Phage exposure causes dynamic shifts in the expression states of specific phase-variable genes of Campylobacter jejuni.

Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacterjejuni this reversible adaptive process is mediated by mutations in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase for this CPS modification, cj1421, and two other phase-variable CPS genes generated phage resistance in C. jejuni. Here we investigate the population dynamics of C. jejuni NCTC11168 when exposed to phage F336 in vitro using a newly described method - the 28-locus-CJ11168 PV analysis. Dynamic switching was observed in the ON/OFF states of three phase-variable CPS genes, cj1421, cj1422 and cj1426, during phage F336 exposure, with the dominant phage-resistant phasotype differing between cultures. Although loss of the phage receptor was predominately observed, several other PV events also led to phage resistance, a phenomenon that increases the chance of phage-resistant subpopulations being present in any growing culture. No other PV genes were affected and exposure to phage F336 resulted in a highly specific response, only selecting for phase variants of cj1421, cj1422 and cj1426. In summary, C. jejuni may benefit from modification of the surface in multiple ways to inhibit or reduce phage binding, thereby ensuring the survival of the population when exposed to phages.

Campylobacter jejuni motility is required for infection of the flagellotropic bacteriophage F341.

Previous studies have identified a specific modification of the capsular polysaccharide as receptor for phages that infect Campylobacter jejuni. Using acapsular kpsM mutants of C. jejuni strains NCTC11168 and NCTC12658, we found that bacteriophage F341 infects C. jejuni independently of the capsule. In contrast, phage F341 does not infect C. jejuni NCTC11168 mutants that either lack the flagellar filaments (ΔflaAB) or that have paralyzed, i.e., nonrotating, flagella (ΔmotA and ΔflgP). Complementing flgP confirmed that phage F341 requires rotating flagella for successful infection. Furthermore, adsorption assays demonstrated that phage F341 does not adsorb to these nonmotile C. jejuni NCTC11168 mutants. Taken together, we propose that phage F341 uses the flagellum as a receptor. Phage-host interactions were investigated using fluorescence confocal and transmission electron microscopy. These data demonstrate that F341 binds to the flagellum by perpendicular attachment with visible phage tail fibers interacting directly with the flagellum. Our data are consistent with the movement of the C. jejuni flagellum being required for F341 to travel along the filament to reach the basal body of the bacterium. The initial binding to the flagellum may cause a conformational change of the phage tail that enables DNA injection after binding to a secondary receptor.

Strategies for developing phages into novel antimicrobial tailocins.

Tailocins are high-molecular-weight bacteriocins produced by bacteria to kill related environmental competitors by binding and puncturing their target. Tailocins are promising alternative antimicrobials, yet the diversity of naturally occurring tailocins is limited. The structural similarities between phage tails and tailocins advocate using phages as scaffolds for developing new tailocins. This article reviews three strategies for producing tailocins: disrupting the capsid-tail junction of phage particles, blocking capsid assembly during phage propagation, and creating headless phage particles synthetically. Particularly appealing is the production of tailocins through synthetic biology using phages with contractile tails as scaffolds to unlock the antimicrobial potential of tailocins.

Diverse bacteriophages for biocontrol of ESBL- and AmpC-ß-lactamase-producing E. coli.

Novel solutions are needed to reduce the risk of transmission of extended spectrum ß-lactamase (ESBL) and AmpC ß-lactamase producing Escherichia coli (ESBL/AmpC E. coli) from livestock to humans. Given that phages are promising biocontrol agents, a collection of 28 phages that infect ESBL/AmpC E. coli were established. Whole genome sequencing showed that all these phages were unique and could be assigned to 15 different genera. Host range analysis showed that 82% of 198 strains, representing the genetic diversity of ESBL/AmpC E. coli, were sensitive to at least one phage. Identifying receptors used for phage binding experimentally as well as in silico predictions, allowed us to combine phages into two different cocktails with broad host range targeting diverse receptors. These phage cocktails efficiently inhibit the growth of ESBL/AmpC E. coli in vitro, thus suggesting the potential of phages as promising biocontrol agents.

A Representative Collection of Commensal Extended-Spectrum- and AmpC-ß-Lactamase-Producing Escherichia coli of Animal Origin for Phage Sensitivity Studies.

Introduction: Extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (AmpC)-producing Escherichia coli from livestock and meat represent a zoonotic risk and biocontrol solutions are needed to prevent transmission to humans. Methods: In this study, we established a representative collection of animal-origin ESBL/AmpC E. coli as target to test the antimicrobial potential of bacteriophages. Results: Bioinformatic analysis of whole-genome sequence data of 198 ESBL/AmpC E. coli from pigs, broilers, and broiler meat identified strains belonging to all known E. coli phylogroups and 65 multilocus sequence types. Various ESBL/AmpC genes and plasmid types were detected with expected source-specific patterns. Plaque assay using 15 phages previously isolated using the E. coli reference collection demonstrated that Warwickvirus phages showed the broadest host range, killing up to 26 strains. Conclusions: 154/198 strains were resistant to infection by all phages tested, suggesting a need for isolating phages specific for ESBL/AmpC E. coli. The strain collection described in this study is a useful resource fulfilling such need.

Bacteriophage F336 recognizes the capsular phosphoramidate modification of Campylobacter jejuni NCTC11168.

Bacteriophages infecting the food-borne human pathogen Campylobacter jejuni could potentially be exploited to reduce bacterial counts in poultry prior to slaughter. This bacterium colonizes the intestinal tract of poultry in high numbers, and contaminated poultry meat is regarded as the major source of human campylobacteriosis. In this study, we used phage F336 belonging to the Myoviridae family to select a C. jejuni NCTC11168 phage-resistant strain, called 11168R, with the aim of investigating the mechanisms of phage resistance. We found that phage F336 has reduced adsorption to 11168R, thus indicating that the receptor is altered. While proteinase K-treated C. jejuni cells did not affect adsorption, periodate treatment resulted in reduced adsorption, suggesting that the phage binds to a carbohydrate moiety. Using high-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy, we found that 11168R lacks an O-methyl phosphoramidate (MeOPN) moiety attached to the GalfNAc on the capsular polysaccharide (CPS), which was further confirmed by mass spectroscopy. Sequence analysis of 11168R showed that the potentially hypervariable gene cj1421, which encodes the GalfNAc MeOPN transferase, contains a tract of 10 Gs, resulting in a nonfunctional gene product. However, when 11168R reverted back to phage sensitive, cj1421 contained 9 Gs, and the GalfNAc MeOPN was regained in this strain. In summary, we have identified the phase-variable MeOPN moiety, a common component of the diverse capsular polysaccharides of C. jejuni, as a novel receptor of phages infecting this bacterium.

Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages.

Phages belonging to the Ackermannviridae family encode up to four tail spike proteins (TSPs), each recognizing a specific receptor of their bacterial hosts. Here, we determined the TSPs diversity of 99 Ackermannviridae phages by performing a comprehensive in silico analysis. Based on sequence diversity, we assigned all TSPs into distinctive subtypes of TSP1, TSP2, TSP3 and TSP4, and found each TSP subtype to be specifically associated with the genera (Kuttervirus, Agtrevirus, Limestonevirus, Taipeivirus) of the Ackermannviridae family. Further analysis showed that the N-terminal XD1 and XD2 domains in TSP2 and TSP4, hinging the four TSPs together, are preserved. In contrast, the C-terminal receptor binding modules were only conserved within TSP subtypes, except for some Kuttervirus TSP1s and TSP3s that were similar to specific TSP4s. A conserved motif in TSP1, TSP3 and TSP4 of Kuttervirus phages may allow recombination between receptor binding modules, thus altering host recognition. The receptors for numerous uncharacterized phages expressing TSPs in the same subtypes were predicted using previous host range data. To validate our predictions, we experimentally determined the host recognition of three of the four TSPs expressed by kuttervirus S117. We confirmed that S117 TSP1 and TSP2 bind to their predicted host receptors, and identified the receptor for TSP3, which is shared by 51 other Kuttervirus phages. Kuttervirus phages were thus shown encode a vast genetic diversity of potentially exchangeable TSPs influencing host recognition. Overall, our study demonstrates that comprehensive in silico and host range analysis of TSPs can predict host recognition of Ackermannviridae phages.

Campylobacter phages use hypermutable polyG tracts to create phenotypic diversity and evade bacterial resistance.

Phase variation is a common mechanism for creating phenotypic heterogeneity of surface structures in bacteria important for niche adaptation. In Campylobacter, phase variation occurs by random variation in hypermutable homonucleotide 7-11 G (polyG) tracts. To elucidate how phages adapt to phase-variable hosts, we study Fletchervirus phages infecting Campylobacter dependent on a phase-variable receptor. Our data demonstrate that Fletcherviruses mimic their host and encode hypermutable polyG tracts, leading to phase-variable expression of two of four receptor-binding proteins. This creates phenotypically diverse phage populations, including a sub-population that infects the bacterial host when the phase-variable receptor is not expressed. Such population dynamics of both phage and host promote co-existence in a shared niche. Strikingly, we identify polyG tracts in more than 100 phage genera, infecting more than 70 bacterial species. Future experimental work may confirm phase variation as a widespread strategy for creating phenotypically diverse phage populations.

Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein.

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria.

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present Innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysin-engineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.

Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages.

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.