce greater structural stabilization. Then, CTC molecules were adsorbed on NPs surface in various pollutant concentrations. Electrostatic and hydrogen bond were the major types of interactions between CTC molecules and the adsorbent surface. CTC molecules formed a complex with NP surface from their amine side chains; while they were parallel to each other in their aromatic rings and π-π bond between two CTC molecules was formed. Diffusion rate of CTC molecules could predict the adsorption mechanism. At lower concentration of CTC, CTC molecules tend to adsorb on the NP surface. At these concentrations, the diffusion rate of CTC was high. By increasing the CTC concentration, the pollutant agglomeration was enhanced which decreased the diffusion rate. At this time, the surface of NP was saturated. In addition, the results of isotherm curves showed that CTC adsorption on small NPs could be defined with both Langmuir and Freundlich isotherm models, while Freundlich isotherm model was more appropriate for larger NPs. In conclusion, observations confirmed that MD simulation could successfully predict the behavior of CTC adsorption on the Fe₃O₄ NP surface.

Communicated by Ramaswamy H. Sarma     

Virus-inspired design principles of nanoparticle-based bioagents

The highly effectiveness and robustness of receptor-mediated viral invasion of living cells shed lights on the biomimetic design of nanoparticle(NP)-based therapeutics. Through thermodynamic analysis, we elucidate that the mechanisms governing both the endocytic time of a single NP and the cellular uptake can be unified into a general energy-balance framework of NP-membrane adhesion and membrane deformation. Yet the NP-membrane adhesion strength is a globally variable quantity that effectively regulates the NP uptake rate. Our analysis shows that the uptake rate interrelatedly depends on the particle size and ligand density, in contrast to the widely reported size effect. Our model predicts that the optimal radius of NPs for maximal uptake rate falls in the range of 25-30 nm, and optimally several tens of ligands should be coated onto NPs. These findings are supported by both recent experiments and typical viral structures, and serve as fundamental principles for the rational design of NP-based nanomedicine.     

Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.     

Non-toxic nanoparticles from phytochemicals: preparation and biomedical application

Nanoparticles (NPs) have various applications in biomedicine and drug delivery carriers and also are widely used in cosmetics. However, the preparation of biocompatible and non-toxic nanomaterials is a very important issue as most of the starting materials are synthesized using toxic chemical reagents. This review introduces the preparation of biocompatible NPs in a range of their concentrations using phytochemicals for biomedicine and biotechnology. Phytochemicals are natural products that are extracted from plants, vegetables, and fruits. Phytochemicals serve as reducing agents and stabilizers during NP synthesis to convert metal ions to metal NPs in water. Possible applications of such nanomaterials in biomedical sciences are also described in this review.     

Membrane Partitioning of Anionic,Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling,Local Disordering,and Cholesterol Depletion

Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.     

Cellular Uptake and In Vitro Drug Release Studies on Paclitaxel-Loaded Poly(caprolactone)-Grafted Dextran Copolymeric Nanoparticles

Biodegradable and biocompatible polymers play a key role to provide a solution for sustained chemotherapy, when engineered to nanostructure. One such effort has been put forward to engineer self-assembled poly(caprolactone)-grafted dextran (PGD) core–shell micellar vehicle for anticancer drug (paclitaxel) and presented in this study. Paclitaxel-loaded PGD nanoparticles (NPs) were prepared by a modified oil/water emulsion method and characterized by laser light scattering, atomic force microscopy, and zeta potential measurements. The effects of the copolymeric compositions of PGD NPs on drug encapsulation efficiency, in vitro drug release, cellular uptake, and cell viability of NP formulation with paclitaxel were investigated. The drug encapsulation efficiency was determined spectrophotometrically, and in vitro drug release was estimated using dialysis bag. Human gastric cancer cell line (SNU-638) were used to image and measure the cellular uptake of fluorescent PGD NPs. Cancer cell viability of the drug-loaded PGD NPs was measured by crystal violet staining method. From the results obtained on various aspects, we inferred that the above formulated drug-loaded PGD NPs have significant drug encapsulation efficiency, cellular uptake, and the cancer cell mortality.     

Functional Analysis of the Putative Integrin Recognition Motif on Adeno-associated Virus 9

Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system.     

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.