Bacteriophages M13 and T4 Increase the Expression of Anchorage-Dependent Survival Pathway Genes and Down Regulate Androgen Receptor Expression in LNCaP Prostate Cell Line.

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 105 pfu/mL, 1 × 106 pfu/mL, and 1 × 107 pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes AKT, ITGA5, ITGB1, ITGB3, ITGB5, MAPK3, and PI3K were significantly up-regulated, whilst the genes AR, HSPB1, ITGAV, and PGC1A were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with PI3K/AKT pathway inhibitors.

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.

A novel approach to nonsurgical sterilization; application of menadione-modified gonocyte-targeting M13 bacteriophage for germ cell ablation in utero.

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox-cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide 'phage display library proved effective in the isolation of gonocyte-specific targeting clones. One such clone was modified via N-succinimidyl-S-acetylthioacetate (SATA) linkage to the N-terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof-of-concept experiments we applied the modified 'phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species.

Analysis of Microbial Cell Viability in a Liquid Using an Acoustic Sensor.

A method was developed for the rapid analysis and evaluation of the viability of bacteriophage-infected Escherichia coli (E.coli) XL-1 directly in a conducting suspension by using a slot-mode sensor. The method is based on recording the changes in the depth and frequency of resonant absorption peaks in the frequency dependence of the insertion loss of the sensor before and after the biologic interaction of E. coli with specific bacteriophages. The possibility was shown of recording the infection of E. coli with specific bacteriophages and assessing its viability in suspensions with a conductivity of 4.5-30 µS/cm. Сontrol experiments were carried out with non-specific interactions of E. coli cells with bacteriophages, in which no changes in the sensor variables were observed. The optimal informational variable for estimating the number of viable cells was the degree of change in the depth of the resonant peaks in the frequency dependence of the insertion loss of the sensor. The limit of cell detection was ∼102-103 cells mL-1, with an analysis time of about 5 min. An additional advantage of the sensor was the availability of a removable liquid container, which allows one to use it repeatedly and to facilitate the cleaning of the container from spent samples. The results are promising for the detection of bacteria and assessment of their viability in solutions with conductivity in the range 4.5-30 µS/cm.

Visible-light-driven photocatalytic inactivation of bacteriophage f2 by Cu-TiO2 nanofibers in the presence of humic acid.

Pathogenic viruses in drinking water are great threats to public health. Visible-light-driven photocatalysis is a promising technology for virus inactivation. However, the existing photocatalytic antiviral research studies have mostly been carried out in single-component systems, neglecting the effect of natural organic matter, which exists widely in actual water bodies. In this paper, electrospun Cu-TiO2 nanofibers were prepared as photocatalysts, and their photocatalytic antiviral performance in the presence of humic acid (HA) was comprehensively studied for the first time. The properties of the reaction mixture were measured during the reaction. In addition, the safety, reliability and stability of photocatalytic disinfection in the mixed system were evaluated. The results showed that the virus removal efficiency decreased with the increase of the HA concentration. The type of reaction solution, such as PBS buffer solution or water, did not affect the removal efficiency noticeably. Under acidic conditions, the electrostatic forces between photocatalysts and viruses were strengthened, leading to higher virus removal efficiency. As the reaction time went on, the pH value in the solution increased first and then tended to be stable, the conductivity remained stable, and the dissolved oxygen increased first and then decreased. The safety test showed that the concentration of Cu ions released into the solution was lower than specified by the international standards. No photoreactivation was observed, and the addition of HA significantly reduced the reutilization efficiency of the photocatalysts.

High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based Assemblies with a Smectic Liquid Crystalline Orientation.

Polymers are generally considered thermal insulators because the amorphous arrangement of the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies reveal that individual chains of polymers with oriented structures could have high thermal conductivity, because such stretched polymeric chains effectively conduct phonons through polymeric covalent bonds. Previously, we have found that the liquid crystalline assembly composed of one of the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity even though the assembly is based on non-covalent bonds. Despite such potential applicability of biopolymeric assemblies as thermal conductive materials, stability against heating has rarely been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 °C) treatment. The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability against heating processes. Our results provide insight into the future use of biomolecular assemblies for reliable thermal conductive materials.

Phage-based capacitive biosensor for Salmonella detection.

This article reports the detection of Salmonella spp. based on M13 bacteriophage in a capacitive flow injection system. Salmonella-specific M13 bacteriophage was immobilized on a polytyramine/gold surface using glutaraldehyde as a crosslinker. The M13 bacteriophage modified electrode can specifically bind to Salmonella spp. via the amino acid groups on the filamentous phage. An alkaline solution was used to break the binding between the sensing surface and the analyte to allow renewable use up to 40 times. This capacitive system provided good reproducibility with a relative standard deviation (RSD) of 1.1%. A 75 µL min-1 flow rate and a 300 µL sample volume provided a wide linear range, from 2.0 × 102 to 1.0 × 107 cfu mL-1, with a detection limit of 200 cfu mL-1. Bacteria concentration can be analyzed within 40 min after the sample injection. When applied to test real samples (raw chicken meat) it provided good recoveries (100-111%). An enrichment process was also explored to increase the bacteria concentration, enabling a quantitative detection of Salmonella spp. This biosensor opens a new opportunity for the detection of pathogenic bacteria using bacteriophage.

Evolution of mutualism from parasitism in experimental virus populations.

While theory suggests conditions under which mutualism may evolve from parasitism, few studies have observed this transition empirically. Previously, we evolved Escherichia coli and the filamentous bacteriophage M13 in 96-well microplates, an environment in which the ancestral phage increased the growth rate and yield of the ancestral bacteria. In the majority of populations, mutualism was maintained or even enhanced between phages and coevolving bacteria; however, these same phages evolved traits that harmed the ancestral E. coli genotype. Here, we set out to determine if mutualism could evolve from this new parasitic interaction. To do so, we chose six evolved phage populations from the original experiment and used them to establish new infections of the ancestral bacteria. After 20 passages, mutualism evolved in almost all replicates, with the remainder growing commensally. Many phage populations also evolved to benefit both their local, evolving bacteria and the ancestral bacteria, though these phages were less beneficial to their co-occurring hosts than phages that harm the ancestral bacteria. These results demonstrate the rapid recovery of mutualism from parasitism, and we discuss how our findings relate to the evolution of phages that enhance the virulence of bacterial pathogens.

Conformations and membrane-driven self-organization of rodlike fd virus particles on freestanding lipid membranes.

Membrane-mediated interactions and aggregation of colloidal particles adsorbed to responsive elastic membranes are challenging problems relevant for understanding the microscopic organization and dynamics of biological membranes. We experimentally study the behavior of rodlike semiflexible fd virus particles electrostatically adsorbed to freestanding cationic lipid membranes and find that their behavior can be controlled by tuning the membrane charge and ionic strength of the surrounding medium. Three distinct interaction regimes of rodlike virus particles with responsive elastic membranes can be observed. (i) A weakly charged freestanding cationic lipid bilayer in a low ionic strength medium represents a gentle quasi-2D substrate preserving the integrity, structure, and mechanical properties of the membrane-bound semiflexible fd virus, which under these conditions is characterized by a monomer length of 884 ± 4 nm and a persistence length of 2.5 ± 0.2 µm, in perfect agreement with its properties in bulk media. (ii) An increase in the membrane charge leads to the membrane-driven collapse of fd virus particles on freestanding lipid bilayers and lipid nanotubes into compact globules. (iii) When the membrane charge is low, and the mutual electrostatic repulsion of membrane-bound virus particles is screened to a considerable degree, membrane-driven self-organization of membrane-bound fd virus particles into long linear tip-to-tip aggregates showing dynamic self-assembly/disassembly and quasi-semiflexible behavior takes place. These observations are in perfect agreement with the results of recent theoretical and simulation studies predicting that membrane-mediated interactions can control the behavior of colloidal particles adsorbed on responsive elastic membranes.

The Transmembrane Morphogenesis Protein gp1 of Filamentous Phages Contains Walker A and Walker B Motifs Essential for Phage Assembly.

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.

Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production.

The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h-1 and a multiplicity of infection of 0.05 pfu cfu-1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L-1 . Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc.

Simulation of the M13 life cycle II: Investigation of the control mechanisms of M13 infection and establishment of the carrier state.

Bacteriophage M13 is a true parasite of bacteria, able to co-opt the infected cell and control the production of progeny across many cellular generations. Here, our genetically-structured simulation of M13 is applied to quantitatively dissect the interplay between the host cellular environment and the controlling interactions governing the phage life cycle during the initial establishment of infection and across multiple cell generations. Multiple simulations suggest that phage-encoded feedback interactions constrain the utilization of host DNA polymerase, RNA polymerase and ribosomes. The simulation reveals the importance of p5 translational attenuation in controlling the production of phage double-stranded DNA and suggests an underappreciated role for p5 translational self-attenuation in resource allocation. The control elements active in a single generation are sufficient to reproduce the experimentally-observed multigenerational curing of the phage infection. Understanding the subtleties of regulation will be important for maximally exploiting M13 particles as scaffolds for nanoscale devices.

Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation.

To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle.

Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans.

Controlled self-assembly of biomolecular rods on structured substrates.

We report on the evaporative self-assembly and orientational ordering of semi-flexible spherocylindrical M13 phages on asymmetric stranded webs of thin amorphous carbon films. Although the phages were dispersed with a low concentration in the isotropic phase, the substrate edges induced nematic ordering and bending of the phages. As revealed by transmission electron microscopy, phages were aligned parallel to the curved substrate edges. This two-dimensional self-assembly on structured substrates opens a new route to the design of structures of orientationally ordered semi-flexible biomacromolecules.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.

[Determination of a Spectrum of Lytic Activity of Bacteriophages by the Method of Acoustic Analysis].

The changes in the electro-acoustic parameters of cell suspension due to the interaction of cells with bacteriophages both in a pure. culture and in the presence of extraneous microflora were investigated. It has been found that the specific changes in the electroacoustic parameters of cell suspension under the action of bacteriophage occur only in microbial cells which are sensitive to the bacteriophage studied. It has been established that a sensor unit allows of distinguishing a situation when the bacterial cells are infected with specific bacteriophages of the control experiments and a situation with no introduction of infection. An approximate criterion of the presence of specific interactions of bacteriophages and cells in suspension was developed. In accordance with this criterion the change in electrical impedance of the sensor unit must not be less than - 1%. In control experiments a standard microbiological technique, plating the cells infected with bacteriophages on solid nutrient medium, was used. For the first time the possibility of using the method of electroacoustic analysis for determination of a spectrum of lytic activity of bacteriophages was shown. The results obtained may be used for development of a new express method for determining the sensitivity to bacteriophages of the microbial cells.

Virus based Full Colour Pixels using a Microheater.

Mimicking natural structures has been received considerable attentions, and there have been a few practical advances. Tremendous efforts based on a self-assembly technique have been contributed to the development of the novel photonic structures which are mimicking nature's inventions. We emulate the photonic structures from an origin of colour generation of mammalian skins and avian skin/feathers using M13 phage. The structures can be generated a full range of RGB colours that can be sensitively switched by temperature and substrate materials. Consequently, we developed an M13 phage-based temperature-dependent actively controllable colour pixels platform on a microheater chip. Given the simplicity of the fabrication process, the low voltage requirements and cycling stability, the virus colour pixels enable us to substitute for conventional colour pixels for the development of various implantable, wearable and flexible devices in future.

Discovery of catalytic phages by biocatalytic self-assembly.

Discovery of new catalysts for demanding aqueous reactions is challenging. Here, we describe methodology for selection of catalytic phages by taking advantage of localized assembly of the product of the catalytic reaction that is screened for. A phage display library covering 10(9) unique dodecapeptide sequences is incubated with nonassembling precursors. Phages which are able to catalyze formation of the self-assembling reaction product (via amide condensation) acquire an aggregate of reaction product, enabling separation by centrifugation. The thus selected phages can be amplified by infection of Escherichia coli. These phages are shown to catalyze amide condensation and hydrolysis. Kinetic analysis shows a minor role for substrate binding. The approach enables discovery and mass-production of biocatalytic phages.

Synthetic phage for tissue regeneration.

Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.