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21 pages, 8384 KiB

Open AccessArticle

Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis

by Alla K. Golomidova, Eugene E. Kulikov, Nikolai S. Prokhorov, Ricardo С. Guerrero-Ferreira, Yuriy A. Knirel, Elena S. Kostryukova, Karina K. Tarasyan and Andrey V. Letarov

Viruses 2016, 8(1), 26; https://doi.org/10.3390/v8010026 - 21 Jan 2016

Cited by 51 | Viewed by 8425

Abstract

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and [...] Read more.

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host’s O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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9 pages, 1700 KiB

Open AccessCommunication

Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages

by David J. Simpson, Jessica C. Sacher and Christine M. Szymanski

Viruses 2016, 8(1), 17; https://doi.org/10.3390/v8010017 - 11 Jan 2016

Cited by 30 | Viewed by 8516

Abstract

Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs [...] Read more.

Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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14 pages, 9041 KiB

Open AccessCommunication

Development of Phage Lysin LysA2 for Use in Improved Purity Assays for Live Biotherapeutic Products

by Sheila M. Dreher-Lesnick, Jeremy E. Schreier and Scott Stibitz

Viruses 2015, 7(12), 6675-6688; https://doi.org/10.3390/v7122965 - 16 Dec 2015

Cited by 11 | Viewed by 11611

Abstract

Live biotherapeutic products (LBPs), commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, [...] Read more.

Live biotherapeutic products (LBPs), commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, but there is also growing interest in evaluating such products for treatment of specific diseases. While over-the-counter probiotics are generally viewed as very safe, at least in healthy individuals, it must be remembered that clinical studies to assess these products may be done in individuals whose defenses are compromised, such as through a disease process, immunosuppressive clinical treatment, or an immature or aging immune system. One of the major safety criteria for LBPs used in clinical studies is microbial purity, i.e., the absence of extraneous, undesirable microorganisms. The main goal of this project is to develop recombinant phage lysins as reagents for improved purity assays for LBPs. Phage lysins are hydrolytic enzymes containing a cell binding domain that provides specificity and a catalytic domain responsible for lysis and killing. Our approach is to use recombinant phage lysins to selectively kill target product bacteria, which when used for purity assays will allow for outgrowth of potential contaminants under non-selective conditions, thus allowing an unbiased assessment of the presence of contaminants. To develop our approach, we used LysA2, a phage lysin with reported activity against a broad range of Lactobacillus species. We report the lytic profile of a non-tagged recombinant LysA2 against Lactobacillus strains in our collection. We also present a proof-of-concept experiment, showing that addition of partially purified LysA2 to a culture of Lactobacillus jensenii (L. jensenii) spiked with low numbers of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus ) effectively eliminates or knocks down L. jensenii, allowing for clear detection of the contaminating strains. With continued identification and characterization of phage lysins, we hope that the use of recombinant phage lysins in purity assays for products containing live microbials may offer additional tools to help advance product development of LBPs. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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14 pages, 3202 KiB

Open AccessArticle

A Flagellar Glycan-Specific Protein Encoded by Campylobacter Phages Inhibits Host Cell Growth

by Muhammad Afzal Javed, Jessica C. Sacher, Lieke B. Van Alphen, Robert T. Patry and Christine M. Szymanski

Viruses 2015, 7(12), 6661-6674; https://doi.org/10.3390/v7122964 - 16 Dec 2015

Cited by 14 | Viewed by 6363

Abstract

We previously characterized a carbohydrate binding protein, Gp047, derived from lytic Campylobacter phage NCTC 12673, as a promising diagnostic tool for the identification of Campylobacter jejuni and Campylobacter coli. We also demonstrated that this protein binds specifically to acetamidino-modified pseudaminic acid residues [...] Read more.

We previously characterized a carbohydrate binding protein, Gp047, derived from lytic Campylobacter phage NCTC 12673, as a promising diagnostic tool for the identification of Campylobacter jejuni and Campylobacter coli. We also demonstrated that this protein binds specifically to acetamidino-modified pseudaminic acid residues on host flagella, but the role of this protein in the phage lifecycle remains unknown. Here, we report that Gp047 is capable of inhibiting C. jejuni growth both on solid and liquid media, an activity, which we found to be bacteriostatic. The Gp047 domain responsible for bacterial growth inhibition is localized to the C-terminal quarter of the protein, and this activity is both contact- and dose-dependent. Gp047 gene homologues are present in all Campylobacter phages sequenced to date, and the resulting protein is not part of the phage particle. Therefore, these results suggest that either phages of this pathogen have evolved an effector protein capable of host-specific growth inhibition, or that Campylobacter cells have developed a mechanism of regulating their growth upon sensing an impending phage threat. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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11 pages, 4552 KiB

Open AccessArticle

Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

by Nicholas R. Stambach, Stephanie A. Carr, Christopher R. Cox and Kent J. Voorhees

Viruses 2015, 7(12), 6631-6641; https://doi.org/10.3390/v7122962 - 14 Dec 2015

Cited by 44 | Viewed by 9114

Abstract

A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. [...] Read more.

A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 10⁶ pfu·mL⁻¹, offering detection below that obtainable by the naked eye (LOD 6 × 10⁷ pfu·mL⁻¹). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 10⁷ colony forming units (cfu)·mL⁻¹, 5 × 10⁶ cfu·mL⁻¹, 5 × 10⁵ cfu·mL⁻¹ and 5 × 10⁴ cfu·mL⁻¹ was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 10⁵ pfu·mL⁻¹) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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20 pages, 714 KiB

Open AccessArticle

What Can We Learn from a Metagenomic Analysis of a Georgian Bacteriophage Cocktail?

by Henrike Zschach, Katrine G. Joensen, Barbara Lindhard, Ole Lund, Marina Goderdzishvili, Irina Chkonia, Guliko Jgenti, Nino Kvatadze, Zemphira Alavidze, Elizabeth M. Kutter, Henrik Hasman and Mette V. Larsen

Viruses 2015, 7(12), 6570-6589; https://doi.org/10.3390/v7122958 - 12 Dec 2015

Cited by 40 | Viewed by 11938

Abstract

Phage therapy, a practice widespread in Eastern Europe, has untapped potential in the combat against antibiotic-resistant bacterial infections. However, technology transfer to Western medicine is proving challenging. Bioinformatics analysis could help to facilitate this endeavor. In the present study, the Intesti phage cocktail, [...] Read more.

Phage therapy, a practice widespread in Eastern Europe, has untapped potential in the combat against antibiotic-resistant bacterial infections. However, technology transfer to Western medicine is proving challenging. Bioinformatics analysis could help to facilitate this endeavor. In the present study, the Intesti phage cocktail, a key commercial product of the Eliava Institute, Georgia, has been tested on a selection of bacterial strains, sequenced as a metagenomic sample, de novo assembled and analyzed by bioinformatics methods. Furthermore, eight bacterial host strains were infected with the cocktail and the resulting lysates sequenced and compared to the unamplified cocktail. The analysis identified 23 major phage clusters in different abundances in the cocktail, among those clusters related to the ICTV genera T4likevirus, T5likevirus, T7likevirus, Chilikevirus and Twortlikevirus, as well as a cluster that was quite distant to the database sequences and a novel Proteus phage cluster. Examination of the depth of coverage showed the clusters to have different abundances within the cocktail. The cocktail was found to be composed primarily of Myoviridae (35%) and Siphoviridae (32%), with Podoviridae being a minority (15%). No undesirable genes were found. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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14 pages, 2835 KiB

Open AccessArticle

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

by Amanda Donnelly, Teerapong Yata, Kaoutar Bentayebi, Keittisak Suwan and Amin Hajitou

Viruses 2015, 7(12), 6476-6489; https://doi.org/10.3390/v7122951 - 8 Dec 2015

Cited by 10 | Viewed by 8135

Abstract

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential [...] Read more.

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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17 pages, 4760 KiB

Open AccessArticle

Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone

by Carmela Garcia-Doval, José R. Castón, Daniel Luque, Meritxell Granell, José M. Otero, Antonio L. Llamas-Saiz, Madalena Renouard, Pascale Boulanger and Mark J. Van Raaij

Viruses 2015, 7(12), 6424-6440; https://doi.org/10.3390/v7122946 - 8 Dec 2015

Cited by 40 | Viewed by 8387

Abstract

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the [...] Read more.

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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18 pages, 2144 KiB

Open AccessArticle

Bio-Control of Salmonella Enteritidis in Foods Using Bacteriophages

by Hongduo Bao, Pengyu Zhang, Hui Zhang, Yan Zhou, Lili Zhang and Ran Wang

Viruses 2015, 7(8), 4836-4853; https://doi.org/10.3390/v7082847 - 24 Aug 2015

Cited by 148 | Viewed by 13148

Abstract

Two lytic phages, vB_SenM-PA13076 (PA13076) and vB_SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages [...] Read more.

Two lytic phages, vB_SenM-PA13076 (PA13076) and vB_SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages in liquid culture showed 104 multiplicity of infection (MOI) was the best in inhibiting bacteria, with PC2184 exhibiting more activity than PA13076. The two phages exhibited broad host range within the genus Salmonella. Phage PA13076 and PC2184 had a lytic effect on 222 (71.4%) and 298 (95.8%) of the 311 epidemic Salmonella isolates, respectively. We tested the effectiveness of phage PA13076 and PC2184 as well as a cocktail combination of both in three different foods (chicken breast, pasteurized whole milk and Chinese cabbage) contaminated with SE. Samples were spiked with 1 × 10⁴ CFU individual SE or a mixture of strains (ATCC13076 and CVCC2184), then treated with 1 × 10⁸ PFU individual phage or a two phage cocktail, and incubated at 4 °C or 25 °C for 5 h. In general, the inhibitory effect of phage and phage cocktail was better at 4 °C than that at 25 °C, whereas the opposite result was observed in Chinese cabbage, and phage cocktail was better than either single phage. A significant reduction in bacterial numbers (1.5–4 log CFU/sample, p < 0.05) was observed in all tested foods. The two phages on the three food samples were relatively stable, especially at 4 ºC, with the phages exhibiting the greatest stability in milk. Our research shows that our phages have potential effectiveness as a bio-control agent of Salmonella in foods. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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17 pages, 636 KiB

Open AccessArticle

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

by Joanna Majewska, Weronika Beta, Dorota Lecion, Katarzyna Hodyra-Stefaniak, Anna Kłopot, Zuzanna Kaźmierczak, Paulina Miernikiewicz, Agnieszka Piotrowicz, Jarosław Ciekot, Barbara Owczarek, Agnieszka Kopciuch, Karolina Wojtyna, Marek Harhala, Mateusz Mąkosa and Krystyna Dąbrowska

Viruses 2015, 7(8), 4783-4799; https://doi.org/10.3390/v7082845 - 20 Aug 2015

Cited by 137 | Viewed by 12640

Abstract

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 [...] Read more.

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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31 pages, 4281 KiB

Open AccessArticle

Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4

by Sergey A. Buth, Laure Menin, Mikhail M. Shneider, Jürgen Engel, Sergei P. Boudko and Petr G. Leiman

Viruses 2015, 7(8), 4676-4706; https://doi.org/10.3390/v7082839 - 18 Aug 2015

Cited by 9 | Viewed by 8240

Abstract

Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender β-helix that is formed by three polypeptide chains wrapped around a [...] Read more.

Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender β-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helix is small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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22 pages, 2543 KiB

Open AccessArticle

Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

by Angela V. Holguín, Guillermo Rangel, Viviana Clavijo, Catalina Prada, Marcela Mantilla, María Catalina Gomez, Elizabeth Kutter, Corinda Taylor, Peter C. Fineran, Andrés Fernando González Barrios and Martha J. Vives

Viruses 2015, 7(8), 4602-4623; https://doi.org/10.3390/v7082835 - 12 Aug 2015

Cited by 46 | Viewed by 9452

Abstract

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability [...] Read more.

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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19 pages, 1241 KiB

Open AccessArticle

Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

by Rebekah A. Frampton, Elena Lopez Acedo, Vivienne L. Young, Danni Chen, Brian Tong, Corinda Taylor, Richard A. Easingwood, Andrew R. Pitman, Torsten Kleffmann, Mihnea Bostina and Peter C. Fineran

Viruses 2015, 7(7), 3361-3379; https://doi.org/10.3390/v7072776 - 24 Jun 2015

Cited by 27 | Viewed by 8716

Abstract

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use [...] Read more.

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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16 pages, 848 KiB

Open AccessArticle

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

by Steven M. Swift, Bruce S. Seal, Johnna K. Garrish, Brian B. Oakley, Kelli Hiett, Hung-Yueh Yeh, Rebekah Woolsey, Kathleen M. Schegg, John Eric Line and David M. Donovan

Viruses 2015, 7(6), 3019-3034; https://doi.org/10.3390/v7062758 - 12 Jun 2015

Cited by 52 | Viewed by 9584

Abstract

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added [...] Read more.

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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23 pages, 2256 KiB

Open AccessArticle

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

by Jens Andre Hammerl, Nicole Roschanski, Rudi Lurz, Reimar Johne, Erich Lanka and Stefan Hertwig

Viruses 2015, 7(6), 2771-2793; https://doi.org/10.3390/v7062746 - 2 Jun 2015

Cited by 6 | Viewed by 6659

Abstract

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a [...] Read more.

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O(_{ m{R}})3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O(_{ m{R}})3) upstream of the prophage repressor gene. The O(_{ m{R}})3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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19 pages, 1328 KiB

Open AccessArticle

by Harald Eriksson, Barbara Maciejewska, Agnieszka Latka, Grazyna Majkowska-Skrobek, Marios Hellstrand, Öjar Melefors, Jin-Town Wang, Andrew M. Kropinski, Zuzanna Drulis-Kawa and Anders S. Nilsson

Viruses 2015, 7(4), 1804-1822; https://doi.org/10.3390/v7041804 - 7 Apr 2015

Cited by 38 | Viewed by 10209

Abstract

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and [...] Read more.

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1. Full article

(This article belongs to the Special Issue 21st Evergreen International Phage Biology Meeting - Emerging Research)

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