RÉSUMÉ
A new modality of targeting therapeutic drugs based on the use of bacteriophage (virus), as an emerging tool for specific targeting and for vaccine development, has been an area of interest for genetic and cancer research. The approach is based on genetic manipulation and modification in the chemical structure of a filamentous bacteriophage that facilitates its application not only for in vivo imaging but also for therapeutic purpose, as a gene delivery vehicle, as drug carriers, and also as an immunomodulatory agent. Filamentous bacteriophage on account of its high surface holding ability with adaptable genetic engineering properties can effectively be used in loading of chemical and genetic drugs specifically on to the targeted lesion location. Moreover, the specific peptides/proteins exhibited on the phage surface can be applied directly as self-navigating drug delivery nanovehicles. The present review article has been framed with an objective to summarize the importance of bacteriophage in phage cancer therapy and to understand the possible future prospective of this approach in developing new tools for biotechnological and genetic research, especially in phage -mediated cancer therapy. Importantly, the peptides or proteins emerging from the surface of a nano carrier will make the expense of such peptides economically more effective as compared to other immunological tools, and this seems to be a potential approach for developing a new nanodrug carrier platform.
Sujet(s)
Antinéoplasiques/administration et posologie , Vecteurs de médicaments/composition chimique , Vecteurs génétiques/génétique , Inovirus/génétique , Tumeurs/thérapie , Animaux , Génie génétique , Thérapie génétique/méthodes , Vecteurs génétiques/composition chimique , Vecteurs génétiques/immunologie , Humains , Inovirus/composition chimique , Inovirus/immunologie , Nanoparticules , Tumeurs/génétique , Tumeurs/immunologieRÉSUMÉ
Filamentous bacteriophages, also known as filamentous bacterial viruses or Inoviruses, have been studied extensively over the years. They are interesting paradigms in structural molecular biology and offer insight into molecular assembly, a process that remains to be fully understood. In this chapter, an overview on filamentous bacteriophages will be provided. In particular, we review the constituent proteins of filamentous bacteriophage and discuss assembly by examining the structure of the major coat protein at various stages of the process. The minor coat proteins will also be briefly reviewed. Structural information provides key snapshots into the dynamic process of assembly.
Sujet(s)
Protéines de capside , Inovirus , Assemblage viral/physiologie , Protéines de capside/composition chimique , Protéines de capside/génétique , Protéines de capside/métabolisme , Inovirus/composition chimique , Inovirus/physiologieRÉSUMÉ
BACKGROUND: Filamentous M13 phages have recently been utilized as components for developing novel functional soft materials in various fields such as sensor, device, and biomedical applications. Recently, we have developed liquid crystalline hydrogels composed of M13 phages and gold nanoparticles (GNPs) based on specific interactions between the components. OBJECTIVES: The main objective of this study was to clarify the self-healing capability of the hydrogels composed of M13 phages and GNPs. METHODS: M13 phages displaying tag peptides with a sequence of YPYDVPDYA (HA phages) were genetically constructed through general molecular biology. The mechanical strength of hydrogels composed of the HA phages and anti-HA peptide antibodies-immobilized GNPs (HA-GNPs) was measured by indentation tests. The rupture point of the hydrogels was visually observed. An aliquot of buffer solution was added into the rupture point of the hydrogels after the indentation test. After incubation for 2 days, self-healing of the rupture point was checked visually. The indentation test was also performed after self-healing. To clarify the assembled structures of the components in the hydrogels, transmission electron microscopy (TEM) observation was performed by transferring the hydrogel onto a TEM grid before and after healing. RESULTS: The strength of the original hydrogel (before self-healing) required for rupture was approximately 55 mN. Self-healing of the rupture point was confirmed visually, and the hydrogels behaved as uniform hydrogels again during the vial inversion tests. As a result of the indentation test for the self-healed points of the hydrogels, the rupture force of approximately 45 mN was detected, indicating the self-healing capability of the hydrogels. TEM observation of the before and after self-healing exhibited the regularly assembled structures composed of the HA-GNPs, suggesting that the ruptured networks were recovered into regularly assembled network structures. Importantly, control of the concentration of the HA-GNPs resulted in suppression of decreasing the rupture forces during the repetitive self-healing processes. CONCLUSION: Our results demonstrated the self-healing capability of structurally regular hybrid hydrogels composed of genetically engineered filamentous viruses displaying antigen peptides and antibody-immobilized GNPs. The results indicated that supramolecular hydrogels containing filamentous viruses would expand the applicability of virus-based soft materials.
Sujet(s)
Bactériophage M13/composition chimique , Or/composition chimique , Hydrogels/composition chimique , Maladies à complexes immuns/traitement médicamenteux , Inovirus/composition chimique , Nanoparticules métalliques/composition chimique , Oligopeptides/composition chimique , Séquence d'acides aminés , Complexe antigène-anticorps , Bactériophage M13/génétique , Escherichia coli , Inovirus/génétique , Microscopie électronique à transmission/méthodes , Oligopeptides/génétique , Banque de peptidesRÉSUMÉ
Biological systems often generate unique and useful structures, which can have industrial relevance either as direct components or as an inspiration for biomimetic materials. For fabrication of nanoscale silica structures, we explored the use of the silaffin R5 peptide from Cylindrotheca fusiformis expressed on the surface of the fd bacteriophage. By utilizing the biomineralizing peptide component displayed on the bacteriophage surface, we found that low concentrations (0.09 mg/mL of the R5 bacteriophage, below the concentration range used in other studies) could be used to create silica nanofibers. An additional benefit of this approach is the ability of our R5-displaying phage to form silica materials without the need for supplementary components, such as aminopropyl triethoxysilane, that are typically used in such processes. Because this method for silica formation can occur under mild conditions when implementing our R5 displaying phage system, we may provide a relatively simple, economical, and environmentally friendly process for creating silica nanomaterials.
Sujet(s)
Inovirus/composition chimique , Nanofibres/composition chimique , Fragments peptidiques/composition chimique , Précurseurs de protéines/composition chimique , Silice/composition chimique , Inovirus/métabolismeRÉSUMÉ
BACKGROUND: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. OBJECTIVE: In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. RESULTS: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. CONCLUSION: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents.
Sujet(s)
Immunocontraception , Inovirus/métabolisme , Vaccins contraceptifs/immunologie , Animaux , Vecteurs génétiques/génétique , Vecteurs génétiques/métabolisme , Inovirus/composition chimique , Inovirus/immunologie , Banque de peptides , Vaccins sous-unitaires/immunologie , Vaccins à pseudo-particules virales/immunologieRÉSUMÉ
In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of Escherichia coli, the protein gp4 forms a pore-like structure, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggests that gp1 might be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.
Sujet(s)
Bactériophage M13/génétique , Bactériophage M13/physiologie , Inovirus/génétique , Inovirus/physiologie , Protéines virales/génétique , Protéines virales/métabolisme , Assemblage viral , Motifs d'acides aminés , Bactériophage M13/composition chimique , Séquence conservée , Analyse de mutations d'ADN , Escherichia coli/métabolisme , Escherichia coli/virologie , Inovirus/composition chimiqueRÉSUMÉ
The preparation of thiamethoxam (TMX) organic crystals with high morphological uniformity was achieved by controlled aggregation-driven crystallization of primitive TMX crystals and phage using the filamentous M13 bacteriophage. The development of a regular, micrometer-sized, tetragonal-bipyramidal crystal structure was dependent on the amount of phage present. The phage appears to affect the supersaturation driving force for crystallization. The phage adsorption isotherm to TMX was well-fitted by the Satake-Yang model, which suggests a cooperative binding between neighboring phages as well as a binding of phage with the TMX crystal surface. This study shows the potential of phage additives to control the morphology and morphological uniformity of organic crystals.
Sujet(s)
Bactériophage M13/composition chimique , Bactériophage M13/ultrastructure , Cristallisation/méthodes , Nanoparticules/composition chimique , Nanoparticules/ultrastructure , Composés nitrés/composition chimique , Oxazines/composition chimique , Thiazoles/composition chimique , Inovirus/composition chimique , Inovirus/ultrastructure , Test de matériaux , Conformation moléculaire , Néonicotinoïdes , Propriétés de surface , ThiaméthoxameRÉSUMÉ
Filamentous bacteriophages are nanowire-like virion molecules consisting of a single stranded DNA (ssDNA) as the genomic material packed in a protein cage. In this study, Tyr containing 5-mer peptides were displayed on phage filaments for enhanced Au binding and reduction properties. Wild type fd (AEGDD) and engineered YYYYY, AYSSG and AYGDD phages were investigated by Quartz crystal microbalance (QCM), Atomic force microscopy (AFM), Scanning electron microscopy (SEM) and Energy dispersive X-ray spectroscopy (EDX) analyses. Presence of only one Tyr unit on five aa flexible region of p8 coat proteins increased Au binding affinities of engineered phages. YYYYY phages were shown to have the strongest Au surface and AuNP binding affinities. Recombinant phages were shown to be coated with Au clusters after one-step metallization reaction. With further genetic modifications, phages can be programmed to function as site specific self-assembling biotemplates for bottom-up manufacturing in nanoelectronics and biosensor application studies.
Sujet(s)
Techniques de biocapteur/méthodes , Régulation de l'expression des gènes viraux , Or/composition chimique , Inovirus/composition chimique , Oligopeptides/composition chimique , Tyrosine/composition chimique , Protéines de capside/biosynthèse , Protéines de capside/composition chimique , Protéines de capside/génétique , ADN simple brin/génétique , ADN simple brin/métabolisme , ADN viral/génétique , ADN viral/métabolisme , Génie génétique/méthodes , Inovirus/génétique , Inovirus/métabolisme , Nanotechnologie/méthodes , Oligopeptides/biosynthèse , Oligopeptides/génétique , Oxydoréduction , Banque de peptides , Propriétés de surface , Tyrosine/métabolismeRÉSUMÉ
Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1.
Sujet(s)
Vaccins contre le SIDA/immunologie , Vecteurs génétiques/composition chimique , Infections à VIH/immunologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , Inovirus/composition chimique , Vaccins contre le SIDA/génétique , Animaux , Vecteurs génétiques/génétique , Vecteurs génétiques/métabolisme , Infections à VIH/prévention et contrôle , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Humains , Immunité cellulaire , Immunité humorale , Inovirus/génétique , Inovirus/métabolismeRÉSUMÉ
A filamentous bacteriophage (Ï), ÏRS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ÏRS603 was found to have a circular single-stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ÏRS603 genome showed strong similarity with those of Ralstonia phages ÏRSM1 and ÏRSM3, as reported by Askora et al. The ÏRS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ÏRSM3. ÏRS603 had an ORF that was homologous to other Ralstonia phages ÏRSS0 and ÏRSS1; however, ÏRSM1 and ÏRSM3 did not.
Sujet(s)
ADN simple brin/génétique , ADN viral/génétique , Génome viral , Inovirus/génétique , Ralstonia solanacearum/virologie , Protéines virales/génétique , Séquence d'acides aminés , ADN simple brin/composition chimique , ADN viral/composition chimique , Inovirus/composition chimique , Inovirus/isolement et purification , Données de séquences moléculaires , Cadres ouverts de lecture , Alignement de séquences , Similitude de séquences d'acides aminés , Protéines virales/composition chimiqueRÉSUMÉ
Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium. The bacteriophage is a semi-flexible worm-like nucleoprotein filament. The virion comprises a tube of several thousand identical major coat protein subunits around a core of single-stranded circular DNA. Each protein subunit is a polymer of about 50 amino-acid residues, largely arranged in an α-helix. The subunits assemble into a helical sheath, with each subunit oriented at a small angle to the virion axis and interdigitated with neighbouring subunits. A few copies of "minor" phage proteins necessary for infection and/or extrusion of the virion are located at each end of the completed virion. Here we review both the structure of the virion and aspects of its function, such as the way the virion enters the host, multiplies, and exits to prey on further hosts. In particular we focus on our understanding of the way the components of the virion come together during assembly at the membrane. We try to follow a basic rule of empirical science, that one should chose the simplest theoretical explanation for experiments, but be prepared to modify or even abandon this explanation as new experiments add more detail.
Sujet(s)
Inovirus/composition chimique , Inovirus/métabolisme , Animaux , Membrane cellulaire/virologie , ADN viral/biosynthèse , ADN viral/génétique , ADN viral/métabolisme , Humains , Inovirus/génétique , Inovirus/physiologie , Modèles moléculaires , Protéines virales/composition chimique , Protéines virales/métabolisme , Virion/composition chimique , Virion/métabolismeRÉSUMÉ
The fd bacteriophage is a filamentous virus consisting of a circular single-stranded DNA (ssDNA) wrapped by thousands of copies of a major coat protein subunit (the capsid). The coat protein subunits are mostly α-helical and curved, and are arranged in the capsid in consecutive pentamers related by a translation along the main viral axis and a rotation of ~36° (C5S2 symmetry). The DNA is right-handed and helical, but information on its structure and on its interface with the capsid is incomplete. We present here an approach for assigning the DNA nucleotides and studying its interactions with the capsid by magic-angle spinning solid-state NMR. Capsid contacts with the ssDNA are obtained using a two-dimensional (13)C-(13)C correlation experiment and a proton-mediated (31)P-(13)C polarization transfer experiment, both acquired on an aromatic-unlabeled phage sample. Our results allow us to map the residues that face the interior of the capsid and to show that the ssDNA-capsid interactions are sustained mainly by electrostatic interactions between the positively charged lysine side chains and the phosphate backbone. The use of natural abundance aromatic amino acids in the growth media facilitated the complete assignment of the four nucleotides and the observation of internucleotide contacts. Using chemical shift analysis, our study shows that structural features of the deoxyribose carbons reporting on the sugar pucker are strikingly similar to those observed recently for the Pf1 phage. However, the ssDNA-protein interface is different, and chemical shift markers of base pairing are different. This experimental approach can be utilized in other filamentous and icosahedral bacteriophages, and also in other biomolecular complexes involving structurally and functionally important DNA-protein interactions.
Sujet(s)
Bactériophage M13/composition chimique , Bactériophage M13/métabolisme , Capside/composition chimique , ADN simple brin/composition chimique , Inovirus/composition chimique , Séquence nucléotidique , Capside/métabolisme , Spectroscopie par résonance magnétique , Conformation moléculaire , Données de séquences moléculairesRÉSUMÉ
While elastin-like polypeptides and peptides (ELPs) have been used for various stimulus-responsive applications, details of their switching remain unclear. We therefore constructed a novel series of filamentous phage particles displaying a high surface density of short ELPs. The surface display of ELPs did not disrupt either particle shape or dimensions, and the resulting ELP-phage particles were colloidally stable over several weeks. However, in spite of a saturating surface density, macroscopic aggregation of ELP-phages cannot be triggered in water. To investigate the underlying mechanisms we examined conformational changes in the secondary structure of the phage proteins by circular dichroism and tryptophan fluorescence, which indicate partial protein unfolding in ELP-phage particles. To gain further insight into the ELP itself, analogous "free" ELP peptides were synthesized and characterized. Circular dichroism of these peptides shows the onset of ß-type conformations with increasing temperature, consistent with the accepted view of the microscopic transition that underlies the inverse phase behavior of ELPs. Increased guest residue hydrophobicity was found to depress the microscopic transition temperature of the peptides, also consistent with a previously proposed intramolecular hydrogen-bonding mechanism. Importantly, our results indicate that although the nanoscale presentation state can suppress macroscopic aggregation of ELPs, microscopic transitions of the ELP can still occur. Given the growing use of ELPs within supra-molecular scaffolds, such effects are important design considerations for future applications.
Sujet(s)
Techniques d'exposition à la surface cellulaire , Élastine/métabolisme , Inovirus/composition chimique , Peptides/métabolisme , Dichroïsme circulaire , Élastine/composition chimique , Élastine/génétique , Fluorescence , Interactions hydrophobes et hydrophiles , Inovirus/génétique , Peptides/composition chimique , Peptides/génétique , Pliage des protéines , Structure secondaire des protéines , Protéines virales/composition chimiqueRÉSUMÉ
Very large quantities of tailings are produced as a result of processing oil sands. After the sand particles settle out, a dense stable mixture of clay, silt, water with residual bitumen, salts, and organics called mature fine tailings (MFT) can remain in suspension for decades. Research into developing methods that would allow consolidation and sedimentation of the suspended particles is ongoing. We have studied the ability of a filamentous bacteriophage (called VP12 bearing the peptide DSQKTNPS at the N-terminus of the major coat protein pVIII) to aggregate MFT. To understand the biophysical basis of the aggregation, phage-induced aggregation of diluted MFT was measured at room temperature under varying conditions of pH, salt, detergent. Phage at concentrations of 5.0 × 10(11)/mL to 10(12)/mL induced rapid settling of the diluted MFT. The addition of sodium chloride (10 mM) lowered the concentration of phage required to induce aggregation. Since the non-ionic detergents Triton-X 100 and Tween-20, and the ionic detergent sodium deoxycholate had little effect, hydrophobic interactions do not appear to be a major contributor to the phage-induced aggregation of MFT. However, aggregation was prevented at pH values higher than 9.0 suggesting that positively charged amino acid residues are required for MFT aggregation by phage. Genetic engineering of the pVIII peptide sequence indicated that hydrogen bonding also contributes to phage-induced aggregation. In addition, replacing the basic residue lysine with an alanine in the recombinant peptide of VP12 completely prevented phage-induced aggregation. Three other phage displaying different amino acid sequences but all containing a lysine in the same position had variable aggregation efficiencies, ranging from no aggregation to rapid aggregation. We conclude that not only are the functional groups of the amino acids important, but the conformation that is adopted by the variable pVIII peptide is also important for phage-induced MFT aggregation.
Sujet(s)
Floculation , Déchets industriels , Inovirus/composition chimique , Protéines virales/composition chimique , Liaison hydrogène , Concentration en ions d'hydrogène , Inovirus/génétique , Électricité statique , Protéines virales/génétiqueRÉSUMÉ
We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.
Sujet(s)
Marqueurs biologiques tumoraux/analyse , Tumeurs du sein/métabolisme , Protéines de capside/pharmacocinétique , Produits de contraste/synthèse chimique , Inovirus/composition chimique , Microscopie de fluorescence multiphotonique/méthodes , Tumeurs du sein/anatomopathologie , Lignée cellulaire tumorale , Humains , Imagerie moléculaire/méthodes , Coloration et marquage/méthodesRÉSUMÉ
BACKGROUND: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS(6) or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. CONCLUSIONS/SIGNIFICANCE: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations.
Sujet(s)
Protéines de capside/métabolisme , Banque de peptides , Peptides/métabolisme , Protéines de fusion recombinantes/métabolisme , Séquence d'acides aminés , Bactériophage M13/génétique , Bactériophage M13/métabolisme , Séquence nucléotidique , Protéines de capside/analyse , Protéines de capside/composition chimique , Clonage moléculaire/méthodes , Rendement , Inovirus/composition chimique , Inovirus/génétique , Inovirus/métabolisme , Données de séquences moléculaires , Peptides/analyse , Peptides/composition chimique , Signaux de triage des protéines/génétique , Protéines de fusion recombinantes/analyse , Protéines de fusion recombinantes/composition chimiqueRÉSUMÉ
Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.
Sujet(s)
Oxyde ferrosoferrique/composition chimique , /composition chimique , Magnétisme/méthodes , Techniques de culture de tissus/méthodes , Astrocytes , Lignée cellulaire tumorale , Glioblastome , Or/composition chimique , Humains , Inovirus/composition chimique , Microscopie de fluorescence , Protéines/métabolismeRÉSUMÉ
The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA [Liu, D. J., and Day, L. A. (1994) Science 265, 671-674] to develop a novel molecular model for the Pf1 assembly.
Sujet(s)
ADN simple brin/composition chimique , ADN simple brin/génétique , ADN viral/composition chimique , ADN viral/génétique , Génome viral/génétique , Inovirus/composition chimique , Inovirus/génétique , Modèles moléculaires , Analyse spectrale Raman , Virion/composition chimique , Virion/génétiqueRÉSUMÉ
During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.
Sujet(s)
Inovirus/composition chimique , Inovirus/physiologie , Nanotechnologie , Séquence d'acides aminés , Biotechnologie , Membrane cellulaire/métabolisme , Inovirus/métabolisme , Données de séquences moléculaires , Coloration et marquage , Protéines virales/composition chimique , Protéines virales/métabolismeRÉSUMÉ
In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Young's modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to study the response of E. coli under load before and after M13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Young's modulus of E. coli, are negligible after M13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.