Martine C. Holst Sørensen

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.

Phase Variable Expression of Capsular Polysaccharide Modifications Allows Campylobacter jejuni to Avoid Bacteriophage Infection in Chickens

Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage–host co-evolution.

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.

Campylobacter jejuni Motility Is Required for Infection of the Flagellotropic Bacteriophage F341

ABSTRACT 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.

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.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Phase Variable Expression of a Single Phage Receptor in Campylobacter jejuni NCTC12662 Influences Sensitivity Toward Several Diverse CPS-Dependent Phages

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS composition of 12662R by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) showed a diminished signal for O-methyl phosphoramidate (MeOPN), a phase variable modification of the CPS. This suggested that the majority of the 12662R population did not express this phase variable modification in the CPS, indicating that MeOPN serves as a phage receptor in NCTC12662. Whole genome analysis of 12662R showed a switch in the length of the phase variable homopolymeric G tract of gene 06810, encoding a putative MeOPN-transferase located in the CPS locus, resulting in a non-functional protein. To confirm the role of 06810 in phage resistance development of NCTC12662, a 06810 knockout mutant in NCTC12662 was constructed and analyzed by HR-MAS NMR demonstrating the absence of MeOPN in the CPS of the mutant. Plaque assays using NCTC12662Δ06810 demonstrated that seven of our CPS-dependent Campylobacter phages are dependent on the presence of MeOPN for successful infection of C. jejuni, whereas the remaining 29 phages infect independently of MeOPN, although with reduced efficiencies. Our data indicate that CPS-dependent phages uses diverse mechanisms for their initial interaction with their C. jejuni host.

Methods for Initial Characterization of Campylobacter jejuni Bacteriophages

Here we describe an initial characterization of Campylobacter jejuni bacteriophages by host range analysis, genome size determination by pulsed-field gel electrophoresis, and receptor-type identification by screening mutants for phage sensitivity.

Whole-Genome Sequence of the Bacteriophage-Sensitive Strain Campylobacter jejuni NCTC12662

ABSTRACT Campylobacter jejuni NCTC12662 has been the choice bacteriophage isolation strain due to its susceptibility to C. jejuni bacteriophages. This trait makes it a good candidate for studying bacteriophage–host interactions. We report here the whole-genome sequence of NCTC12662, allowing future elucidation of the molecular mechanisms of phage–host interactions in C. jejuni.

Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. coli is needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria. IMPORTANCE The extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria responsible for infections causing more than 50,000 deaths per year across Europe and the US. In response, the World Health Organization has stressed an urgent need to discover new antimicrobials to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug resistance. However, the outer membrane of Gram-negative bacteria limits the access of many antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor binding proteins. As proof of concept, we constructed Innolysins against E. coli using the endolysin and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding proteins and their different binding specificities, our proof of concept paves the route for creating an arsenal of pathogen specific alternative antimicrobials.