Antivirulence Activity and Therapeutic Potential of Jumbo Vibrio Phage pVa-22 against Vibrio alginolyticus

Phages are the most abundant biological entity on Earth. There are many variants in phage virion sizes, morphology, and genome sizes. Large virion sized phages, with genome sizes greater than 200 kbp have been identified and termed as Jumbo phages. These phages exhibit certain characteristics that have not been reported in phages with smaller genomes. In this work, a jumbo phage named MIJ3 (vB_PaeM_MIJ3) that infects Pseudomonas aeruginosa PAO1 was isolated from an equine livery yard in Leicestershire, United Kingdom. The genome and biological characteristics of this phage have been investigated. MIJ3 is a Myovirus with multiple long tail fibers. Assessment of the host range of MIJ3 revealed that it has the ability to infect many clinical isolates of P. aeruginosa. Bioinformatics analysis of the phage genome indicated that MIJ3 is closely related to the Pseudomonas phage, PA5oct. MIJ3 possesses several unusual features that are either rarely present in other phages or have not yet been reported. In particular, MIJ3 encodes a FtsH-like protein, and a putative lysidine synthase, TilS. These two proteins have not been reported in phages. MIJ3 also possesses a split DNA polymerase B with a novel intein. Of particular interest, unlike other jumbo phages infecting Pseudomonas spp., MIJ3 lacks the genetic elements required for the formation of the phage nucleus, which was believed to be conserved across jumbo Pseudomonas phages.

BackgroundPseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections, and belongs to the group of ESKAPE pathogens that are the leading cause of healthcare-associated infections and have high level of antibiotic resistance. The treatment of infections caused by antibiotic-resistant P. aeruginosa is challenging, which makes it a common target for phage therapy. The successful utilization of phage therapy requires a collection of well characterized phages.MethodsPhage fMGyn-Pae01 was isolated from a commercial phage therapy cocktail. The phage morphology was studied by transmission electron microscopy and the host range was analyzed with a liquid culture method. The phage genome was sequenced and characterized, and the genome was compared to closest phage genomes. Phage resistant bacterial mutants were isolated and whole genome sequencing and motility, phage adsorption and biofilm formation assays were performed to the mutants and host bacterium.ResultsThe genomic analysis revealed that fMGyn-Pae01 is a lytic, phiKZ-like jumbo phage with genome size of 277.8 kb. No genes associated with lysogeny, bacterial virulence, or antibiotic resistance were identified. Phage fMGyn-Pae01 did not reduce biofilm formation of P. aeruginosa, suggesting that it may not be an optimal phage to be used in monophage therapy in conditions where biofilm formation is expected. Host range screening revealed that fMGyn-Pae01 has a wide host range among P. aeruginosa strains and its infection was not dependent on O-serotype. Whole genome sequencing of the host bacterium and phage resistant mutants revealed that the mutations had inactivated either a flagellar or rpoN gene, thereby preventing the biosynthesis of a functional flagellum. The lack of functional flagella was confirmed in motility assays. Additionally, fMGyn-Pae01 failed to adsorb on non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor.ConclusionfMGyn-Pae01 is a phiKZ-like jumbo phage infecting P. aeruginosa. fMGyn-Pae01 uses the flagellum as its phage-binding receptor, supporting earlier suggestions that flagellum might be utilized by phiKZ but differs from some other previous findings showing that phiKZ-like phages use the type-IV pili as the phage-binding receptor.

Characterization of a novel genus of jumbo phages and their application in wastewater treatment

Campylobacter jejuni is the leading bacterial cause of foodborne human gastroenteritis worldwide. The first C. jejuni genome (strain NCTC11168) was sequenced in 2000. This original annotation was a milestone in Campylobacter research, but soon became outdated. A re-annotation and re-analysis of this genome sequence was performed resulting in updates to over 90% of coding sequences (CDSs) and modification of 18.2% of CDS product functions (Gundogdu et al., 2007). Following this re-annotation, 15 uncharacterised CDSs with revised functions relating to virulence, signal transduction or regulation of gene expression were selected for further investigation. Defined isogenic C. jejuni 11168H mutants were constructed and after preliminary analysis, the Cj1556 and Cj0248 mutants were selected for further characterisation. Cj1556 was originally annotated as a hypothetical protein and was updated to a MarR family transcriptional regulator. Further bioinformatic analysis indicated a putative role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic (O2) stress, decreased ability for intracellular survival in both Caco-2 intestinal epithelial cells (IECs) and J774A.1 mouse macrophages and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant compared to the wild-type strain indicated negative autoregulation of Cj1556 expression and down-regulation of genes associated with oxidative and aerobic (O2) stress responses. Cj0248 was originally annotated as a hypothetical protein however the re-annotation identified a HD domain linked to a superfamily of metal-dependent phosphohydrolases with roles in signal transduction in bacteria. Previously a C. jejuni 81-176 Cj0248 mutant was shown to be deficient for motility and chick colonisation, however the exact function of Cj0248 was not investigated. The C. jejuni 11168H Cj0248 mutant also possessed a reduced motility phenotype and exhibited reduced interaction and invasion when co-cultured with Caco-2 IECs compared to the wild-type strain. However the Cj0248 mutant showed no difference in autoagglutination compared to the wild-type strain and TEM analysis indicated the mutant possessed intact flagella. Higher magnification TEM indicated the possibility of an altered flagella basal body region in the Cj0248 mutant. Secretion profile analysis identified no differences in the protein profile of the Cj0248 mutant compared to the wild-type strain. The exact function of Cj0248 remains unclear.

The pandemic dissemination of KPC carbapenemase-producing Klebsiella pneumoniae (KPC-KP) represents a major public health problem, given their extensive multidrug resistance profiles and primary role in causing healthcare-associated infections. This phenomenon has largely been contributed by strains of Clonal Group (CG) 258, mostly of clade II, which in some areas represent the majority of KPC-KP isolates. Here we have characterized a newly discovered lytic Podoviridae, named φBO1E, targeting KPC-KP strains of clade II lineage of CG258. Genomic sequencing revealed that φBO1E belongs to the Kp34virus genus (87% nucleotide identity to vB_KpnP_SU552A). ΦBO1E was stable over a broad pH and temperature range, exhibited strict specificity for K. pneumoniae strains of clade II of CG258, and was unable to establish lysogeny. In a Galleria mellonella infection model, φBO1E was able to protect larvae from death following infection with KPC-KP strains of clade II of CG258, including one colistin resistant strain characterized by a hypermucoviscous phenotype. To our best knowledge φBO1E is the first characterized lytic phage targeting K. pneumoniae strains of this pandemic clonal lineage. As such, it could be of potential interest to develop new agents for treatment of KPC-KP infections and for decolonization of subjects chronically colonized by these resistant superbugs.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Time-lapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.

My PhD thesis aims to obtain the structure of several bacteriophages, including so-called “jumbo” phages, using the electron cryo-microscopy (cryo-EM) and to study the structural transformation of these viruses as they attach and infect the host cell. We aim to understand the process of host cell membrane penetration at the molecular level and to achieve the level of knowledge necessary to understand how the genome and proteins, which are originally packaged into the capsid, are delivered into the host cell. This structural information can be of great importance since bacteriophage therapy is a potentially promising alternative to antibiotics, which are urgently needed. Bacteriophages, or phages are the most abundant and diverse form of life on Earth, and have developed various strategies through which to infect a susceptible bacterial host. A vast majority of phages have evolved to use a special organelle, called a “tail”, for host recognition, attachment and genome delivery into the cell. The host cell envelope is penetrated with the help of the tail. Unlike most eukaryotic viruses, infection of a host by tailed bacteriophages usually requires only one virion per bacterium, indicating that tailed bacteriophages have evolved an extremely efficient infection mechanism. Bacteriophages with contractile tails are complex viruses that infect Bacteria and Archaea. They share a common evolutionary origin and some functional aspect with other large macromolecular machines, such as the Serratia entomophila antifeeding prophage, the Photorhabdus virulence cassette, and the type VI secretion system (T6SS), which function to attack other cells by translocating toxic effector proteins into the target cell’s cytoplasm. The T6SS and the so-called “jumbo” phages are the most complex of these contractile injection systems. Jumbo phages have genomes exceeding 250kb and virus particle sizes of 300 nm and greater. Hundreds of different proteins are involved in the morphogenesis and assembly of a jumbo phage particle. A number of bacteriophages infecting important pathogens were isolated and characterized by means of cryo-EM and bioinformatics tools. Bacteriophage ɸ92 is a virus infecting a wide variety of acapsular Escherichia coli laboratory strains, Salmonella strains, and pathogenic E.Coli strains carrying a polysialic acid, which cause severe invasive infections including meningitis, pneumonia, septicaemia, osteomyelitis, septic arthritis and pyelonephritis. A cryo-EM reconstructions of the ɸ92 capsid, baseplate and long contractile tail were calculated, and complete three-dimensional model of the phage virion was built. Two bacteriophages, ɸEco32 infecting mastitis-causing strains of E.Coli and 7-11 infecting Salmonella strains, were isolated. They are a members of the Podoviridae family with the rare C3 morphotype, which occurs in <1% of phage virions. Two functional states of mature ɸEco32 virion, before and after DNA ejection, and mature 7-11 tail were characterized by cryo-EM and cryo-ET. Bacteriophage AR9 is a flagella-specific Bacillus subtilis jumbo phage with a tail of remarkable complexity. The AR9 adsorption apparatus could be used for attachment and “riding on” the motile B. subtilis flagellum. A three-dimensional model of the jumbo virion was built and flagella-specific adsorption apparatus was characterized. Bacterial phage CBA120 belongs to the Myoviridae family distinct from T4 in morphology and behavior and specifically infects E.Coli strain O157, an important food-borne pathogen. CBA120 represents a newly described group of phages. Extended and contracted conformations of CBA120 tail were characterized by cryo-EM. Mutant bacteriophage T4 lacking the tip protein of the cell-puncturing device responsible for the first interaction with the host cell membrane was isolated. Three-dimensional cryo-EM reconstruction of the baseplate and a tail fragment was calculated and compared with that of the wild type T4 phage and the distant member of T4-superfamily phage RB43.

Hypervirulent and multidrug-resistant Klebsiella pneumoniae (hvKP and MDR-KP) are significant public health threats. This study aimed to isolate a lytic bacteriophage targeting these high-risk strains, systematically characterize its biological properties, genomic features, and therapeutic efficacy, and establish a foundation for clinical phage therapy and novel antimicrobial development. The phage vB_Kp_XP4 was isolated from river water using the double-layer agar plate method with the clinically isolated strain P4 as the host. Morphology was analyzed via transmission electron microscopy (TEM). Host range, pH, and thermal stability were assessed using spot assays and OD630 measurements. One-step growth curves determined the latent period and burst size. Whole-genome sequencing and phylogenetic analysis were performed. Therapeutic efficacy and safety were evaluated in a Galleria mellonella infection model. TEM revealed Phage vB_Kp_XP4 as a tailed phage with an icosahedral head and a long, flexible tail. It lysed an hvKP strain (carrying rmp, peg, iuc, iro genes) and an MDR-KP strain (resistant to carbapenems, fluoroquinolones, etc.), with an optimal MOI of 0.1 and latent period <10 minutes. Stability was maintained at pH 4-11 and ≤70°C. Whole-genome sequencing revealed a linear double-stranded DNA genome of 44,344 bp with a G+C content of 53.80%. The genome comprised 54 coding sequences and lacked lysogenic, virulence, or antibiotic resistance genes. Phylogenetic analysis positioned phage vB_Kp_XP4 as a novel species within the genus Drulisvirus, family Autographiviridae. In the Galleria mellonella model, vB_Kp_XP4 prolonged survival of P4-infected larvae (P < 0.001). Phage vB_Kp_XP4 exhibits high stability, specificity, potent lytic activity, and no undesirable genes, demonstrating effective in vivo therapeutic efficacy, suggest its potential for clinical applications against Klebsiella pneumoniae infections. The presence of multiple halos during plaque formation further enhances its research value. The complete genome sequence has been submitted to GenBank under accession number PP663283.

Vibrio alginolyticus is a zoonotic pathogenic bacterium that causes various diseases in marine animals, including Penaeus vannamei. Phage therapy, as a natural antibiotic, offers a novel approach to the prevention and control of vibriosis. In this study, we isolated, identified, and characterized a novel bacteriophage targeting V. alginolyticus, designated as PV48. PV48, a short-tailed bacteriophage isolated from the Ningbo aquaculture farm, possesses the ability to lyse V. alginolyticus strains that affect shrimp. It displays stability over a range of temperatures (30-50°C) and pH values (4-10). The latent period and burst size for phage PV48 were determined to be 20 minutes and 15.6 PFU/cell, respectively. Whole-genome sequencing revealed that PV48 has a dsDNA genome with a length of 43,398 bp and a G + C content of 49.54%. The phage genome contains 44 open reading frames (ORFs). Comparative genomic analysis suggested that PV48 might represent a new species within the genus Maculvirus. Phylogenetic analysis indicated a close evolutionary link between PV48 and other Vibrio-targeting phages, suggesting a shared viral lineage. This study lays the groundwork for the application of V. alginolyticus bacteriophage in aquaculture.

There is an increasing emergence of antibiotic-resistant Vibrio alginolyticus, a zoonotic pathogen that causes mass mortality in aquatic animals and infects humans; therefore, there is a demand for alternatives to antibiotics for the treatment and prevention of infections caused by this pathogen. One possibility is through the exploitation of bacteriophages. In the present study, the novel bacteriophage pVa-21 was classified as Myoviridae and characterised as a candidate biocontrol agent against V. alginolyticus. Its morphology, host range and infectivity, growth characteristics, planktonic or biofilm lytic activity, stability under various conditions, and genome were investigated. Its latent period and burst size were estimated to be approximately 70 min and 58 plaque-forming units/cell, respectively. In addition, phage pVa-21 can inhibit bacterial growth in both the planktonic and biofilm states. Furthermore, phylogenetic and genome analysis revealed that the phage is closely related to the giant phiKZ-like phages and can be classified as a new member of the phiKZ-like bacteriophages that infect bacteria belonging to the family Vibrionaceae.

A jumbo phage infecting Ralstonia solanacearum species complex strains, designated RsoM2USA, was isolated from soil of a tomato field in Florida, United States, and belongs to the family Myoviridae. The phage has a long latent period of 270 min and completed its infection cycle in 360 min with a burst size of approximately 32 particles per cell. With a genome size of 343,806 bp, phage RsoM2USA is the largest Ralstonia-infecting phage sequenced and reported to date. Out of the 486 ORFs annotated for RsoM2USA, only 80 could be assigned putative functions in replication, transcription, translation including 44 tRNAs, and structure with the main structural proteins experimentally confirmed. Phylogenetic analyses placed RsoM2USA in the same clade as Xanthomonas phage XacN1, prompting a proposal of a new genus for the two jumbo phages. Jumbo phage RsoM2USA is a lytic phage and has a wide host range, infecting each of the three newly established Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of its susceptible R. solanacearum strain RUN302 in tomato plants, suggesting that this jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages

Aeromonas hydrophila is a zoonotic pathogen displaying resistance to multiple antibiotics. Here, we aim to develop a candidate biocontrol agent against A. hydrophila. In this study, we isolated and characterized the phage vB-AhyM-AP1 from sewage. It showed lytic activity against A. hydrophila strains. One-step growth curve revealed that the latent period lasted for 40min. The burst size of one lytic cycle was 1413 PFU per infected cell. Temperature stability studies showed that the phage vB-AhyM-AP1 was active over temperatures ranging from 4 to 45°C for 1h. pH stability studies indicated that the phage remained active within a pH range of 5-10 after 24h of incubation. Stability tests in salt solutions showed that the phage was stable at salinities ranging from 0·1 to 2%. The phage also showed stabilities in organic solvents when incubated for 10min. The Illumina Hiseq sequencing of its genome indicated that the phage vB-AhyM-AP1was a jumbo phage with a genome size of 2, 54490bp and GC content of 40·3%. The phylogenetic analysis of the terminase large subunit and major capsid protein indicated that the phage closely clustered with other Tevenvirinae phages. The genome encoded 455 ORFs and 22 tRNAs. The phage resulted in a reduction of 0·8 log units of viable A. hydrophila cells in biofilms grown on PVC coupons maintained in a low nutrient medium for 10days. The phage showed lytic activity against planktonic and biofilm cells of A. hydrophila. Genome-based prediction showed it to be a strictly lytic phage without any virulence or antibiotic resistance genes indicating safety for environmental and clinical applications. The multidrug-resistant strains of A. hydrophila pose a significant health risk to both cultured fishes and consumers leaving few options for treatment. Phage vB-AhyM-AP1 may be used as a candidate biocontrol agent against A. hydrophila strains.

Biochemical characterisation of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6

Tailed phages with genomes larger than 200kbp are classified as jumbo phage and exhibit extremely high diversity. In this study, a novel jumbo phage, vB_BpuM_BpSp, infecting pathogenic Bacillus pumilus, the cause of ginger rhizome rot disease, was isolated. Notable features of phage vB_BpuM_BpSp are the large phage capsid of 137nm and baseplate-attached curly tail fibers. The genome of the phage is 255,569bp in size with G+C content of 25.9%, and it shows low similarity to known biological entities. The phage genome contains 318 predicted coding sequences. Among these predicted coding sequences, 26 genes responsible for nucleotide metabolism were found, and seven structural genes could be identified. The findings of this study provide new understanding of the genetic diversity of phages.