Special Issue "Biotechnological Applications of Phage and Phage-Derived Proteins 2021"

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

Deadline for manuscript submissions: closed (30 June 2021).

Special Issue Editors

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

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.

Prof. Dr. Joana Azeredo
Dr. Sílvio Santos
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 2200 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 (8 papers)

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Research

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Article
Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies
Viruses 2021, 13(8), 1462; https://doi.org/10.3390/v13081462 - 27 Jul 2021
Viewed by 311
Abstract
Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the [...] Read more.
Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium’s close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction. Full article
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Article
Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity
Viruses 2021, 13(6), 1034; https://doi.org/10.3390/v13061034 - 31 May 2021
Viewed by 860
Abstract
Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of [...] Read more.
Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage’s host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4–85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0–12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria. Full article
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Article
Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay
Viruses 2021, 13(5), 750; https://doi.org/10.3390/v13050750 - 24 Apr 2021
Viewed by 1082
Abstract
Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The [...] Read more.
Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules. Full article
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Article
Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans
Viruses 2021, 13(5), 725; https://doi.org/10.3390/v13050725 - 21 Apr 2021
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Abstract
Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in [...] Read more.
Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area. Full article
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Review

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Review
Bacteriophages as Fecal Pollution Indicators
Viruses 2021, 13(6), 1089; https://doi.org/10.3390/v13061089 - 07 Jun 2021
Cited by 1 | Viewed by 625
Abstract
Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), [...] Read more.
Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), have been proposed as alternatives or complements to fecal indicator bacteria. Here, we provide a general overview of the potential use of enteric bacteriophages as fecal and viral indicators in different environments, as well as the available methods for their detection and enumeration, and the regulations for their application. Full article
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Review
Viruses with U-DNA: New Avenues for Biotechnology
Viruses 2021, 13(5), 875; https://doi.org/10.3390/v13050875 - 10 May 2021
Viewed by 612
Abstract
Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations [...] Read more.
Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA–protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes. Full article
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Review
The Advantages and Challenges of Using Endolysins in a Clinical Setting
Viruses 2021, 13(4), 680; https://doi.org/10.3390/v13040680 - 15 Apr 2021
Cited by 1 | Viewed by 809
Abstract
Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases [...] Read more.
Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that act to lyse bacterial cells by targeting their cell’s wall, particularly in Gram-positive bacteria due to their naturally exposed peptidoglycan layer. These lytic enzymes have received much interest from the scientific community in recent years for their specificity, mode of action, potential for engineering, and lack of resistance mechanisms. Over the past decade, a renewed interest in endolysin therapy has led to a number of successful applications. Recombinant endolysins have been shown to be effective against prominent pathogens such as MRSA, Listeria monocytogenes, Staphylococcus strains in biofilm formation, and Pseudomonas aeruginosa. Endolysins have also been studied in combination with other antimicrobials, giving a synergistic effect. Although endolysin therapy comes with some regulatory and logistical hurdles, the future looks promising, with the emergence of engineered “next-generation” lysins. This review will focus on the likelihood that endolysins will become a viable new antimicrobial therapy and the challenges that may have to be overcome along the way. Full article
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Review
Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy
Viruses 2021, 13(4), 649; https://doi.org/10.3390/v13040649 - 09 Apr 2021
Viewed by 516
Abstract
Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides [...] Read more.
Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy. Full article
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