Additional Phages Research Articles

Multi-interface licensing of protein import into a phage nucleus.

Bacteriophages use diverse mechanisms to evade antiphage defence systems. ΦKZ-like jumbo phages assemble a proteinaceous, nucleus-like compartment that excludes antagonistic host nucleases and also internalizes DNA replication and transcription machinery1-4. The phage factors required for protein import and the mechanisms of selectivity remain unknown, however. Here we uncover an import system comprising proteins highly conserved across nucleus-forming phages, together with additional cargo-specific contributors. Using a genetic selection that forces the phage to decrease or abolish the import of specific proteins, we determine that the importation of five different phage nuclear-localized proteins requires distinct interfaces of the same factor, Imp1 (gp69). Imp1 localizes early to the nascent phage nucleus and forms discrete puncta in the mature phage nuclear periphery, probably in complex with direct interactor Imp6 (gp67), a conserved protein encoded in the same locus. The import of certain proteins, including a host topoisomerase, additionally requires Imp3 (gp59), aconserved factor necessary for proper Imp1 function. Three additional non-conserved phage proteins (Imp2and Imp4/Imp5) are required for the import of two queried nuclear cargos (nuclear-localized protein 1 and host topoisomerase, respectively), perhaps acting as specific adaptors. We therefore propose a core import system that includes Imp1, Imp3 and Imp6, with multiple interfaces ofImp1 licensing transport through a protein lattice.

A Phage-Based Approach to Identify Antivirulence Inhibitors of Bacterial Type IV Pili.

The increasing threat of antibiotic resistance underscores the urgent need for innovative strategies to combat infectious diseases, including the development of antivirulants. Microbial pathogens rely on their virulence factors to initiate and sustain infections. Antivirulants are small molecules designed to target virulence factors, thereby attenuating the virulence of infectious microbes. The bacterial type IV pilus (T4P), an extracellular protein filament that depends on the T4P machinery (T4PM) for its biogenesis, dynamics and function, is a key virulence factor in many significant bacterial pathogens. While the T4PM presents a promising antivirulence target, the systematic identification of inhibitors for its multiple protein constituents remains a considerable challenge. Here we report a novel high-throughput screening (HTS) approach for discovering T4P inhibitors. It uses Pseudomonas aeruginosa, a high-priority pathogen, in combination with its T4P-targeting phage, φKMV. Screening of a library of 2168 compounds using an optimised protocol led to the identification of tuspetinib, based on its deterrence of the lysis of P. aeruginosa by φKMV. Our findings show that tuspetinib also inhibits two additional T4P-targeting phages, while having no effect on a phage that recognises lipopolysaccharides as its receptor. Additionally, tuspetinib impedes T4P-mediated motility in P. aeruginosa and Acinetobacter species without impacting growth or flagellar motility. This bacterium-phage pairing approach is applicable to a broad range of virulence factors that are required for phage infection, paving ways for the development of advanced chemotherapeutics against antibiotic-resistant infections.

A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery

Biopanning methods to select target-specific Nanobodies® (Nbs) involve presenting the antigen, immobilized on plastic plates or magnetic beads, to Nb libraries displayed on phage. Most routines are operator-dependent, labor-intensive and often material- and time-consuming. Here we validate an improved panning strategy that uses biosensors to present the antigen to phage-displayed Nbs in a well. The use of automated Octet biolayer interferometry sensors (Sartorius) enables high throughput and precise control over each step. By playing with association and dissociation times and buffer composition, one can efficiently decrease the background of aspecific and low-affinity Nbs, reducing the rounds of panning needed for the enrichment of high-affinity binders. Octet panning also enables the use of unpurified target proteins and unpurified phage from a bacterial culture supernatant. Additionally, downscaling to a 384-well format significantly reduces the amount of protein required. Moreover, enrichment of binders can be quantified by monitoring phage binding to the target by interferometry, omitting additional phage titration steps. Routinely, up to three rounds of Octet panning can be performed in only five days to deliver target-specific binders, ready for screening and characterization using the same Octet instrument.

Proportion of patients with prosthetic joint infection eligible for adjuvant phage therapy: a French single-centre retrospective study

BackgroundBone and joint infections represent a major public health issue due to their increasing prevalence, their functional prognosis and their cost to society. Phage therapy has valuable anti-biofilm properties against prosthetic joint infections (PJI). The aim of this study was to establish the proportion of patients eligible for phage therapy and to assess their clinical outcome judged against all patients presenting with PJI.Method. Patients admitted for periprosthetic joint infection (PJI) at a French general hospital between 2015 and 2019 were retrospectively included. Eligibility for phage therapy was determined based on French recommendations, with polymicrobial infections serving as exclusion criteria. Patients were categorized into two groups: those eligible and those ineligible for phage therapy. We analyzed their characteristics and outcomes, including severe adverse events, duration of intravenous antibiotic therapy, length of hospitalization, and relapse rates.Results. In this study, 96 patients with PJI were considered in multidisciplinary medical meetings. Of these, 44% patients (42/96) were eligible for additional phage therapy. This group of patients had a longer duration of intravenous therapy (17 days vs. 10 days, p = 0.02), more severe adverse events (11 vs. 3, p = 0.08) and had a longer hospital stay (43 days vs. 18 days, p < 0.01).Conclusion. A large number of patients met eligibility criteria for phage therapy and treatment and follow-up is more complex. A larger epidemiological study would more accurately describe the prognosis of eligible patients.

Genome sequences of two Arthrobacter phages isolated from soil.

The isolation and characterization of additional phages is crucial for adding reliable viral sequences with relevant biological information to viral databases. In this study, we present the complete genomes of two Arthrobacter phages obtained from different soil samples.

Isolation and Characterization of a Novel Virulent Phage ASG01 of Aeromonas salmonicida and Its Cell Wall Hydrolase Activity.

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

Phage Therapy as a Novel Therapeutic for the Treatment of Bone and Joint Infections.

Solutions for bone and joint infection (BJI) are needed where conventional treatments are inadequate. Bacteriophages (phages) are naturally occurring viruses that infect bacteria and have been harnessed for refractory bone and joint infections (BJI) in many case reports. Here we examine the safety and efficacy of English-language published cases of BJI since 2010 with phage therapy. From 33 reported cases of BJI treated with phage therapy, 29 (87%) achieved microbiological or clinical success, 2 (5.9%) relapsed with the same organisms, and 2 (5.9%) with a different organism. Of these 4 relapses, all but 1 had eventual clinical resolution with additional surgery or phage treatments. Eight out of 33 cases (24%) reported mild, transient adverse events with no serious events reported. Further work is needed to understand the true efficacy of phages and the role of phages in BJI. Opportunities lay ahead for thoughtfully designed clinical trials adapted to individualized therapies.

Diversity and Current Classification of dsRNA Bacteriophages.

Half a century has passed since the discovery of Pseudomonas phage phi6, the first enveloped dsRNA bacteriophage to be isolated. It remained the sole known dsRNA phage for a quarter of a century and the only recognised member of the Cystoviridae family until the year 2018. After the initial discovery of phi6, additional dsRNA phages have been isolated from globally distant locations and identified in metatranscriptomic datasets, suggesting that this virus type is more ubiquitous in nature than previously acknowledged. Most identified dsRNA phages infect Pseudomonas strains and utilise either pilus or lipopolysaccharide components of the host as the primary receptor. In addition to the receptor-mediated strictly lytic lifestyle, an alternative persistent infection strategy has been described for some dsRNA phages. To date, complete genome sequences of fourteen dsRNA phage isolates are available. Despite the high sequence diversity, similar sets of genes can typically be found in the genomes of dsRNA phages, suggesting shared evolutionary trajectories. This review provides a brief overview of the recognised members of the Cystoviridae virus family and related dsRNA phage isolates, outlines the current classification of dsRNA phages, and discusses their relationships with eukaryotic RNA viruses.

An efficient, scarless, selection-free technology for phage engineering

ABSTRACT Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1–14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50–201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.

Computational design of phage cocktails based on phage-bacteria infection networks

The misuse and overuse of antibiotics have boosted the proliferation of multidrug-resistant (MDR) bacteria, which are considered a major public health issue in the twenty-first century. Phage therapy may be a promising way in the treatment of infections caused by MDR pathogens, without the side effects of the current available antimicrobials. Phage therapy is based on phage cocktails, that is, combinations of phages able to lyse the target bacteria. In this work, we present and explain in detail two innovative computational methods to design phage cocktails taking into account a given phage-bacteria infection network. One of the methods (Exhaustive Search) always generates the best possible phage cocktail, while the other method (Network Metrics) always keeps a very reduced runtime (a few milliseconds). Both methods have been included in a Cytoscape application that is available for any user. A complete experimental study has been performed, evaluating and comparing the biological quality, runtime, and the impact when additional phages are included in the cocktail.

Genome sequence analysis of Cronobacter phage PF-CE2 and proposal of a new species in the genus Pseudotevenvirus.

The genome of a Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work, and a new species named "Cronobacter virus PF-CE2", in the genus Pseudotevenvirus of the subfamily Tevenvirinae of the family Myoviridae is proposed. The Gp190 gene of phage PF-CE2 is predicted to encode a bacteriophage-borne glycanase that is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Furthermore, we propose changing the taxonomic status of eight additional phages based on nucleotide sequence comparisons. This work provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.

A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents.

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

Bacteroides thetaiotaomicron-Infecting Bacteriophage Isolates Inform Sequence-Based Host Range Predictions

Our emerging view of the gut microbiome largely focuses on bacteria, while less is known about other microbial components, such as bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the gut's "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

Lactococcus Ceduovirus Phages Isolated from Industrial Dairy Plants—From Physiological to Genomic Analyses

LactococcusCeduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new Lactococcus lactis Ceduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.

A cornucopia of Shigella phages from the Cornhusker State

Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.

Comparative genomics reveals structural and functional features specific to the genome of a foodborne Escherichia coli O157:H7

BackgroundEscherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains.ResultsThe chromosome of NADC 6564 contained 5466 kb compared to reference strains Sakai (5498 kb) and EDL933 (5547 kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32–33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains.ConclusionsThese results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.

Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters

Natural products are a rich source of potential drugs for many applications. Discovery of natural products through the activation of cryptic gene clusters encoding their biosynthetic pathways, engineering of those biosynthetic pathways and optimization of production yields often rely on the expression of these gene clusters in suitable heterologous host strains. Streptomyces albus J1074 provides high success rates of heterologous cluster expression with high levels of metabolite production, rapid growth and amenability to genetic manipulations. Here, we report the construction of S. albus chassis strains optimized for the discovery of natural products through heterologous expression of secondary metabolite clusters. 15 clusters encoding secondary metabolite biosynthetic pathways were deleted in the chromosome of S. albus Del14. This strain provides a substantially improved compound detection limit, owing to the lack of native secondary metabolites. Furthermore, the production yield of natural products heterologously expressed in S. albus Del14 was higher than in commonly used S. albus J1074 and S. coelicolor hosts. S. albus strains B2P1 and B4 were generated by introduction of additional phage phiC31 attB sites into the chromosome of S. albus Del14, allowing integration of up to four copies of a heterologous gene cluster. Amplification of gene clusters in the chromosome of the constructed strains further improved production yields of the encoded compounds. One cryptic cluster from Streptomyces spp. and two clusters from distantly related Frankia spp. strains were successfully activated in these new chassis strains, leading to the isolation of a new compound fralnimycin.

Recognition of six additional cystoviruses: Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae.

Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens.

To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic.IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infect A. tumefaciens may provide insights for designing preventative strategies against bacterial pathogens.

Simulation of phage dynamics in multi-reactor models of complex wastewater treatment systems

Existing population models of bacteria and phages have most commonly described single well-mixed reactors. Whilst crucial to gain an understanding of population dynamics, these models are not directly applicable to scenarios requiring multi-reactor models. Using standard approaches we have formulated these models to describe behaviour within multiple interconnected reactors. A model incorporating phage dynamics in wastewater treatment systems was then developed to investigate the potential use of phages as a tool to reduce foaming or bulking. The model couples wastewater treatment dynamics within the commercial software GPS-X (Hydromantis Inc.) with phage dynamics through a newly developed add-on called PhageDyn. Simulations predict that immediately after phage dosing there is a lag period during which no apparent changes are observed, which is followed by a sudden and quick increase in the phage concentration and reduction in foaming biomass. “Normalization” without foaming is achieved within 1–2 weeks after dosing. The system may subsequently relapse back to foaming, requiring additional phage dosing, or be “cured” such that the added phages keep the problematic foaming biomass indefinitely at bay. Kinetic parameters describing the behaviour of the phages, as well as reactor configuration, are key determinants of the final outcome. The model is useful for predicting potential behaviour and for determining the characteristics of phages that are likely to be successful, assisting in isolation and screening.