Single Phage Particle Research Articles

Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining.

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Enumeration of Bacteriophages by Plaque Assay.

Enumeration of bacteriophages by plaque assay requires the mixing of host-specific bacteria with a lytic bacteriophage of interest in a soft agar overlay (top agar) to prevent the spread of viral infection in the medium; the mixture is then spread on a solid bottom agar. An infection of a single lytic phage particle with a bacterium results in the lysis of the host bacterium and the release of new phage progeny. The new phage progeny released from each bacterium will infect/lyse neighboring bacteria to form a "plaque," which is a clear visible area (that can be counted) with the naked eye. If phages are not present in the mixture, the host bacterium will form a "lawn" in the soft agar overlay and grow to a stationary phase.

Targeted Single-Phage Isolation Reveals Phage-Dependent Heterogeneous Infection Dynamics.

Due to rising antibiotic resistance, there is an urgent need for different treatment options for multidrug-resistant infections. One alternative under investigation is phage therapy, which uses phages to treat bacterial infections. Although phages are highly abundant in the environment, not all phages are suitable for phage therapy, and finding efficient phages that lack undesirable traits such as bacterial virulence factors is challenging. Here, we developed a targeted single-phage isolation method to detect and isolate phages of interest and to characterize their kinetics in a high-throughput manner. This assay has also revealed cell-to-cell variations at a single-cell level among cells infected with the same phage species, as well as among cells infected with different phage species. IMPORTANCE The spread of multidrug-resistant bacteria is a global human health threat, and without immediate action we are fast approaching a postantibiotic era. One possible alternative to antibiotics is the use of phages, that is, bacterial viruses. However, the isolation of phages that effectively kill their target bacteria has proven challenging. In addition, isolated phages must go through significant characterization before their efficacy is measured. The method developed in this work can isolate single phage particles on the basis of their similarity to previously characterized phages while excluding those with known undesirable traits, such as bacterial toxins, as well as characterizing their kinetics. Using this method, we revealed significant cell-to-cell variations in phage kinetics at a single-cell level among highly virulent phages. These results shed some light on unknown phage-bacterium interactions at the single-cell level.

Bacteriophage therapy for challenging bacterial infections: achievements, limitations and prospects for future clinical use by veterinary dermatologists.

Bacteriophages were discovered just over 100 years ago and have been used to treat bacterial infections in animals since the 1920s. The antimicrobial resistance crisis has led to a new surge of interest in the use of bacteriophage therapy as an alternative or supplement to antimicrobial therapy in humans and other animals. To describe the nature of bacteriophages and provide a critical review and update on the clinical use of bacteriophages in the treatment of challenging bacterial infections, with an emphasis on companion animal veterinary applications. The scientific literature on the subject was critically evaluated. Findings from the most pertinent papers have been presented in summary form and critiqued. Over the last 20 years there has been a considerable increase in the volume and quality of publications dealing with bacteriophage therapy. Some recent papers build on excellent work published in the 1980s and describe promising veterinary applications. Challenges related particularly to the registration and approval of phage remedies will need to be overcome before phage therapy can become a mainstream tool for use in veterinary settings. Considerably more research, particularly controlled clinical trials, needs to be done. Bacteriophage therapy is one of the most promising approaches to tackling the looming antimicrobial resistance crisis, yet substantial regulatory challenges will need to be overcome before it enters widespread use. Phage therapy also may, in the future, improve the management of challenging bacterial infections that are not necessarily multidrug-resistant.

Phage-Display Methodology for the Study of Protein-Protein Interactions: Overview

INTRODUCTIONIn recent years, phage display has evolved into a powerful tool providing opportunities to define natural protein-protein interactions and to mold novel ligand receptors. The essential advantages of phage-display approaches originate in the incorporation of the protein and genetic components into a single phage particle. By providing a direct physical link between the expressed protein and the encoding genetic information, clones with desirable functional capacities can be efficiently subjected to iterative rounds of selection, followed by amplification of the selected sublibrary. Hence, during library selection (or panning), specific phage clones are progressively enriched on the basis of their specificity and affinity for ligand. Thus, relatively rare ligand-binding clones can be rescued rapidly and efficiently from large libraries. As these expression cloning systems have matured, versatile selection methods have been reported that are based on the functional properties of displayed proteins in diverse immunochemical and biological settings. This article summarizes phage-display methodology, including relevant biology, nucleotide-doping strategies, and considerations for library design.

Single-molecule selection and recovery of structure-specific antibodies using atomic force microscopy

Protein misfolding and aggregation are a common thread in numerous diseases including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, diabetes, and prion-related diseases. Elucidation of the role played by the various protein forms in these diseases requires reagents that can target specific protein forms. Here we present a method to isolate antibodies that bind to a specific protein form. We combined the imaging and nanomanipulation capabilities of atomic force microscopy (AFM) with the protein diversity of phage display antibody libraries to develop a technology that allows us to recover a single antibody molecule that is bound to a single protein molecular target. The target protein-antibody complex is first imaged by AFM, the AFM tip is then manipulated by nanolithography over the target antibody to recover the associated phage, and the antibody gene is recovered from the single phage particle by polymerase chain reaction.

DNA ejection from single phage particles

DNA ejection from single phage particles

Diversity and dynamics of bacteriophages in horse feces

The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5-11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (10(3)-10(7) plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain.

Encapsidation and transfer of phage DNA into host cells: From in vivo to single particles studies

A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.

Real-Time Imaging of DNA Ejection from Single Phage Particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

Reaction Kinetics of Protease with Substrate Phage: KINETIC MODEL DEVELOPED USING STROMELYSIN

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.

Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene.

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition. We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal. The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes. tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function.

Characterization of Fv fragments expressed on phage surface.

We characterized a phage antibody in which an Fv fragment, namely, a free VH fragment noncovalently associated with a VL fragment that is fused with a truncated cpIII molecule (VL-DeltacpIII), is expressed on the phage surface. D1.3 antibody specific for hen egg-white lysozyme was used as a model system. Both VH and VL-DeltacpIII fragments were stably expressed and associated with each other to form a faithful antigen-binding site. The results of Western blotting indicated that more than 5% of phages expressed the Fv fragment on their surface. Analysis of the kinetics of binding of the phage antibody to the antigen suggested the possibility of presence of phages having multiple-binding sites on a single phage particle.

Interaction of bacteriophage T4 with reconstituted cell envelopes of Escherichia coli K-12

The interaction with bacteriophage T4 of the cell surface of Escherichia coli K-12 reconstituted from outer membrane protein O-8, lipopolysaccharide, and the lipoprotein-bearing peptidoglycan sacculus was studied. The reconstituted cell surface was active as a receptor for the phage, resulting in the contraction of the tail sheath, a morphological change in the base plate which was accompanied by the extension of short tail pins down to the cell surface and the penetration of the needle through the cell surface. However, the ejection of phage deoxyribonucleic acid did not take place. Both O-8 and lipopolysaccharide were essential for the interaction. In the reconstitution, the wild-type lipopolysaccharide could not be replaced by either heptoseless lipopolysaccharide or lipid A. The lipoprotein-bearing peptidoglycan sacculus was also found to be an active component for the phage adsorption. The sacculus most likely functioned as a basal framework on which O-8 and lipopolysaccharide assembled to form a flat sheet which is large enough to interact with individual distal ends of long tail fibers of a single phage particle.

SPECIALIZED TRANSDUCTION BY BACTERIOPHAGE P22 IN SALMONELLA TYPHIMURIUM: GENETIC AND PHYSICAL STRUCTURE OF THE TRANSDUCING GENOMES AND THE PROPHAGE ATTACHMENT SITE

P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.--Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.--A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.

Recombination between phage S1 and the TC-resistant gene on Staphylococcus aureus plasmid.

Abstract When bacteriophage S1 is grown in a Staphylococcus aureus strain carrying the nonconjugative plasmid rms7 that encodes tetracycline (TC) resistance, a phage lysate capable of transducing TC resistance at an extremely high frequency was obtained. A linear relationship was found between transduction frequency and multiplicity of phage infection and a single phage particle can form a plaque containing TC lysogenic cells in its center. Treatment with anti-S1 phage serum and heating at 63° eliminated both transducing and plaque-forming activities of the lysate. These results indicated that a single recombinant of S1 particle (called S1 ptet ) carries the tet gene(s) of the rms7 plasmid and is responsible for the transduction.

A bacteriophage S1 derivative that transduces tetracycline resistance to Staphylococcus aureus

Nonconjugative tetracycline-resistant plasmid r ms7( tet) was transduced with bacteriophage S1. The product, obtained at an extremely high frequency, was a phage lysate capable of transducing the character of tetracycline resistance (TC r). Lysogenization always accompanied transduction of TC r. The transduction frequency was correlated linearly with multiplicity of infection. Infection with a single phage particle allowed the formation of plaques as well as TC r lysogenic cells at the center of the plaque. Anti-S1 serum and heating at 63° eliminated both transducing and plaque-forming activities of the lysate. We conclude that we have isolated a derivative of S1 phage that transduces TC r and infects productively and, futhermore, the TC r gene ( tet) of r ms7( tet) plasmid is part of the genome of bacteriophage S1.

Host-Induced Changes in Lactic Streptococcal Bacteriophages

Quantitative differences in lytic activity of a given phage against various susceptible host bacteria are common among lactic streptococcal bacteriophages. Changes in activity pattern against a range of hosts were quickly established by propagating a given phage on a particular host. Activity patterns determined by the parent host were highly reproducible, did not change with repeated propagations on the same host, and reflected virulence of a homogeneous progeny from replication of a single phage particle. A second kind of host-induced change was calcium requirements of phages for rapid proliferation. Three out of eight phage strains were altered in calcium requirements by a single propagation on a different host.

Antibody Formation

Summary Avidity of guinea pig antibodies to bacteriophage φX 174 was studied by means of the decision-tube and reactivation methods of Jerne and Avegno. The avidity of both γM and γG antibodies increased with time after immunization. γM antibody obtained 1 week after immunization usually had a higher avidity than γG antibody obtained 2 weeks after immunization. The possibility that the relatively high avidity of γM antibody is caused by multiple binding between a single phage particle and one γM antibody molecule is discussed.

Physical and chemical properties of a bacterial virus as related to its inhibition by streptomycin

Streptomycin specifically and reversibly inhibits the injection mechanism of a bacterial virus (P9) which attacks Streptococcus faecium. Dihydrostreptomycin does not inhibit injection, but reverses the inhibition brought about by streptomycin. Another virus of this same host (P3) is resistant to inhibition, and it is also possible to isolate mutants of P9 which are partially resistant. Streptomycin, but not dihydrostreptomycin, is able to cross-link and precipitate polyanions, and it is likely that the cross-linking function of the antibiotic is involved in the inhibitory process. Evidence is presented that there is no marked difference in the DNA characteristics of the streptomycin-sensitive and streptomycin-resistant viruses. Indirect evidence from methylene blue-mediated photodynamic inactivation studies suggests that neither SM or DHSM penetrates to the DNA of the virus. Evidence from dark inactivation of the viruses by dyes or protamine sulfate, and its reversal by the antibiotics, suggests that the antibiotics combine in some way with the protein coat of both P3 and P9. Therefore resistance to SM 4 is not a consequence of inability to bind antibiotic. SM precipitates P3, probably by cross-linking between adjacent phage particles. SM does not precipitate P9, showing that the antiviral effect is not a consequence of interparticle precipitation. However, SM, but not DHSM, causes a marked increase in the sedimentation constant of P9 under conditions in which SM is biologically active. Under the same conditions SM, but not DHSM, causes a shrinkage of the head and tail of the phage particle as viewed under the electron microscope. It is postulated that SM causes a shrinkage of the phage particle by cross-linking between adjacent protein subunits of a single phage particle, and thus pulling the protein subunits closer together. Since SM, but not DHSM, causes both the shrinkage of the phage particle and the inhibition of injection, it is postulated that the inhibition of injection is a consequence of the ability of the antibiotic to cross-link the protein subunits of the virus particle. Neomycin, which is structurally unrelated to SM, but is an effective cross-linking agent, behaves in a manner similar to SM.