Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0 × 107  pfu mL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

Filamentous bacteriophages, natural nanoparticles, for viral vaccine strategies.

Filamentous bacteriophages are natural nanoparticles formed by the self-assembly of structural proteins that have the capability of replication and infection. They are used as a highly efficient vaccine platform to enhance immunogenicity and effectively stimulate the innate and adaptive immune response. Compared with traditional vaccines, phage-based vaccines offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. This review summarizes recent research on phage-based vaccines in virus prevention. In addition, the expression systems of filamentous phage-based virus vaccines and their application principles are discussed. Moreover, the prospect of the prevention of emerging infectious diseases, such as coronavirus 2019 (COVID-19), is also discussed.

A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

Filamentous Bacteriophage-A Powerful Carrier for Glioma Therapy.

Glioma is a life-threatening malignant tumor. Resistance to traditional treatments and tumor recurrence present major challenges in treating and managing this disease, consequently, new therapeutic strategies must be developed. Crossing the blood-brain barrier (BBB) is another challenge for most drug vectors and therapy medications. Filamentous bacteriophage can enter the brain across the BBB. Compared to traditional drug vectors, phage-based drugs offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. Tumor-targeting peptides from phage library and phages displaying targeting peptides are ideal drug delivery agents. This review summarized recent studies on phage-based glioma therapy and shed light on the developing therapeutics phage in the personalized treatment of glioma.

Association of host proteins with the broad host range filamentous phage NgoΦ6 of Neisseria gonorrhoeae.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.

NMR "Crystallography" for Uniformly (13 C, 15 N)-Labeled Oriented Membrane Proteins.

In oriented-sample (OS) solid-state NMR of membrane proteins, the angular-dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only 1 H-15 N dipolar couplings and 15 N chemical shifts have been routinely assessed in oriented 15 N-labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (15 N, 13 C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1 Hα -13 Cα dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α-helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α-helical transmembrane structure for Pf1 protein.

Multiple Mechanisms Are Involved in Repression of Filamentous Phage SW1 Transcription by the DNA-Binding Protein FpsR.

SW1 is the first filamentous phage isolated from a deep-sea environment. Nevertheless, the mechanism by which the SW1 genetic switch is controlled is largely unknown. In this study, the function of the phage-encoded FpsR protein was characterized by molecular biological and biochemical analyses. The deletion of fpsR increased the copy number of SW1 ssDNA and mRNA, indicating that FpsR functions as a repressor. In addition, transcription from the fpsR promoter was shown to be increased in an fpsR deletion mutant, suggesting self-repression by FpsR. Purified FpsR bound to four adjacent operator sites (O1-O4) embedded within the fpsA promoter and the fpsA-fpsR intergenic region. A surface plasmon resonance experiment showed that FpsR can bind to the O1-O4 operators separately and with different binding affinity, and the dissociation constants of FpsR with O2 and O3 were found to be lower at 4 °C than at 20 °C. A gel permeation chromatography assay revealed that FpsR oligomerized to form tetramers. Point mutation analysis indicated that the C-terminal domain influenced the binding affinity and regulatory function of FpsR. Collectively, these data support a model in which FpsR actively regulates phage production by interacting with the corresponding operators, thus playing a crucial role in the SW1 genetic switch.

Filamentous Bacteriophage Viruses: Preparation, Magic-Angle Spinning Solid-State NMR Experiments, and Structure Determination.

Filamentous bacteriophages are elongated semi-flexible viruses that infect bacteria. They consist of a circular single-stranded DNA (ssDNA) wrapped by a capsid consisting of thousands of copies of a major coat protein subunit. Given the increasing number of discovered phages and the existence of only a handful of structures, the development of methods for phage structure determination is valuable for biophysics and structural virology. In recent years, we developed and applied techniques to elucidate the 3D atomic-resolution structures of intact bacteriophages using experimental magic-angle spinning (MAS) solid-state NMR data. The flexibility in sample preparation - precipitated homogeneous solids - and the fact that ssNMR presents no limitation on the size, weight or morphology of the system under study makes it an ideal approach to study phage systems in detail.In this contribution, we describe approaches to prepare isotopically carbon-13 and nitrogen-15 enriched intact phage samples in high yield and purity, and we present experimental MAS NMR methods to study the capsid secondary and tertiary structure, and the DNA-capsid interface. Protocols for the capsid structure determination using the Rosetta modeling software are provided. Specific examples are given from studies of the M13 and fd filamentous bacteriophage viruses.

Silica formation with nanofiber morphology via helical display of the silaffin R5 peptide on a filamentous bacteriophage.

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.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development.

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.

Engineering filamentous bacteriophages for enhanced gold binding and metallization properties.

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.

Architectural insight into inovirus-associated vectors (IAVs) and development of IAV-based vaccines inducing humoral and cellular responses: implications in HIV-1 vaccines.

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.

Structure and assembly of filamentous bacteriophages.

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.

Construction of a filamentous phage display peptide library.

The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.

Affinity selection using filamentous phage display.

Display of peptides on filamentous phage, phage display, is an in vitro selection technique well suited for identification of therapeutic peptide binders for a huge variety of protein targets. The peptides are identified in a process where phage libraries are subjected to affinity selection towards a particular protein target. A successful outcome of an affinity selection is dependent on proper surveillance of the phage life cycle, to make sure that the selection is based on affinity for the target, not on bias in phage propagation rate. In this chapter we present two approaches for protein target presentation and a protocol for phage rescue and propagation, which includes several controls to ensure that all phages initially eluted from the protein target are given equal conditions during the following amplification and selection steps.

Precipitation of filamentous bacteriophages for their selective recovery in primary purification.

Filamentous bacteriophages and their derivatives are showing great promise as a whole new class of industrial agents, such as biologically based nano-materials and viral vectors. This raises challenges for their large-scale manufacture, principally due to the lack of bioprocessing knowledge. This article addresses what will be a potentially important option in the primary purification of the bacteriophages. Polyethylene glycol (PEG)-salt dual precipitants, calcium ions, spermidine, and isoelectric precipitation were first examined for their potential suitability for bacteriophage concentration under both pure and broth conditions. Successful precipitants were further studied on the basis of their selective purification ability from DNA and protein contaminants in a clarified broth system. Both PEG-based and isoelectric precipitations resulted in bacteriophage purity improvements, and PEG-based precipitations offered the highest selectivities. This work shows that precipitation of bacteriophages can be an effective primary purification step in a large-scale bioprocess.

Dynamic disulfide scanning of the membrane-inserting Pf3 coat protein reveals multiple YidC substrate contacts.

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of ATP synthase and respiratory complexes is catalyzed by YidC. To investigate how YidC interacts with membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate Pf3 coat protein. The single cysteine mutants were expressed and analyzed for disulfide formation during 30 s of synthesis. The results show that the substrate contacts different YidC residues in four of the six transmembrane regions. The residues are located either in the region of the inner leaflet, in the center, as well as in the periplasmic leaflet, consistent with the hypothesis that YidC presents a hydrophobic platform for inserting membrane proteins. In a YidC mutant where most of the contacting residues were mutated to serines, YidC function was severely disturbed and no longer active in a complementation test, suggesting that the residues are important for function. In addition, a Pf3 mutant with a defect in membrane insertion was deficient to contact the periplasmic residues of YidC.

Similarities and differences within members of the Ff family of filamentous bacteriophage viruses.

The filamentous bacteriophage viruses of the Ff family, fd and M13, slightly differ in their genome, and their 50-residue-long major capsid proteins have a single site difference: the uncharged asparagine-12 in M13 is replaced with a negatively charged aspartate in fd. We have used magic-angle spinning solid-state NMR spectroscopy to site-specifically assign the resonances belonging to the capsid protein of M13. Assignment of several mobile residues was facilitated by using J-based spectroscopy, which in addition provided sugar-base contacts in the M13-DNA stemming from two-bond scalar couplings. A comparison between M13 and fd bacteriophages reveals that the two virions have a very conserved and stable structure, manifested in negligibly small chemical shift differences and similar dynamic properties for nearly all resonances. The principal difference between the two phages involves residues in the vicinity of residue 12. We suggest that the elimination of the single charge at position 12 throughout the entire assembly affects the electrostatic and hydrogen-bonding interaction network governing inter- and intraresidue contacts, mainly by the rearrangement of the positively charged lysine residue at position 8.