Deoxyinosine and 7-Deaza-2-Deoxyguanosine as Carriers of Genetic Information in the DNA of Campylobacter Viruses.

Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.

Data to Power Precision Phage Therapy: A Look at the Phage Directory-Phage Australia Partnership.

Phage Directory has recently partnered with Phage Australia to help optimize Australia's data-centric standardized approach to personalized phage therapy. Here is a behind-the-scenes look at the genesis of Phage Australia, how the Phage Directory-Phage Australia partnership started, and what it is working toward.

inPhocus: "State of Phage" Survey Highlights Widespread Diverse Phage Isolation and Research in 40+ Countries.

The phage field is clearly growing around the world, but no one had compiled a bird's eye view of the phages being collected and the phage research and development work being done. To fill this gap, and help establish a baseline for the current state of phage research so we can track and cultivate its growth and evolution over time, we created the State of Phage 2020 Survey. We aimed to get a snapshot of the strains and phages being collected around the world, and to capture activities and opinions of phage laboratories, including go-to methods, definitions of what phage characterization looks like, level of characterization of phages, preferred phage sharing practices, and more. Here we present an overview of selected results from our State of Phage 2020 survey, along with our interpretations and perspective on where to go from here as a field to maximize the impact of phage work being done around the globe. We conclude that given the magnitude of the task at hand (developing a robust understanding of the phages we have, which span hundreds of phages targeting hundreds of species), and given the large yet diverse collective phage expertise around the world, the best way forward is to develop systems that allow the field to combine forces, including making room for domain experts to focus on their specific strengths and investing in phage and data sharing practices.

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress.

Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.

Phage Biobank: Present Challenges and Future Perspectives.

After a century of use in human infection, the preparation and administration of therapeutic bacteriophages (phages) still relies on ad hoc partnerships of researchers, biotech companies, clinicians and regulators. There is a clear need to improve the reproducibility, safety and speed of the provision of suitable phages. Here we discuss the specific characteristics and challenges of a sustainable phage biobank and, as we build a national consortium aimed at delivering phage therapeutics, suggest a roadmap toward national biobanking and phage therapy initiatives using the Australian context as a model.

Binding of Phage-Encoded FlaGrab to Motile Campylobacter jejuni Flagella Inhibits Growth, Downregulates Energy Metabolism, and Requires Specific Flagellar Glycans.

Many bacterial pathogens display glycosylated surface structures that contribute to virulence, and targeting these structures is a viable strategy for pathogen control. The foodborne pathogen Campylobacter jejuni expresses a vast diversity of flagellar glycans, and flagellar glycosylation is essential for its virulence. Little is known about why C. jejuni encodes such a diverse set of flagellar glycans, but it has been hypothesized that evolutionary pressure from bacteriophages (phages) may have contributed to this diversity. However, interactions between Campylobacter phages and host flagellar glycans have not been characterized in detail. Previously, we observed that Gp047 (now renamed FlaGrab), a conserved Campylobacter phage protein, binds to C. jejuni flagella displaying the nine-carbon monosaccharide 7-acetamidino-pseudaminic acid, and that this binding partially inhibits cell growth. However, the mechanism of this growth inhibition, as well as how C. jejuni might resist this activity, are not well-understood. Here we use RNA-Seq to show that FlaGrab exposure leads C. jejuni 11168 cells to downregulate expression of energy metabolism genes, and that FlaGrab-induced growth inhibition is dependent on motile flagella. Our results are consistent with a model whereby FlaGrab binding transmits a signal through flagella that leads to retarded cell growth. To evaluate mechanisms of FlaGrab resistance in C. jejuni, we characterized the flagellar glycans and flagellar glycosylation loci of two C. jejuni strains naturally resistant to FlaGrab binding. Our results point toward flagellar glycan diversity as the mechanism of resistance to FlaGrab. Overall, we have further characterized the interaction between this phage-encoded flagellar glycan-binding protein and C. jejuni, both in terms of mechanism of action and mechanism of resistance. Our results suggest that C. jejuni encodes as-yet unidentified mechanisms for generating flagellar glycan diversity, and point to phage proteins as exciting lenses through which to study bacterial surface glycans.

Current State of Compassionate Phage Therapy.

There is a current unmet medical need for the treatment of antibiotic-resistant infections, and in the absence of approved alternatives, some clinicians are turning to empirical ones, such as phage therapy, for compassionate treatment. Phage therapy is ideal for compassionate use due to its long-standing historical use and publications, apparent lack of adverse effects, and solid support by fundamental research. Increased media coverage and peer-reviewed articles have given rise to a more widespread familiarity with its therapeutic potential. However, compassionate phage therapy (cPT) remains limited to a small number of experimental treatment centers or associated with individual physicians and researchers. It is possible, with the creation of guidelines and a greater central coordination, that cPT could reach more of those in need, starting by increasing the availability of phages. Subsequent steps, particularly production and purification, are difficult to scale, and treatment paradigms stand highly variable between cases, or are frequently not reported. This article serves both to synopsize cPT publications to date and to discuss currently available phage sources for cPT. As the antibiotic resistance crisis continues to grow and the future of phage therapy clinical trials remains undetermined, cPT represents a possibility for bridging the gap between current treatment failures and future approved alternatives. Streamlining the process of cPT will help to ensure high quality, therapeutically-beneficial, and safe treatment.

Cj1388 Is a RidA Homolog and Is Required for Flagella Biosynthesis and/or Function in Campylobacter jejuni.

Campylobacter jejuni is the leading bacterial cause of acute gastroenteritis worldwide and thus significant to public health. C. jejuni primarily lives in the gastrointestinal tracts of poultry and can contaminate meat during processing. Despite a small genome, the metabolic plasticity of C. jejuni allows proliferation in chicken ceca and mammalian host intestines, and survival in environments with a variety of temperatures, pH, osmotic conditions, and nutrient availabilities. The exact mechanism of C. jejuni infection is unknown, however, virulence requires motility. Our data suggest the C. jejuni RidA homolog, Cj1388, plays a role in flagellar biosynthesis, regulation, structure, and/or function and, as such is expected to influence virulence of the organism. Mutants lacking cj1388 have defects in motility, autoagglutination, and phage infectivity under the conditions tested. Comparison to the RidA paradigm from Salmonella enterica indicates the phenotypes of the C. jejuni cj1388 mutant are likely due to the inhibition of one or more pyridoxal 5'-phosphate-dependent enzymes by the reactive enamine 2-aminoacrylate.

Bacterial AB5 toxins inhibit the growth of gut bacteria by targeting ganglioside-like glycoconjugates.

The AB5 toxins cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are notorious for their roles in diarrheal disease, but their effect on other intestinal bacteria remains unexplored. Another foodborne pathogen, Campylobacter jejuni, can mimic the GM1 ganglioside receptor of CT and LT. Here we demonstrate that the toxin B-subunits (CTB and LTB) inhibit C. jejuni growth by binding to GM1-mimicking lipooligosaccharides and increasing permeability of the cell membrane. Furthermore, incubation of CTB or LTB with a C. jejuni isolate capable of altering its lipooligosaccharide structure selects for variants lacking the GM1 mimic. Examining the chicken GI tract with immunofluorescence microscopy demonstrates that GM1 reactive structures are abundant on epithelial cells and commensal bacteria, further emphasizing the relevance of this mimicry. Exposure of chickens to CTB or LTB causes shifts in the gut microbial composition, providing evidence for new toxin functions in bacterial gut competition.

Transcriptomic Analysis of the Campylobacter jejuni Response to T4-Like Phage NCTC 12673 Infection.

Campylobacter jejuni is a frequent foodborne pathogen of humans. As C. jejuni infections commonly arise from contaminated poultry, phage treatments have been proposed to reduce the C. jejuni load on farms to prevent human infections. While a prior report documented the transcriptome of C. jejuni phages during the carrier state life cycle, transcriptomic analysis of a lytic C. jejuni phage infection has not been reported. We used RNA-sequencing to profile the infection of C. jejuni NCTC 11168 by the lytic T4-like myovirus NCTC 12673. Interestingly, we found that the most highly upregulated host genes upon infection make up an uncharacterized operon (cj0423⁻cj0425), which includes genes with similarity to T4 superinfection exclusion and antitoxin genes. Other significantly upregulated genes include those involved in oxidative stress defense and the Campylobactermultidrug efflux pump (CmeABC). We found that phage infectivity is altered by mutagenesis of the oxidative stress defense genes catalase (katA), alkyl-hydroxyperoxidase (ahpC), and superoxide dismutase (sodB), and by mutagenesis of the efflux pump genes cmeA and cmeB. This suggests a role for these gene products in phage infection. Together, our results shed light on the phage-host dynamics of an important foodborne pathogen during lytic infection by a T4-like phage.

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

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.

Exploring the interactions between bacteriophage-encoded glycan binding proteins and carbohydrates.

There is an unprecedented interest in glycobiology due to the increasing appreciation of its impact on all aspects of life. Likewise, bacteriophage biology is enjoying a new renaissance as the post-antibiotic era fuels the search for novel ways to control harmful bacteria. Phages have spent the last 3 billion years developing ways of recognizing and manipulating bacterial surface glycans. Therefore, phages comprise a massive reservoir of glycan-binding and -hydrolyzing proteins with the potential to be exploited for glycan analysis, bacterial diagnostics and therapeutics. We discuss phage tail proteins that recognize bacterial surface polysaccharides, endolysins that bind and cleave peptidoglycan, Ig-like proteins that attach to mucin glycans, and phage effector proteins that recognize both bacterial and eukaryotic oligosaccharides.

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

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.