Special Issue "Biotechnological Applications of Phage and Phage-Derived Proteins"

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Bacterial Viruses".

Deadline for manuscript submissions: closed (30 April 2018).

Special Issue Editors

Guest Editor
Dr. Sílvio Santos Website E-Mail
Centre of Biological Engineering, University of Minho, Braga, Portugal
Interests: bacteriophages; genetic and protein engineering; protein functional analysis; biotechnology; pharmaceutical
Guest Editor
Prof. Dr. Joana Azeredo Website E-Mail
Centre of Biological Engineering, University of Minho, Braga, Portugal
Interests: bacteriophages; biofilms; bacteria and yeast virulence; biotechnology

Special Issue Information

Dear Colleagues,

Bacteriophages, the viruses of bacteria, are recognized for their potential as antimicrobial agents since their discovery, roughly a century ago, but the early inadequately controlled trials, the poor knowledge on their biology and the discovery of antibiotics has slowed phage research. In recent years, the increasing problem of multidrug-resistant bacteria has renewed and heightened interest in the use of phages as antimicrobial agents.

The recent progress in sequencing technologies, DNA manipulation and synthetic biology has equipped scientists with the necessary tools to disclose and use the powerful armamentarium of proteins that phages possess to parasite bacteria. These proteins can be used ex-phage (isolated from the phage particle) or modified/added to design phages with improved and superior characteristics and functionalities which enabled the development of new powerful applications of phages and their proteins not only in therapeutics (as new source of antimicrobials, drug delivery systems and vaccines) but also in diagnostics and materials science (for the assembly of new materials). As new phage proteins are being discovered, new valuable biotechnological applications are envisaged.

With the wide array of possibilities offered by genetic engineering and its attracting intense interest coupled with the high potential of phages, this Special Issue will focus on new biotechnological applications of phage and their derived proteins, as well on the new strategies to obtain them. Bacteriophage therapy through the use of wild type phages (without any genetic modification) is out of focus of this special issue.

Dr. Sílvio Santos
Prof. Dr. Joana Azeredo
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • bacteriophage
  • biotechnological applications
  • genetic engineering
  • antimicrobials
  • diagnostic

Published Papers (16 papers)

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Editorial

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Open AccessEditorial
Bacteriophage-Based Biotechnological Applications
Viruses 2019, 11(8), 737; https://doi.org/10.3390/v11080737 - 10 Aug 2019
Abstract
Phages have shown a high biotechnological potential with numerous applications. The advent of high-resolution microscopy techniques aligned with omic and molecular tools are revealing innovative phage features and enabling new processes that can be further exploited for biotechnological applications in a wide variety [...] Read more.
Phages have shown a high biotechnological potential with numerous applications. The advent of high-resolution microscopy techniques aligned with omic and molecular tools are revealing innovative phage features and enabling new processes that can be further exploited for biotechnological applications in a wide variety of fields. This special issue is a collection of original and review articles focusing on the most recent advances in phage-based biotechnology with applications for human benefit. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)

Research

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Open AccessArticle
Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens
Viruses 2019, 11(3), 284; https://doi.org/10.3390/v11030284 - 21 Mar 2019
Cited by 1
Abstract
The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting [...] Read more.
The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol
Viruses 2019, 11(3), 241; https://doi.org/10.3390/v11030241 - 11 Mar 2019
Cited by 1
Abstract
The ‘Appelmans protocol’ is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this [...] Read more.
The ‘Appelmans protocol’ is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3
Viruses 2019, 11(1), 50; https://doi.org/10.3390/v11010050 - 11 Jan 2019
Cited by 2
Abstract
The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in [...] Read more.
The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10–1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal
Viruses 2018, 10(11), 638; https://doi.org/10.3390/v10110638 - 15 Nov 2018
Cited by 1
Abstract
Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity [...] Read more.
Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detection
Viruses 2018, 10(11), 626; https://doi.org/10.3390/v10110626 - 13 Nov 2018
Cited by 1
Abstract
The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox [...] Read more.
The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessShort Note
Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening
Viruses 2018, 10(10), 552; https://doi.org/10.3390/v10100552 - 10 Oct 2018
Cited by 2
Abstract
Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, a vaginal–rectal sample is recommended to be [...] Read more.
Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, a vaginal–rectal sample is recommended to be enriched, followed by bacterial identification. In some cases, Enterococcus faecalis overgrows in the enrichment culture. Consequently, the identification test yields false-negative results. Bacteriophages (phages) can be used as antimicrobial materials. Here, we explored the feasibility of using phages to minimize false-negative results in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of vaginal E. faecalis strains. The simple coculture of E. faecalis and S. agalactiae was used as an experimental enrichment model. Phage mixture treatment led to suppression of E. faecalis growth and facilitation of S. agalactiae growth. In addition, testing several sets of S. agalactiae and E. faecalis strains, the treatment with phage mixture in the enrichment improved S. agalactiae detection on chromogenic agar. Our results suggest that the phage mixture can be usefully employed in the S. agalactiae culture test to increase test accuracy. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Strategies to Encapsulate the Staphylococcus aureus Bacteriophage phiIPLA-RODI
Viruses 2018, 10(9), 495; https://doi.org/10.3390/v10090495 - 13 Sep 2018
Cited by 2
Abstract
The antimicrobial properties of bacteriophages make them suitable food biopreservatives. However, such applications require the development of strategies that ensure stability of the phage particles during food processing. In this study, we assess the protective effect of encapsulation of the Staphylococcus aureus bacteriophage [...] Read more.
The antimicrobial properties of bacteriophages make them suitable food biopreservatives. However, such applications require the development of strategies that ensure stability of the phage particles during food processing. In this study, we assess the protective effect of encapsulation of the Staphylococcus aureus bacteriophage phiIPLA-RODI in three kinds of nanovesicles (niosomes, liposomes, and transfersomes). All these systems allowed the successful encapsulation of phage phiIPLA-RODI with an efficiency ranged between 62% and 98%, regardless of the concentration of components (like phospholipids and surfactants) used for vesicle formation. Only niosomes containing 30 mg/mL of surfactants exhibited a slightly lower percentage of encapsulation. Regarding particle size distribution, the values determined for niosomes, liposomes, and transfersomes were 0.82 ± 0.09 µm, 1.66 ± 0.21 µm, and 0.55 ± 0.06 µm, respectively. Importantly, bacteriophage infectivity was maintained during storage for 6 months at 4 °C for all three types of nanovesicles, with the exception of liposomes containing a low concentration of components. In addition, we observed that niosomes partially protected the phage particles from low pH. Thus, while free phiIPLA-RODI was not detectable after 60 min of incubation at pH 4.5, titer of phage encapsulated in niosomes decreased only 2 log units. Overall, our results show that encapsulation represents an appropriate procedure to improve stability and, consequently, antimicrobial efficacy of phages for application in the food processing industry. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessCommunication
Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens
Viruses 2018, 10(8), 431; https://doi.org/10.3390/v10080431 - 15 Aug 2018
Cited by 2
Abstract
Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen [...] Read more.
Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers
Viruses 2018, 10(8), 427; https://doi.org/10.3390/v10080427 - 14 Aug 2018
Cited by 9
Abstract
The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin [...] Read more.
The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains—two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Novel T7 Phage Display Library Detects Classifiers for Active Mycobacterium Tuberculosis Infection
Viruses 2018, 10(7), 375; https://doi.org/10.3390/v10070375 - 19 Jul 2018
Cited by 2
Abstract
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and transmitted through inhalation of aerosolized droplets. Eighty-five percent of new TB cases occur in resource-limited countries in Asia and Africa and fewer than 40% of TB cases are diagnosed due to the lack of [...] Read more.
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and transmitted through inhalation of aerosolized droplets. Eighty-five percent of new TB cases occur in resource-limited countries in Asia and Africa and fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays. Currently, diagnosis relies on the demonstration of the bacterium in clinical specimens by serial sputum smear microscopy and culture. These methods lack sensitivity, are time consuming, expensive, and require trained personnel. An alternative approach is to develop an efficient immunoassay to detect antibodies reactive to MTB antigens in bodily fluids, such as serum. Sarcoidosis and TB have clinical and pathological similarities and sarcoidosis tissue has yielded MTB components. Using sarcoidosis tissue, we developed a T7 phage cDNA library and constructed a microarray platform. We immunoscreened our microarray platform with sera from healthy (n = 45), smear positive TB (n = 24), and sarcoidosis (n = 107) subjects. Using a student t-test, we identified 192 clones significantly differentially expressed between the three groups at a False Discovery Rate (FDR) <0.01. Among those clones, we selected the top ten most significant clones and validated them on independent test set. The area under receiver operating characteristics (ROC) for the top 10 significant clones was 1 with a sensitivity of 1 and a specificity of 1. Sequence analyses of informative phage inserts recognized as antigens by active TB sera may identify immunogenic antigens that could be used to develop therapeutic or prophylactic vaccines, as well as identify molecular targets for therapy. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Self-Assembled Nanoporous Biofilms from Functionalized Nanofibrous M13 Bacteriophage
Viruses 2018, 10(6), 322; https://doi.org/10.3390/v10060322 - 12 Jun 2018
Cited by 5
Abstract
Highly periodic and uniform nanostructures, based on a genetically engineered M13 bacteriophage, displayed unique properties at the nanoscale that have the potential for a variety of applications. In this work, we report a multilayer biofilm with self-assembled nanoporous surfaces involving a nanofiber-like genetically [...] Read more.
Highly periodic and uniform nanostructures, based on a genetically engineered M13 bacteriophage, displayed unique properties at the nanoscale that have the potential for a variety of applications. In this work, we report a multilayer biofilm with self-assembled nanoporous surfaces involving a nanofiber-like genetically engineered 4E-type M13 bacteriophage, which was fabricated using a simple pulling method. The nanoporous surfaces were effectively formed by using the networking-like structural layers of the M13 bacteriophage during self-assembly. Therefore, an external template was not required. The actual M13 bacteriophage-based fabricated multilayered biofilm with porous nanostructures agreed well with experimental and simulation results. Pores formed in the final layer had a diameter of about 150–500 nm and a depth of about 15–30 nm. We outline a filter application for this multilayered biofilm that enables selected ions to be extracted from a sodium chloride solution. Here, we describe a simple, environmentally friendly, and inexpensive fabrication approach with large-scale production potential. The technique and the multi-layered biofilms produced may be applied to sensor, filter, plasmonics, and bio-mimetic fields. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Clostridium perfringens Virulent Bacteriophage CPS2 and Its Thermostable Endolysin LysCPS2
Viruses 2018, 10(5), 251; https://doi.org/10.3390/v10050251 - 11 May 2018
Cited by 5
Abstract
Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The [...] Read more.
Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The CPS2 phage contains a 17,961 bp double-stranded DNA genome with 25 putative ORFs, and belongs to the Picovirinae, subfamily of Podoviridae. Bioinformatic analysis of the CPS2 genome revealed a putative endolysin, LysCPS2, which is homologous to the endolysin of Clostridium phage phiZP2 and phiCP7R. The enzyme showed strong lytic activity against C. perfringens with optimum conditions at pH 7.5–10, 25–65 °C, and over a broad range of NaCl concentrations. Interestingly, LysCPS2 was found to be highly thermostable, with up to 30% of its lytic activity remaining after 10 min of incubation at 95 °C. The cell wall binding domain in the C-terminal region of LysCPS2 showed a binding spectrum specific to C. perfringens strains. This is the first report to characterize highly thermostable endolysin isolated from virulent C. perfringens bacteriophage. The enzyme can be used as an alternative biocontrol and detection agent against C. perfringens. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Open AccessArticle
Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell
Viruses 2018, 10(4), 168; https://doi.org/10.3390/v10040168 - 01 Apr 2018
Cited by 2
Abstract
Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, [...] Read more.
Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Review

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Open AccessReview
Landscape Phage: Evolution from Phage Display to Nanobiotechnology
Viruses 2018, 10(6), 311; https://doi.org/10.3390/v10060311 - 07 Jun 2018
Cited by 8
Abstract
The development of phage engineering technology has led to the construction of a novel type of phage display library—a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the “landscape phage”, serve as [...] Read more.
The development of phage engineering technology has led to the construction of a novel type of phage display library—a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the “landscape phage”, serve as a huge resource of diagnostic/detection probes and versatile construction materials for the preparation of phage-functionalized biosensors and phage-targeted nanomedicines. Landscape-phage-derived probes interact with biological threat agents and generate detectable signals as a part of robust and inexpensive molecular recognition interfaces introduced in mobile detection devices. The use of landscape-phage-based interfaces may greatly improve the sensitivity, selectivity, robustness, and longevity of these devices. In another area of bioengineering, landscape-phage technology has facilitated the development and testing of targeted nanomedicines. The development of high-throughput phage selection methods resulted in the discovery of a variety of cancer cell-associated phages and phage proteins demonstrating natural proficiency to self-assemble into various drug- and gene-targeting nanovehicles. The application of this new “phage-programmed-nanomedicines” concept led to the development of a number of cancer cell-targeting nanomedicine platforms, which demonstrated anticancer efficacy in both in vitro and in vivo experiments. This review was prepared to attract the attention of chemical scientists and bioengineers seeking to develop functionalized nanomaterials and use them in different areas of bioscience, medicine, and engineering. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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Other

Open AccessOpinion
Nanomedicine and Phage Capsids
Viruses 2018, 10(6), 307; https://doi.org/10.3390/v10060307 - 06 Jun 2018
Cited by 2
Abstract
Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate [...] Read more.
Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate states of DNA injection and DNA packaging (dynamic states). We discuss and test the idea that some T3 dynamic states include extensive α-sheet in subunits of the capsid’s shell. Second, dynamic states of pathogenic viral capsids are possible targets of innate immune systems. Specifically, α-sheet-rich innate immune proteins would interfere with dynamic viral states via inter-α-sheet co-assembly. A possible cause of neurodegenerative diseases is excessive activity of these innate immune proteins. Third, some phage capsids appear to have characteristics useful for improved drug delivery vehicles (DDVs). These characteristics include stability, uniformity and a gate-like sub-structure. Gating by DDVs is needed for (1) drug-loading only with gate opened; (2) closed gate-DDV migration through circulatory systems (no drug leakage-generated toxicity); and (3) drug release only at targets. A gate-like sub-structure is the connector ring of double-stranded DNA phage capsids. Targeting to tumors of phage capsid-DDVs can possibly be achieved via the enhanced permeability and retention effect. Full article
(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)
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