Special Issue "100 Years of Bacteriophage: From Basic Research to Medical Application"

A special issue of Methods and Protocols (ISSN 2409-9279).

Deadline for manuscript submissions: closed (31 December 2018)

Special Issue Editor

Guest Editor
Dr. habil. Hansjörg Lehnherr

PTC Phage Technology Center GmbH, Im Kompetenzzentrum Bio-Security, Siemensstrasse 42, D-59199 Bönen
Website | E-Mail
Interests: bacteriophage; genetics; gene expression; plasmid

Special Issue Information

Dear Colleagues,

A century ago, Félix d’Hérelle coined the term bacteriophage. Since then, bacteriophage research has paved the way for major scientific breakthroughs such as the identification of DNA as the genetic material, the triplet nature of the genetic code, and the elucidation of the structure of DNA. The phenomenon of "restriction-modification", a phage defense mechanism, gave rise to the field of molecular biology, which, at present, is being revolutionized by the application of "CRISPR", which is also a phage defense mechanism. Molecular tools based on various phage-encoded proteins or whole phage particles, initially developed for basic research, are moving towards practical applications such as phage display for antibody engineering or diagnostics, capsid-based nanoparticles as drug-delivery vehicles and next-generation therapeutics, nano-films with anti-bacterial properties, bio-imaging, and rapid pathogen identification. Furthermore, the therapeutic use of bacteriophages to fight pathogenic bacteria, an idea as old as the discovery of phages, turned out to be a valid treatment alternative when antibiotics fail.

For this Special Issue of Methods and Protocols, we invite submissions of research manuscripts, protocols and review articles that focus on methods and techniques for basic bacteriophage research and phage applications.

Dr. habil. Hansjörg Lehnherr
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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. Methods and Protocols is an international peer-reviewed open access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) is waived for well-prepared manuscripts submitted to this issue. 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
  • plaque assay
  • phage therapy
  • phage display
  • diagnostics
  • nano-particles
  • nano-films
  • drug delivery
  • animal model
  • antimicrobial
  • agriculture
  • animal husbandry
  • aquaculture
  • food processing
  • human health
  • sanitation

Published Papers (5 papers)

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Open AccessReview
Campylobacter Phage Isolation and Characterization: What We Have Learned So Far
Methods Protoc. 2019, 2(1), 18; https://doi.org/10.3390/mps2010018
Received: 4 January 2019 / Revised: 5 February 2019 / Accepted: 6 February 2019 / Published: 15 February 2019
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Abstract
Lytic Campylobacter phages, which can be used to combat this pathogen in animals and on food products, have been studied for more than 30 years. Though, due to some peculiarities of the phages, which hampered their isolation and particularly their molecular analysis for [...] Read more.
Lytic Campylobacter phages, which can be used to combat this pathogen in animals and on food products, have been studied for more than 30 years. Though, due to some peculiarities of the phages, which hampered their isolation and particularly their molecular analysis for a long time, progress in this research field was rather slow. Meanwhile, the situation has changed and much more is known about the biology and genetics of those phages. In this article, we address specific issues that should be considered when Campylobacter phages are studied, starting with the isolation and propagation of the phages and ending with a thorough characterization including whole-genome sequencing. The basis for advice and recommendations given here is a careful review of the scientific literature and experiences that we have had ourselves with Campylobacter phages. Full article
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Open AccessReview
Function of the RNA Coliphage Qβ Proteins in Medical In Vitro Evolution
Methods Protoc. 2018, 1(2), 18; https://doi.org/10.3390/mps1020018
Received: 27 March 2018 / Revised: 16 May 2018 / Accepted: 28 May 2018 / Published: 31 May 2018
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Abstract
Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four [...] Read more.
Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A2) and the read-through or minor coat protein (A1), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A1 protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A1 is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A1 protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A1 proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses. Full article
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Other

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Open AccessProtocol
Reverse Genetic Systems for Pseudomonas aeruginosa Leviphages
Methods Protoc. 2019, 2(1), 22; https://doi.org/10.3390/mps2010022
Received: 27 January 2019 / Revised: 27 February 2019 / Accepted: 27 February 2019 / Published: 5 March 2019
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Abstract
Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary [...] Read more.
Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy. Full article
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Open AccessProtocol
A Rapid Bacteriophage DNA Extraction Method
Methods Protoc. 2018, 1(3), 27; https://doi.org/10.3390/mps1030027
Received: 1 June 2018 / Revised: 6 July 2018 / Accepted: 24 July 2018 / Published: 27 July 2018
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Abstract
Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole [...] Read more.
Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 1010 plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform. Full article
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Open AccessProtocol
Practical Method for Isolation of Phage Deletion Mutants
Methods Protoc. 2018, 1(1), 6; https://doi.org/10.3390/mps1010006
Received: 12 December 2017 / Revised: 9 January 2018 / Accepted: 15 January 2018 / Published: 17 January 2018
Cited by 1 | PDF Full-text (4502 KB) | HTML Full-text | XML Full-text
Abstract
The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth [...] Read more.
The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus). Full article
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