Special Issue "Recent Progress in Bacteriophage Research"

Guest Editor
Prof. Dr. Graham F. Hatfull
Chair, Department of Biological Sciences, HHMI Professor, Eberly Family Professor of Biotechnology, 376 Crawford Hall, Department of Biological Sciences, University of Pittsburgh, 4249 5th Avenue, Pittsburgh, PA 15260
Website: http://www.pitt.edu/~gfh/
E-Mail: gfh@pitt.edu
Phone: Tel: (412) 624 4350
Fax: FAX: (412) 624 4870

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Andrew Chibeu, Erika J. Lingohr, Luke Masson, Amee Manges, Josée Harel, Hans-W. Ackermann, Andrew M. Kropinski and Patrick Boerlin Article: Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms Viruses 2012, 4(4), 471-487; doi:10.3390/v4040471 Received: 16 February 2012; in revised form: 20 March 2012 / Accepted: 23 March 2012 / Published: 10 April 2012 Show/Hide Abstract | Download PDF Full-text (1128 KB) | View HTML Full-text | Download PMC-XML Full-text |  Abstract: Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages’ genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2–12 h of incubation.

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Stephen T. Abedon Article: Spatial Vulnerability: Bacterial Arrangements, Microcolonies, and Biofilms as Responses to Low Rather than High Phage Densities Viruses 2012, 4(5), 663-687; doi:10.3390/v4050663 Received: 2 February 2012; in revised form: 13 April 2012 / Accepted: 19 April 2012 / Published: 26 April 2012 Show/Hide Abstract | Download PDF Full-text (453 KB) | View HTML Full-text | Download PMC-XML Full-text Abstract: The ability of bacteria to survive and propagate can be dramatically reduced upon exposure to lytic bacteriophages. Study of this impact, from a bacterium’s perspective, tends to focus on phage-bacterial interactions that are governed by mass action, such as can be observed within continuous flow or similarly planktonic ecosystems. Alternatively, bacterial molecular properties can be examined, such as specific phage‑resistance adaptations. In this study I address instead how limitations on bacterial movement, resulting in the formation of cellular arrangements, microcolonies, or biofilms, could increase the vulnerability of bacteria to phages. Principally: (1) Physically associated clonal groupings of bacteria can represent larger targets for phage adsorption than individual bacteria; and (2), due to a combination of proximity and similar phage susceptibility, individual bacteria should be especially vulnerable to phages infecting within the same clonal, bacterial grouping. Consistent with particle transport theory—the physics of movement within fluids—these considerations are suggestive that formation into arrangements, microcolonies, or biofilms could be either less profitable to bacteria when phage predation pressure is high or require more effective phage-resistance mechanisms than seen among bacteria not living within clonal clusters. I consider these ideas of bacterial ‘spatial vulnerability’ in part within a phage therapy context.

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Type of Paper: Review
Title: Photodynamic inactivation of bacteriophages as surrogates for mammalian viruses
Authors: Liliana Costa ¹, Maria Amparo F. Faustino ², Maria Graça P. M. S. Neves ², Ângela Cunha ¹, Adelaide Almeida ^1,*
Affiliations: ¹ Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal; E-Mails: lcosta@ua.pt (L.C.); acunha@ua.pt (A.C.); ² Department of Chemistry and QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal; E-Mails: faustino@ua.pt (M.A.F.F.); gneves@ua.pt (M.G.P.M.S.N.);* Author to whom correspondence should be addressed; E-Mail: aalmeida@ua.pt; Tel.: +351-234-370-350; Fax: +351-234-372-587.
Abstract: Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of bacteriophages by photosensitization was applied with success since the first decades of the last century. However, due to the fact that bacteriophages are frequently used as models of enteric viruses, which are known to pose a threat to public health, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process
Keywords: bacteriophages; mammalian viruses; photodynamic therapy; photosensitizer; viral photoinactivation process

Type of Paper: Review
Title: CRISPR/Cas-based Phage Resistance Mechanisms and their Regulation
Authors: Corinna Richter, James T. Chang and Peter C. Fineran
Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand; E-mail: peter.fineran@otago.ac.nz
Abstract: Phage are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts by participating in an estimated 10²⁵ infections per second. Thus, bacteria have evolved numerous ‘innate’ mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the newly discovered CRISPR/Cas systems provide acquired, yet heritable, sequence-specific ‘adaptive’ resistance against phage. Resistance is acquired following viral infection when a short sequence of phage genome is added to the CRISPR array. CRISPRs are then transcribed and processed by Cas proteins into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of the CRISPR/Cas mechanism. In this review, we will present the current model(s) of the molecular events involved in the acquisition of immunity and interference and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

Type of Paper: Article
Title: Adaptive Trajectories of Thermotolerance Evolvability in the RNA phage phi-6
Authors: Daniel Goldhill and Paul E. Turner
Affiliation: Dept. Ecology and Evolutionary Biology, Yale University, New Haven, CT  06520;  http://www.yale.edu/turner/home/index.htm; E-Mail: paul.turner@yale.edu; daniel.goldhill@yale.edu
Abstract: Genetic robustness is phenotypic constancy, despite mutational input. The RNA phage phi-6 was shown to evolve decreased robustness when grown for hundreds of generations under frequent co-infection; because complementation (buffering of mutations) occurred under co-infection, viruses experienced weakened selection to retain individual-level capacity for robustness. It was later shown that lineages founded by robust genotypes of phage phi-6 evolved faster under heat-shock selection, demonstrating that a link exists between robustness and evolvability of thermotolerance. However, it is unknown whether robust and brittle populations of the virus both harbor genotypes capable of rapid evolution under heat-shock.  To examine the effect of standing genetic variation on evolvability of thermotolerance, we manipulated initial population size of robust and brittle viruses and examined their rate and extent of adaptation under heat-shock conditions. We showed that robust populations were relatively advantaged in evolvability of thermotolerance at all population sizes, suggesting that increased standing genetic variation does not allow brittle populations to keep “adaptive pace” with their robust counterparts. In addition, we used whole-genome sequencing to identify molecular changes that separate thermotolerant viruses from their ancestors, suggesting candidate loci that may explain the evolution of heat-shock resistance in phage phi-6.

Type of Paper: Article
Title: Networks of Shared Genes in 1,000 Bacteriophage Genomes
Authors: David M. Kristensen¹, Arcady R. Mushegian^2,3, Eugene V. Koonin¹
Affiliations: ¹ National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA; ² Stowers Institute for Medical Research, Kansas City, MO 64110, USA; ³ Department of Microbiology, Molecular Genetics and Immunology, Kansas University Medical Center, Kansas City, KS 66160, USA
Abstract: Despite the fact that bacteriophages are extremely active players in the global ecosystem, as well as the most abundant biological entities on the planet, much remains unknown about how these viruses function in their natural environments. To facilitate their comparative study, we assembled a collection of >2,000 orthologous gene clusters from 500 double-stranded DNA (dsDNA) phages (Phage Orthologous Groups, or POGs). These gene families are delineated by examining top-ranked sequence similarities between proteins in complete genomes without the use of arbitrary similarity cutoffs, and thus represent a natural classification system for fast-evolving and slow-evolving proteins alike. Despite the high sensitivity of the approach, more than half of the POGs were found to have no or very few evolutionary connections to their cellular hosts, indicating that dsDNA phages combine the ability to share and transduce host genes with the ability to maintain a large fraction of unique, phage-specific genes. These genes are useful for targeted research strategies, e.g. as fundamental units of systems biology studies or diagnostic markers.
In order to maintain their utility, the POGs were designed to keep pace with the rapid and accelerating pace of growth of full-genome information from sequencing projects, which have recently reached the point at which genomes are available for over 1,000 distinct bacteriophages. Although likely the vast majority of bacteriophages remain to be discovered, given the frequency at which novel genes are continuously discovered in newly-sequenced genomes, analysis of this new genomic collection allows glimpses of an unprecedented level of detail into the molecular evolution of these highly diverse viruses. In addition to incorporating the latest genomes into the publicly available POGs, we have also extended the scope of the database to include single-stranded DNA (ssDNA) as well as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) phage genomes and unclassified phages—in all, more than doubling the number of genomes included since the last release of the POGs. In addition to re-visiting the previously revealed trends in phage genome evolution with this substantially larger POGs database, we present a network analysis of genes shared between various groups of phages. For instance, even though no single gene is found in even a third of all phages, all dsDNA genomes are nevertheless joined together in a giant connected network such that it is possible to trace a path between every genome and every other genome via intermediates that share at least one gene. Leviviridae (ssRNA), Cystoviridae (dsRNA), and Microviridae (ssDNA) likewise form their own connected networks, albeit separated from each other and from that of dsDNA phages at the level of sequence similarity used, although some of the network of Inoviridae (ssDNA) connects to that of the dsDNA phages. Despite the existence of paths between large clusters of phage genomes in the network, analysis of connections that are more likely to be due to the vertical transfer of genetic material from parent to offspring, rather than horizontal gene transfer, reveals the presence of clusters that at least roughly correspond to existing taxa, thus revealing potential for bacteriophage taxonomy schemes that use genomic information rather than purely structural characteristics.

Type of Paper: Article
Title: Mechanistic Roles of the Portal Vertex of the Bacteriophage DNA Packaging Machine
Authors: Song Gao, Bonnie Draper, and Venigalla B. Rao
Affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064, USA.
Abstract: In the assembly of tailed bacteriophages, the viral genome is translocated into a pre-formed capsid by a powerful packaging machine consisting of a pentameric ATP-driven motor and a dodecameric portal channel. Although the essential roles of the motor have been well established, the functions of the portal beyond serving as a DNA conduit are not well understood. Here we present recent structural, biochemical and genetic findings that suggest various mechanistic roles of the portal, as packaging sensor, ATPase coupler, DNA cruncher, or one-way valve. Evidence that bear on these mechanisms are analyzed and discussed in the context of other translocation channels such as FstK/SpoEIII and SecYEG.

Type of Paper: Article
Title: to be added
Authors: Megan Hargreaves and Phillipa Perrot
Affiliation: Science & Engineering Faculty, Queensland University of Technology, Podium room O330, 2 George St, Brisbane, QLD, 4001, E-Mail: p.perrott@qut.edu.au
Abstract: Bacteriophages have played an important part in various types of research, with widely varying purposes. One such area in which their use has been of great significance is their use as models or surrogates. One example of this the use of the E.coli bacteriophage MS2, in studies examining respiratory viruses such as influenza, as it has a similar size and shape, and similar aerosol characteristics to respiratory viruses. This use of bacteriophages in this application is important because they do not pose serious health risks and are relatively easy to propagate in the laboratory. They are also easier to detect and quantify from aerosols than human viruses, which require complicated tissue culture and often does not allow all viable viruses to propagate on the medium, thus giving a false result. The use of MS2 in conjunction with its relatively simple culture techniques allowed the investigation of whether or not viruses remain viable after aerosolisation. Molecular techniques were also used to determine presence of MS2 in the aerosols, in order to then apply these methods to the detection of influenza following aerosolisation. A specialised PCR method was developed for this study in that could detect and quantify MS2 bacteriophage, and subsequently, influenza viruses, in aerosols. This study investigated aerosol characteristics of MS2 in great depth, investigating environmental factors and collection techniques. Additionally, other characteristics were explored, such as factors which impact on infectivity rates of MS2 when held in varying solutions.

Type of Paper: Review
Title: Cell Biology of Bacteriophage Infection
Authors: Lina Jakutyte and Paulo Tavares
Affiliation: Unité de Virologie Moléculaire et Structurale, UPR3296 CNRS and IFR115, Bâtiment 14B, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France
Abstract: During the long co-evolution of viruses and cells, viruses exploited numerous ways to hijack cell machineries for their efficient multiplication and dissemination. Studies on bacterial viruses (bacteriophages or phages) provided a detailed description of the biochemical reactions engaged during the different steps of virus multiplication. However, their temporal program and spatial distribution in the cell remain poorly characterized. Here we review recent progresses on the cell biology of phage entry in the bacterium, gene expression decision, and genome replication. They provide a real time view of the infection cycle progress in vivo and show how phages exploit architectural properties of the “structured bacterial cell” for efficient infection and multiplication. This novel field of research is of significant importance to understand infection and to investigate cellular processes targeted by the virus.
Keywords: Bacteriophage; virus infection; virus entry; bacterial envelope; viral receptor; bacterial cytoskeleton; lysogeny; viral DNA replication; virus assembly

Type of Paper: Article
Title: A Genetic Approach for Development of new Therapeutic Phages to Fight Pseudomonas Aeruginosa in Wound Infections
Authors: Krylov V.N.,  Shaburova O.V.,  Krylov S.V.,  Pleteneva E.A.
Affiliation: I.I. Mechnikov Research Institute for Vaccines & Sera, RAMS, Moscow 105064, Maly Kazenny pereulok, 5A.  Russia; E-Mail: krylov.mech.inst@mail.ru
Abstract: The emergence of  multy-drug resistant (MDR)  strains of pathogenic bacteria  has created significant difficulties in the curing of various infections, including treatment of infected wounds. As a  possible alternative approach the phage therapy has been proposed.  Indeed, infected wounds  are the perfect place for phage therapy applications, since there is ensured a direct contact of bacteriophages and bacteria that are sensitive to them, which is the basic condition for the effectiveness of this approach.
One of the frequent  participants of  wound infections is  Pseudomonas aeruginosa. Plenty of  virulent ("lytic")  and temperate ("lysogenic") bacteriophages are described for  P.aeruginosa. However, the number of  phage species which are acceptable for introduction into  the therapeutic mix  is limited. The most frequently used phages  active on P.aeruginosa are from the species of PB1 - like, KMV-like and phiKZ - like phages. Some of them, as phages of species PB1, have a high capability to modify their  spectrum of lytic activity due to appearance of mutants that can overcome the adsorptional resistance of many clinical isolates. KMV -like phages have short latent period and can lyse (at least in Petri dishes experiment) even quite old bacterial cultures (at least 3-days old). PhiKZ-like phages may be produced with very inexpensive procedures  in enormous quantities. But phiKZ -like phages being also frequent components of therapeutic mixtures, can at certain conditions to induce in infected cells the state of  pseudolysogeny what leads to formation of  highly viscous  products.
It is obvious that the permanent  use of phage therapy on the background of a high frequency of multy-phage resistant (MPR) mutant bacteria will lead to  decrease in the number of bacteriophages that could be available for therapy among the currently used natural phages.  The frequent presence  in clinical strains of P.aeruginosa of  plasmids which inhibit intracellular phage development creates an additional problem for phage therapy of wounds. Thus there is a critical need in new active phages.
In several studies we have  assessed the possibility to overcome  these difficulties. In particular, we have isolated  virulent mutants  of phiKZ-like phages which are capable to lyse bacteria without  effect of pseudolysogeny.  In addition,  mutants of several  temperate  phages  for  P.aeruginosa were isolated which can overcome   the inhibitory  effects  of  plasmids. Addition of such mutants into existing commercial  phage mixtures  expands the range of lytic activity.
As a new approach might be used the recombination of phages belonging to different species with purpose  to create  new  genome structures.  As we have shown some of  these new hybrid  phages can cause  good lysis of clinical isolates with lowered frequency of  survival of  MPR bacterial mutants.
We discuss the obtained results in frame of generally accepted dogma about the role of phages in horizontal genetic transfer (HGT).