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Special Issue "Bacteriophage—Molecular Studies"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (30 May 2020) | Viewed by 20120

Special Issue Editor

Special Issue Information

Dear Colleagues,

Bacteriophages, the viruses infecting bacterial cells, were first described 100 years ago, in 1915, by Frederick Twort. The scientist who introduced the name “bacteriophage” was Felix d’Herelle, who investigated these viruses for many years, leading to new fields of research, including bacteriophage therapy. In the following years, bacteriophages became important model organisms in molecular biology and genetics. Many basic discoveries were made during studies of these viruses, with such spectacular examples as demonstrating that DNA is a genetic material, viruses can encode enzymes, gene expression proceeds through mRNA molecules, the genetic code is based on nucleotide triplets, gene expression can be regulated by transcription antitermination, specific genes encode heat shock proteins, and that specific mechanisms regulate DNA replication initiation based on the formation and rearrangements of protein–DNA complexes. The regulatory processes occurring in bacteriophage-infected cells have been considered as paradigms of the control of developmental pathways. On the other hand, the history of research on bacteriophages also passed through dark times when, at the end of 20th century, there was the collective impression that we knew almost everything there was to know about these simple viruses, and that it was time to investigate more complex organisms instead. Nevertheless, subsequent discoveries have indicated that such an assumption was unequivocally false, and studies on the molecular biology and biotechnology of bacteriophages have once again become extensive. The interest in these viruses has increased dramatically, and it appears that we are far from understanding the biology of the vast majority of bacteriophages.

This Special Issue of International Journal of Molecular Sciences is devoted to publishing papers on studies of bacteriophages at the molecular level. Papers on phage biodiversity, regulation of processes occurring during phage development, as well as the practical use of bacteriophages—including biotechnology and phage therapy—are welcome, providing the studies deal with the molecular level and utilize molecular biology methods. I am hopeful of building a great collection of articles devoted to recent discoveries in the field of bacteriophage molecular biology. Therefore, I invite you to submit manuscripts to this Special Issue as an excellent forum to share your discoveries in this fascinating research field.

Dr. Alicja Wegrzyn
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 submissions that pass pre-check are 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.

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Keywords

  • bacteriophage biodiversity
  • regulation of bacteriophage development
  • molecular processes in bacteriophages
  • bacteriophage-based biotechnology
  • phage therapy

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Published Papers (11 papers)

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Research

Article
Antibiotics Act with vB_AbaP_AGC01 Phage against Acinetobacter baumannii in Human Heat-Inactivated Plasma Blood and Galleria mellonella Models
Int. J. Mol. Sci. 2020, 21(12), 4390; https://doi.org/10.3390/ijms21124390 - 19 Jun 2020
Cited by 27 | Viewed by 1897
Abstract
Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that [...] Read more.
Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that poses antibacterial activity against clinical Acinetobacter baumannii strains. Moreover, besides genomic and phenotypic analysis our study aims to analyze phage–antibiotic combination effectiveness with the use of ex vivo and in vivo models. The phage AGC01 efficiently adsorbs to A. baumannii cells and possesses a bacteriolytic lifecycle resulting in high production of progeny phages (317 ± 20 PFU × cell−1). The broad host range (50.27%, 93 out of 185 strains) against A. baumannii isolates and the inability of AGC01 to infect other bacterial species show its high specificity. Genomic analysis revealed a high similarity of the AGC01 genome sequence with that of the Friunavirus genus from a subfamily of Autographivirinae. The AGC01 is able to significantly reduce the A. baumannii cell count in a human heat-inactivated plasma blood model (HIP-B), both alone and in combination with antibiotics (gentamicin (GEN), ciprofloxacin (CIP), and meropenem (MER)). The synergistic action was observed when a combination of phage treatment with CIP or MER was used. The antimicrobial activity of AGC01 and phage-antibiotic combinations was confirmed using an in vivo larva model. This study shows the greatest increase in survival of G. mellonella larvae when the combination of phage (MOI = 1) and MER was used, which increased larval survival from 35% to 77%. Hence, AGC01 represents a novel candidate for phage therapy. Additionally, our study suggests that phages and antibiotics can act synergistically for greater antimicrobial effect when used as combination therapy. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin
Int. J. Mol. Sci. 2020, 21(11), 4013; https://doi.org/10.3390/ijms21114013 - 04 Jun 2020
Cited by 6 | Viewed by 1286
Abstract
HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence [...] Read more.
HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of β-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide
Int. J. Mol. Sci. 2020, 21(9), 3170; https://doi.org/10.3390/ijms21093170 - 30 Apr 2020
Cited by 3 | Viewed by 1436
Abstract
Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. [...] Read more.
Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Isolation and Characterization of Two Klebsiella pneumoniae Phages Encoding Divergent Depolymerases
Int. J. Mol. Sci. 2020, 21(9), 3160; https://doi.org/10.3390/ijms21093160 - 30 Apr 2020
Cited by 6 | Viewed by 2363
Abstract
The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics [...] Read more.
The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, πVLC5 and πVLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage πVLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage πVLC6 is conferred by a second, divergent depolymerase. Phage πVLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Structural Analysis of Jumbo Coliphage phAPEC6
Int. J. Mol. Sci. 2020, 21(9), 3119; https://doi.org/10.3390/ijms21093119 - 28 Apr 2020
Cited by 7 | Viewed by 2262
Abstract
The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 [...] Read more.
The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence
Int. J. Mol. Sci. 2020, 21(8), 2930; https://doi.org/10.3390/ijms21082930 - 22 Apr 2020
Cited by 6 | Viewed by 1964
Abstract
The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains [...] Read more.
The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains unclear, especially in a field-based production such as for leek plants in Flanders. Therefore, more insight in bacteria–phage interaction is required, along with the evaluation of different application strategies. In this study, we further characterized the infection strategy of two Pspo phages, KIL3b and KIL5. We found that both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium. LPS is an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests showed that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. However, the impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. Our data suggest that the phage cocktail has promising potential to lower the prevalence of Pspo and to be integrated in a pest management strategy. Targeted research is needed to further explore the applicability of the phages in combination with other disease control strategies. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Identification and Characterization of the First Virulent Phages, Including a Novel Jumbo Virus, Infecting Ochrobactrum spp.
Int. J. Mol. Sci. 2020, 21(6), 2096; https://doi.org/10.3390/ijms21062096 - 18 Mar 2020
Cited by 2 | Viewed by 1748
Abstract
The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an [...] Read more.
The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an insightful analysis of that bacterium, its susceptibility to bacteriophages present in WWTPs has not been evaluated. Using raw sewage sample from WWTP and applying the enrichment method, two virulent phages, vB_OspM_OC and vB_OspP_OH, which infect the POC9 strain, were isolated. These are the first virulent phages infecting Ochrobactrum spp. identified so far. Both phages were subjected to thorough functional and genomic analyses, which allowed classification of the vB_OspM_OC virus as a novel jumbo phage, with a genome size of over 227 kb. This phage encodes DNA methyltransferase, which mimics the specificity of cell cycle regulated CcrM methylase, a component of the epigenetic regulatory circuits in Alphaproteobacteria. In this study, an analysis of the overall diversity of Ochrobactrum-specific (pro)phages retrieved from databases and extracted in silico from bacterial genomes was also performed. Complex genome mining allowed us to build similarity networks to compare 281 Ochrobactrum-specific viruses. Analyses of the obtained networks revealed a high diversity of Ochrobactrum phages and their dissimilarity to the viruses infecting other bacteria. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda NcI Prophages
Int. J. Mol. Sci. 2020, 21(5), 1667; https://doi.org/10.3390/ijms21051667 - 28 Feb 2020
Viewed by 1041
Abstract
The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, [...] Read more.
The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, is a transcriptional antiterminator, required for expression of the terminator-distal genes, and thus, λ N mutants are growth-defective. When E. coli is infected with a λ double mutant that is defective in both N and cI (i.e., λN-cI-), at high multiplicities of 50 or more, it forms polylysogens that contain 20–30 copies of the λN-cI- genome integrated in the E. coli chromosome. Early studies revealed that the polylysogens underwent “conversion” to long filamentous cells that form tiny colonies on agar. Here, we report a large set of altered biochemical properties associated with this conversion, documenting an overall degeneration of the bacterial envelope. These properties reverted back to those of nonlysogenic E. coli as the metastable polylysogen spontaneously lost the λN-cI- genomes, suggesting that conversion is a direct result of the multiple copies of the prophage. Preliminary attempts to identify lambda genes that may be responsible for conversion ruled out several candidates, implicating a potentially novel lambda function that awaits further studies. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent
Int. J. Mol. Sci. 2020, 21(4), 1403; https://doi.org/10.3390/ijms21041403 - 19 Feb 2020
Cited by 12 | Viewed by 1400
Abstract
Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been [...] Read more.
Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage—vB_BbrP_BB8—of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Molecular Profile of Phage Infection: A Novel Approach for the Characterization of Erwinia Phages through qPCR
Int. J. Mol. Sci. 2020, 21(2), 553; https://doi.org/10.3390/ijms21020553 - 15 Jan 2020
Cited by 8 | Viewed by 1624
Abstract
Due to the emergence of antibiotic resistance, phage-mediated biocontrol has become an attractive alternative for pathogen management in agriculture. While the infection characteristics of many phages can be adequately described using plaque assays and optical density, the results from phages of the apple [...] Read more.
Due to the emergence of antibiotic resistance, phage-mediated biocontrol has become an attractive alternative for pathogen management in agriculture. While the infection characteristics of many phages can be adequately described using plaque assays and optical density, the results from phages of the apple pathogen Erwinia amylovora have low reproducibility with these techniques. Using quantitative real-time PCR (qPCR), the stage of the lytic cycle was determined through a combination of chloroform-based sampling, centrifugation, and DNase treatment. Monitoring the transition of phage genomes through the lytic cycle generates a molecular profile from which phage infection characteristics such as adsorption rate and burst size can be determined. To our knowledge, this is the first report of qPCR being used to determine these infection parameters. The characteristics of four different genera of Erwinia phages were determined. The phage ΦEa46-1-A1 was able to adsorb at a rate up to 6.6 times faster than ΦEa35-70 and ΦEa9-2. The low enrichment titer of ΦEa9-2 was shown to be due to the absence of lysis. The ΦEa46-1-A1 and ΦEa21-4 phages had the highest productivity, with burst sizes of 57 virions in 38 min and 185 virions in 98 min, respectively, suggesting these genera would make stronger candidates for the phage-mediated biocontrol of E. amylovora. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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Article
Isolation of Four Lytic Phages Infecting Klebsiella pneumoniae K22 Clinical Isolates from Spain
Int. J. Mol. Sci. 2020, 21(2), 425; https://doi.org/10.3390/ijms21020425 - 09 Jan 2020
Cited by 10 | Viewed by 2310
Abstract
The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to [...] Read more.
The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae. Full article
(This article belongs to the Special Issue Bacteriophage—Molecular Studies)
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