Bacteriophage host range evolution through engineered enrichment bias,exploiting heterologous surface receptor expression

Research on the initial phage–host interaction has been conducted on a limited repertoire of phages and their cognate receptors, such as phage λ and the Escherichia coli LamB (EcLamB) protein. Apart from phage λ, little is known about other phages that target EcLamB. Here, we developed a simple method for isolating novel environmental phages in a predictable way, i.e. isolating phages that target a particular receptor(s) of a bacterium, in this case, the EcLamB protein. A plasmid (pMUT13) encoding the EcLamB porin was transferred into three different enterobacterial genera. By enrichment with these engineered bacteria, a number of phages (ZZ phages) that targeted EcLamB were easily isolated from the environment. Interestingly, although EcLamB-dependent in their recombinant heterologous hosts, these newly isolated ZZ phages also targeted OmpC as an alternative receptor when infecting E. coli. Moreover, the phage host range was readily extended within three different bacterial genera with heterologously expressed EcLamB. Unlike phage λ, which is a member of the Siphoviridae family, these newly isolated EcLamB-dependent phages were more commonly members of the Myoviridae family, based on transmission electron microscopy and genomic sequences. Modifications of this convenient and efficient phage enrichment method could be useful for the discovery of novel phages.     

Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-d-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.     

Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.     

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.     

Bacteriophage isolated from non-target bacteria demonstrates broad host range infectivity against multidrug-resistant bacteria

Antibiotic resistance represents a global health challenge. The emergence of multidrug-resistant (MDR) bacteria such as uropathogenic Escherichia coli (UPEC) has attracted significant attention due to increased MDR properties, even against the last line of antibiotics. Bacteriophage, or simply phage, represents an alternative treatment to antibiotics. However, phage applications still face some challenges, such as host range specificity and development of phage resistant mutants. In this study, using both UPEC and non-UPEC hosts, five different phages were isolated from wastewater. We found that the inclusion of commensal Escherichia coli as target hosts during screening improved the capacity to select phage with desirable characteristics for phage therapy. Whole-genome sequencing revealed that four out of five phages adopt strictly lytic lifestyles and are taxonomically related to different phage families belonging to the Myoviridae and Podoviridae. In comparison to single phage treatment, the application of phage cocktails targeting different cell surface receptors significantly enhanced the suppression of UPEC hosts. The emergence of phage-resistant mutants after single phage treatment was attributed to mutational changes in outer membrane protein components, suggesting the potential receptors recognized by these phages. The findings highlight the use of commensal E. coli as target hosts to isolate broad host range phage with infectivity against MDR bacteria.     

Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage–host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage–host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages’ host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

This study presents the BASEL collection of phages that infect the model bacterium Escherichia coli; this resource for the community is representative of natural E. coli phage diversity and has been extensively characterized phenotypically and genomically.     

Bacteriophage Resistance Conferred on Lactic Streptococci by the Conjugative Plasmid pTR2030: Effects on Small Isometric-, Large Isometric-, and Prolate-Headed Phages

A series of reactions between phages, sensitive hosts, and transconjugants where the sensitivity of small isometric-, large isometric-, and prolate-headed phages to pTR2030-induced phage resistance was evaluated in Streptococcus lactis and Streptococcus cremoris strains. Phage-resistant transconjugants were constructed in the desired host by conjugal transfer of lactose-fermenting ability (Lac⁺, pTR1040) and phage resistance (Hsp⁺, pTR2030) from S. lactis TEK1. S. lactis and S. cremoris transconjugants harboring pTR2030 were resistant to all small isometric-headed phages examined. In contrast, prolate- and large isometric-headed phages were either not inhibited in the pTR2030 transconjugants or exhibited a reduction in plaque size without a reduction in the efficiency of plaquing. Small isometric-headed phages subject to pTR2030 induced inhibition shared no significant DNA homology with pTR2030, suggesting that phage immunity genes are not harbored on the plasmid or responsible for resistance. The general effectiveness of pTR2030 against small isometric-headed phages was highly significant since these are the phages which have been isolated most commonly from dairy fermentation plants.     

Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the Leuconostoc genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50 Leuconostoc pseudomesenteroides [Ln. pseudomesenteroides] phages, 4 Ln. mesenteroides phages) and from 3 external phage collections (17 Ln. pseudomesenteroides phages, 12 Ln. mesenteroides phages). All phages belonged to the Siphoviridae family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all Ln. mesenteroides phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all Ln. pseudomesenteroides phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All Ln. mesenteroides and all Ln. pseudomesenteroides phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all Leuconostoc phage types.     

Genomic Investigation of Lysogen Formation and Host Lysis Systems of the Salmonella Temperate Bacteriophage SPN9CC

To understand phage infection and host cell lysis mechanisms in pathogenic Salmonella, a novel Salmonella enterica serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the Podoviridae family was isolated and characterized. The phage infects S. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and Escherichia coli hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.     

Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.     

Isolation and characterization of phage–host systems from the Baltic Sea ice

In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.     

Vibriophages and Their Interactions with the Fish Pathogen Vibrio anguillarum

Vibrio anguillarum is an important pathogen in aquaculture, responsible for the disease vibriosis in many fish and invertebrate species. Disease control by antibiotics is a concern due to potential development and spread of antibiotic resistance. The use of bacteriophages to control the pathogen may offer a non-antibiotic-based approach to reduce vibriosis. A detailed understanding of the phage-host interaction is needed to evaluate the potential of phages to control the pathogen. In this study, we examined the diversity and interactions of 11 vibriophages, 24 V. anguillarum strains, and 13 Vibrio species strains. Together, the host ranges of the 11 phages covered all of the tested 37 Vibrio sp. host strains, which represented considerable temporal (20 years) and geographical (9 countries) differences in their origins of isolation. Thus, despite the occurrence of unique susceptibility patterns of the individual host isolates, key phenotypic properties related to phage susceptibility are distributed worldwide and maintained in the global Vibrio community for decades. The phage susceptibility pattern of the isolates did not show any relation to the physiological relationships obtained from Biolog GN2 profiles, demonstrating that similar phage susceptibility patterns occur across broad phylogenetic and physiological differences in Vibrio strains. Subsequent culture experiments with two phages and two V. anguillarum hosts demonstrated an initial strong lytic potential of the phages. However, rapid regrowth of both phage-resistant and phage-sensitive cells following the initial lysis suggested that several mechanisms of protection against phage infection had developed in the host populations.     

Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.     

Escherichia coli phage receptors. Minor porins and proteins participating in the specific transport as phage receptors

Except for the main porin proteins OmpC and OmpF there exist the membrane proteins participating in the transport of specific substrates: phosphates, nucleosides, iron, vitamin B12, maltose and maltodextrins, that also play the role of phage receptors. Some phages use as receptors the porins determined by the genes of lambdoid prophages. LamB protein that serves receptor for phage lambda exposes the amino acids sequence on the outer surface of membranes that participates in phage adsorption. The sequence is similar to tetrapeptide of fibronectin responsible for binding with the surface of cellular receptor in eucaryotes.     

Selection and Characterization of Cyanophage Resistance in Marine Synechococcus Strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

The costs of evolving resistance in heterogeneous parasite environments

The evolution of host resistance to parasites, shaped by associated fitness costs, is crucial for epidemiology and maintenance of genetic diversity. Selection imposed by multiple parasites could be a particularly strong constraint, as hosts either accumulate costs of multiple specific resistances or evolve a more costly general resistance mechanism. We used experimental evolution to test how parasite heterogeneity influences the evolution of host resistance. We show that bacterial host populations evolved specific resistance to local bacteriophage parasites, regardless of whether they were in single or multiple-phage environments, and that hosts evolving with multiple phages were no more resistant to novel phages than those evolving with single phages. However, hosts from multiple-phage environments paid a higher cost, in terms of population growth in the absence of phage, for their evolved specific resistances than those from single-phage environments. Given that in nature host populations face selection pressures from multiple parasite strains and species, our results suggest that costs may be even more critical in shaping the evolution of resistance than previously thought. Furthermore, our results highlight that a better understanding of resistance costs under combined control strategies could lead to a more 'evolution-resistant' treatment of disease.