Dual functional AgNPs-M13 phage composite serves as antibacterial film and sensing probe for monitoring the corrosion of chromium-containing dental alloys

The preparation of silver nanoparticles(AgNPs)with microbe or plant tissues as bio-template offers green approach,while it suffers from low harvest and purification is needed.Herein,we propose a facile protocol for one-pot preparation of AgNPs using M13 phage as bio-template by simply mixing AgN03 solution with alkali M13 phage.In the obtained AgNPs-M13 phage composite,Cr(Ⅲ)selectively coordinates with the amino residues on phage surface and leads to the aggregation of AgNPs through the bridging of M13 phages.This makes it feasible for colorimetric sensing of Cr(Ⅲ)by measuring the absorbance ratio of AgNPs at 600 and 405 nm,which provides a LOD of 14 nmol/L.The composite also showed favorable bactericidal activity for both Gram-positive and Gram-negative bacteria,making it a promising candidate as antibacterial film in chromium-containing dental alloys and meanwhile serve as a sensing probe for monitoring the corrosion of the dental alloys.     

Phage-chips for novel optically readable tissue engineering assays

We report novel phage-based array chips that are optically readable for cell proliferation and morphology assays. Using M13 phages that were engineered to display RGD on its major coat proteins and/or immobilize FGFb on its minor coat proteins, we prepared arrays of phage spot matrices composed of self-assembled nanofibrous network structures. We cultured fibroblasts on the arrays and, using surface plasmon resonance (SPR) spectroscopy, monitored the effects of the biochemical cues displayed by the phage on cell proliferation and morphology. This study demonstrates the utility of engineered phages as promising coating materials for lab-on-a-chip (LOC) platforms, allowing sensitive monitoring of the effects of functional peptides on cell growth. Phage-chips have great potential for use as high-throughput screening systems for biochemical assays and biosensors and the discovery of novel drugs.     

Analyzing and monitoring of phage–bacteria interaction using CE

The utilization of CE for monitoring bacteria–phage interaction was investigated in this study. Streptococcus thermophilus and Lactobacillus bulgaricus strains and their phages were used as model bacteria and phages for the purpose of validation in this study. CE with heterogeneous polymer polyethylene oxide was utilized for the separation of intact bacteria and investigation of phage–bacteria interaction. An intact phage detection was carried out with CZE by adding SDS in the running buffer. Calibration graphs of bacteria and phages were obtained with R² values of 0.963 and 0.937, respectively. S. thermophilus strain was infected with its virulent phage B3‐X18 for investigation of phage–bacteria interaction. It was observed in capillary electropherogram that the culture was lysed depending on the multiplicity of infection value and it showed to be completely lysed when the multiplicity of infection value was 10. The interaction of S. thermophilus strain with L. bulgaricus phage was also investigated by using a CE and a microbiological method and it was observed that the L. bulgaricus phage attached itself to the cell wall of S. thermophilus strain without damaging the cell.     

Bacteriophage reporter technology for sensing and detecting microbial targets

Bacteriophages (phages) are bacterial viruses evolutionarily tuned to very specifically recognize, infect, and propagate within only a unique pool of host cells. Knowledge of these phage host ranges permits one to devise diagnostic tests based on phage–host recognition profiles. For decades, fundamental phage typing assays have been used to identify bacterial pathogens on the basis of the ability of phages to kill, or lyse, the unique species, strain, or serovar to which they are naturally targeted. Over time, and with a better understanding of phage–host kinetics and the realization that there exists a phage specific for nearly any bacterial pathogen of clinical, foodborne, or waterborne consequence, a variety of improved, rapid, sensitive, and easy-to-use phage-mediated detection assays have been developed. These assays exploit every stage of the phage recognition and infection cycle to yield a wide variety of pathogen monitoring, detection, and enumeration formats that are steadily advancing toward new biosensor integrations and advanced sensing technologies.     

Atomic force microscopy investigation of phage infection of bacteria

Atomic force microscopy (AFM) was used to study the process of infection of bacterial cells by bacteriophages, for which purpose experimental protocols were elaborated. Three types of bacteriophages were characterized with AFM and transmission electron microscopy (TEM). Bacteriophage interaction with cells was studied for three bacterial hosts: Gram-negative Escherichia coli 057 and Salmonella enteritidis 89 and Gram-positive Bacillus thuringiensis 393. Depending on the phase of lytic cycle, different cell surface changes are observed in AFM images of infected cells in comparison with intact cells: from phage adsorption on the cells and flagella to complete lysis of the cells, accompanied by the release of a large number of newly formed phages. Control experiments (cells without phages and cells with nonspecific phages) did not reveal any surface changes. Penetration of phages inside obligate aerobe Bacillus thuringiensis was shown to be oxygen-dependent and required aeration in laboratory conditions. Our results show great potential of using AFM for numerous fundamental and applied tasks connected with pathogen-host interaction.     

OVERLAPPING ACTION OF HOSTCELL REACTIVATION AND PHOTOREACTIVATION IN BACTERIA

Abstract— The photoreactivation rate of U.V. irradiated phages is decreased in u.v. irradiated bacteria. In contrast, the normal photoreactivation rate is observed if the irradiated bacteria are photoreactivated before phage infection. The decrease of the photoreactivation ratc is understood as a competing effect of the u.v. lesions in the bacterial nucleic acids for the photoreactivation enzyme. This competitive inhibition can be diminished not only by photoreactivation of the bacteria before phage infection but also by hostcell reactivation of the u.v. lesions in the bacterium. The results provide strong evidence that hostcell reactivation and photoreactivation revert the same u.v. photoproducts in bacterial nucleic acids. The experiments show that the hostcell reactivation enzyme is not induced by phage infection or by irradiation, but is normally present in the bacterial cell.     

SUPPRESSION OF PHAGE T4 BY CO-INFECTION WITH U.V.-INACTIVATED HOMOLOGOUS PHAGE

Abstract— Unirradiated phage T4v₁ may fail to produce viable progeny in cells which are co-infected with u.v.-irradiated homologous particles. The extent of this effect, called suppression , is positively correlated with the multiplicity of infection of the irradiated phage and with the U.V. dose. The suppression reaches a maximum level at about 30–600 lethal hits. Quantitative evaluation of the results shows that in some complexes one irradiated phage particle is sufficient to suppress an unirradiated phage. Two hypotheses are discussed to explain the results. (a) Lethal u.v.-damages are incorporated into the unirradiated phage genome by genetic recombination; ( b ) Genetic subunits (e.g. cistrons or operons) of the u.v.-irradiated phages produce informationally incorrect messenger RNA molecules, which compete with the correct ones from the unirradiated phage in the protein-synthetizing system. Hypothesis (6) appears to be more adequate to explain the experimental results.     

Characterisation of methacrylate monoliths for bacteriophage purification

Binding of three different bacteriophages (phages), namely T7, lambda and M13 on methacrylate monoliths was investigated. Phage M13 exhibited the highest dynamic binding capacity of 4.5×10(13) pfu/mL while T7 and lambda showed capacity of 1×10(13) pfu/mL, all corresponding to values of around 1mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2M while there was a constant capacity decrease for T7 and M13 phages. Under optimal conditions, recovery for all three phages approached 100%. Measurement of a pressure drop increase during loading enabled estimation of adsorbed phage layer thickness. At a maximal capacity it was calculated to be around 50 nm for T7 phage and 60 nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80 nm layer thickness was estimated for M13 phage showing its orientation along the pore.     

Screening of phage‐displayed human liver cDNA library against doxorubicin with drug‐immobilized monolithic polyacrylamide cryogel

Monolithic polyacrylamide cryogel was prepared and utilized as a new matrix for drug immobilization to screen against phage‐displayed human liver cDNA library. The macropores and hydrophilic nature of the cryogel made it possible for phage particles to pass unhindered. Doxorubicin, an anticancer drug, was covalently bonded to the monolithic cryogel by the glutaraldehyde method, and after five rounds of affinity selection performed in an SPE cartridge, phage clones that displayed Homo sapiens methyl CpG binding protein 2 (MeCP₂) were selectively enriched. The interaction between doxorubicin and MeCP₂ displayed phages was further validated by studying the retention of doxorubicin on MeCP₂ phage‐coupled cryogel. These results demonstrate that drug‐coupled polyacrylamide cryogel might be a promising kind of matrix for screening target proteins against phage‐displayed library. Copyright © 2013 John Wiley & Sons, Ltd.     

Antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations

The most exciting potential of phage displayed peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. In addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. However, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. This review assesses critically the antigenicity of phage displayed peptides as free peptides and in different molecular environments. The difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phage-derived peptides are discussed. Peptides selected with antibodies from phage displayed random peptide libraries have raised considerable expectations as low molecular weight substitutes of the natural antigen. This review will focus on the results of phage displayed random peptide libraries screened with antibodies specific for proteins, carbohydrates and nucleic acids and critically examine how the above expectations have been met.     

Simultaneous detection of two bacterial pathogens using bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The simultaneous analysis of multiple target microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with bacteriophage amplification is discussed. Following infection of target bacteria with specific bacteriophages, proteins contained in the progeny phage are utilized as a secondary biomarker for the target bacterium. Escherichia coli when mixed with MS2 and MPSS-1 phages specific for E. coli and Salmonella spp., respectively, at levels below their corresponding detection limits, produced only the protein (13.7 kDa) characteristic of the MS2. Likewise, Salmonella spp. when mixed with the two phages only produced a protein (13.5 kDa) characteristic of MPSS-1. When the two bacteria and the two phages were mixed together, proteins characteristic of MS2 and MPSS-1 were observed indicating that both bacteriophages had been amplified. Identification of each bacterium was made based on the presence of the secondary bacteriophage biomarkers. No deleterious effects on bacteriophage amplification were observed because of the presence of multiple bacteria or bacteriophages.     

Investigations on the Interactions of λPhage-Derived Peptides Against the SrtA Mechanism in Bacillus anthracis

Bacillus anthracis is a well-known bioweapon pathogen, which coordinates the expression of its virulence factors in response to a specific environmental signal by its protein architecture. Absences of sortase signal functioning may fail to assemble the surface linked proteins and so B. anthracis cannot sustain an infection with host cells. Targeting the signaling mechanism of B. anthracis can be achieved by inhibition of SrtA enzyme through λphage-derived plyG. The lysin enzyme plyG is experimentally proven as bacteriolytic agent, specifically kill's B. anthracis by inhibiting the SrtA. Here, we have screened the peptides from λphage lysin, and these peptides are having the ability as LPXTG competitive inhibitors. In comparison to the activator peptide LPXTG binding motif, λphage lysin based inhibitor peptides are having much supremacy towards binding of SrtA. Finally, peptide structures extracted from PlyG are free from toxic, allergic abilities and also have the ability to terminate the signal transduction mechanism in B. anthracis.     

Application of protein-coupled liposomes to effective affinity screening from phage library

For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of dipalmitoylphosphatidylcholine (DPPC): dicetylphosphate (DCP) = 10: 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After four rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15% of the selected phages displayed homological peptides in the conventional biopanning. The octapeptide selected by AB-MLVs against the anti-peptide antibody showed comparable binding affinity, which were determined by the competitive ELISA and an immunoaffinity chromatography, to that of the peptide antigen. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.     

Pathogen detection using engineered bacteriophages

Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.     

Tape measure protein having MT3 motif facilitates phage entry into stationary phase cells of Mycobacterium tuberculosis

Tape measure protein (TMP) having MT3 motif in mycobacteriophage TM4 genome has been reported to enable the phage infection of Mycobacterium smegmatis during stationary phase. In the present work looking at eight additional mycobacteriophage genomes by in silico analysis, six of them have been found to possess MT3 motif in TMP. The absence of MT3 motif in Che12 and D29 probably makes them incapable of infecting stationary phase cells of Mycobacterium tuberculosis which was experimentally evaluated by the performance of respective luciferase reporter phage constructs developed from the parental phages Che12, D29 and TM4.     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

Lytic enzymes of staphylococcal phages: Correlation between secondary structure and stability

Lytic enzymes of bacteriophages K, phi11, and phi80α can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus, which makes these enzymes promising antimicrobial agents. The stability of recombinant lysins of phages K, phi11, and phi80α was investigated under the conditions of storage and functioning, and the correlation between the stability and the secondary structure of the enzymes was found. It has been shown that the lower the content of disordered structures in the enzyme molecules, the greater the stability (half-inactivation time) of the lysins. At the storage temperature, the beta-structural lysin of phage phi11 shows the highest stability, while the phage K lysin with an alpha-helical structure and the phi80α lysin with a disordered secondary structure are less stable.     

Phage display of enzymes and in vitro selection for catalytic activity

Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes. The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography. The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.     

利用pⅧ展示系统改进噬菌体抗体芯片

将展示单链抗体的重组噬菌体与羧基终止的硅片偶联, 制成噬菌体抗体芯片, 可用于检测多类蛋白质和蛋白质组. 通常抗体被展示于噬菌体外壳蛋白pⅢ上, 由此制备的芯片灵敏度和信噪比较低. 我们选用凝血酶特异的单链抗体为代表, 比较了pⅢ展示系统和pⅧ展示系统制成芯片的检测效果. 由于pⅧ展示系统的融合蛋白拷贝数多, 所受空间位阻小, 大幅度提高了噬菌体抗体芯片的灵敏度和信噪比, 有望用于制备新型蛋白质芯片.     

Considerations on antibody-phage display methodology

For almost 15 years phage display is being used for the selection of specific antigen-binders from artificial libraries of single chain antibodies. Filamentous phages have been developed in a way to express foreign proteins on the surface and at the same time carrying the genetic information of the surface expressed molecule within the phage capsid. This property guarantees the coupling of phenotype and genotype during phage amplification and affinity selection. The possibility to generate large antibody libraries and the simplified antibody-backbone of a single chain antibody has made antibody-phage display to a powerful tool for the development of new therapeutics against various human diseases. In this review we discuss the general principles and latest developments and applications in antibody phage display technology.