Phage P22 Research Articles

In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles.

Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of Salmonella (S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.

Recognition of an α-helical hairpin in P22 large terminase by a synthetic antibody fragment.

The genome-packaging motor of tailed bacteriophages and herpesviruses is a multisubunit protein complex formed by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal protein vertex of an empty precursor capsid to power the energy-dependent packaging of viral DNA. Both the ATPase and nuclease activities associated with genome packaging reside in TerL. Structural studies of TerL from bacteriophage P22 have been hindered by the conformational flexibility of this enzyme and its susceptibility to proteolysis. Here, an unbiased, synthetic phage-display Fab library was screened and a panel of high-affinity Fabs against P22 TerL were identified. This led to the discovery of a recombinant antibody fragment, Fab4, that binds a 33-amino-acid α-helical hairpin at the N-terminus ofTerL with an equilibrium dissociation constant Kd of 71.5 nM. A 1.51 Å resolution crystal structure of Fab4 bound to the TerL epitope (TLE) together with a 1.15 Å resolution crystal structure of the unliganded Fab4, which is the highest resolution ever achieved for a Fab, elucidate the principles governing the recognition of this novel helical epitope. TLE adopts two different conformations in the asymmetric unit and buries as much as 1250 Å2 of solvent-accessible surface in Fab4. TLE recognition is primarily mediated by conformational changes in the third complementarity-determining region of the Fab4 heavy chain (CDR H3) that take place upon epitope binding. It is demonstrated that TLE can be introduced genetically at the N-terminus of a target protein, where it retains high-affinity binding to Fab4.

Structural Insights into Lactococcal Siphophage p2 Baseplate Activation Mechanism.

Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection.

The lactococcal virulent phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects Lactococcus lactis MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.

Synergistic Effect of Bacteriophage and Antibiotic against Antibiotic-Resistant <i>Salmonella</i> Typhimurium

In this study, we investigated the efficacy of Salmonella phage P22 combined with antibiotics to inhibit antibiotic-resistant S. Typhimurium CCARM 8009. The synergistic effect of phage P22 and antibiotics was evaluated by using disk diffusion and broth dilution assays. The development of Antimicrobial resistance was determined after time-kill assay. The antibiotic susceptibility assay showed the inhibition zone sizes around the antibiotic disks were increased up to 78.8% in the presence of phage (cefotaxime; 13.6%, chloramphenicol; 19.3%, ciprofloxacin; 12.7% and erythromycin; 78.8%). The minimum inhibitory concentration values of the combination treatment significantly decreased from 256 to 64 mg/mL for tetracycline, 8 to 4 mg/mL for chloramphenicol, 0.0156 to 0.0078 mg/mL for ciprofloxacin, 128 to 64 mg/mL for erythromycin and 512 to 256 mg/mL for streptomycin. The number of S. Typhimurium CCARM 8009 was approximately 4-log lower than that of the control throughout the combination treatment with phage P22 and ciprofloxacin delete at 37oC for 20 h. The results indicate that the development of antimicrobial resistance in S. Typhimurium could be reduced in the presence of phage treatment. This study provides promising evidence for the phage-antibiotic combination as an effective treatment to control antibiotic-resistant bacteria.

Virus-Like Particles (VLPs) as a Platform for Hierarchical Compartmentalization.

Hierarchically self-assembled structures are common in biology, but it is often challenging to design and fabricate synthetic analogs. The archetypal cell is defined by hierarchically organized multicompartmentalized structures with boundaries that delineate the interior from exterior environments and is an inspiration for complex functional materials. Here, we have demonstrated an approach to the design and construction of a nested protein cage system that can additionally incorporate the packing of other functional macromolecules and exhibit some of the features of a minimal synthetic cell-like material. We have demonstrated a strategy for controlled co-packaging of subcompartments, ferritin (Fn) cages, together with active cellobiose-hydrolyzing β-glycosidase enzyme macromolecules, CelB, inside the sequestered volume of the bacteriophage P22 capsid. Using controlled in vitro assembly, we were able to modulate the stoichiometry of Fn cages and CelB encapsulated inside the P22 to control the degree of compartmentalization. The co-encapsulated enzyme CelB showed catalytic activity even when packaged at high total macromolecular concentrations comparable to an intracellular environment. This approach could be used as a model to create synthetic protein-based protocells that can confine smaller functionalized proto-organelles and additional macromolecules to support a range of biochemical reactions.

Phylogenetic Grouping of Human Ocular Escherichia coli Based on Whole-Genome Sequence Analysis.

Escherichia coli is a predominant bacterium in the intestinal tracts of animals. Phylogenetically, strains have been classified into seven phylogroups, A, B1, B2, C, D, E, and F. Pathogenic strains have been categorized into several pathotypes such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), Diffusely adherent (DAEC), Uropathogenic (UPEC), Shiga-toxin producing (STEC) or Enterohemorrhagic (EHEC) and Extra-intestinal pathogenic E. coli (ExPEC). E. coli also survives as a commensal on the ocular surface. However, under conditions of trauma and immune-compromised states, E. coli causes conjunctivitis, keratitis, endopthalmitis, dacyrocystitis, etc. The phylogenetic affiliation and the pathotype status of these ocular E. coli strains is not known. For this purpose, the whole-genome sequencing of the 10 ocular E. coli strains was accomplished. Based on whole-genome SNP variation, the ocular E. coli strains were assigned to phylogenetic groups A (two isolates), B2 (seven isolates), and C (one isolate). Furthermore, results indicated that ocular E. coli originated either from feces (enteropathogenic and enterotoxigenic), urine (uropathogenic), or from extra-intestinal sources (extra-intestinal pathogenic). A high concordance was observed between the presence of AMR (Antimicrobial Resistance) genes and antibiotic resistance in the ocular E. coli strains. Furthermore, several virulent genes (fimB to fimI, papB to papX, etc.) and prophages (Enterobacteria phage HK97, Enterobacteria phage P1, Escherichia phage D108 etc.) were unique to ocular E. coli. This is the first report on a whole-genome analysis of ocular E. coli strains.

Application of Recombinase-Based In Vivo Expression Technology to Bifidobacterium longum subsp. longum for Identification of Genes Induced in the Gastrointestinal Tract of Mice

Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.

Bacteriophage P1 does not show spatial preference when infecting Escherichia coli

To begin its infection, a bacteriophage first needs to adsorb to cells. The adsorption site on the cell surface may influence viral DNA injection, gene expression and cell-fate development. Here, we study the early steps of the infection cycle of coliphage P1, focusing on their correlation with spatial locations at the single-cell level. By fluorescently labeling P1 virions, we found that P1 shows no spatial preference on cell surface adsorption. In addition, live-cell phage DNA imaging revealed that adsorption sites do not affect the success rate for P1 in injecting its DNA into the cell. Furthermore, the lysis-lysogeny decision of P1 does not depend on the adsorption site, based on fluorescence reporters for the lytic and lysogenic pathways. These findings highlight the different infection strategies used by the two paradigmatic coliphages differ from those found in the paradigmatic phage lambda, highlighting that different infection strategies are used by phages.

Bi-enzymatic virus-like bionanoreactors for the transformation of endocrine disruptor compounds

Endocrine disruptor compounds (EDCs) are pollutants able to alter both hormone synthesis and their regulation in animals and humans, thus, EDCs represent a risk for public health and for the environment. Cytochrome P450 enzymes (CYPs) are involved in the detoxification of a wide range of compounds, and it has been established that these enzymes produce the initial biotransformation of many EDCs. In this work, a bionanoreactor based on the encapsulation of an enhanced peroxygenase CYPBM321B3 inside the capsid of bacteriophage P22 virus-like particles (VLPs) was designed and characterized. VLPs were functionalized with glucose oxidase to generate in situ hydrogen peroxide necessary to activate the transformation of bisphenol A, nonylphenol, 17β-estradiol, triclosan, and resorcinol. Catalytic parameters, as well as the chemical nature of reaction products are presented. The enzymatic nanoreactors showed specific activities varying from 0.175 to 0.456 min−1 in the transformation of these EDCs, which are equivalent to 22–77% of the activity obtained with free CYP. The capacity to transform structurally diverse compounds, easy production and glucose fueled catalytic activity make these enzymatic nanoreactors an interesting platform for enzyme delivery in the biomedical field.

Characteristics of two myoviruses induced from the coastal photoheterotrophic bacterium Porphyrobacter sp. YT40.

In this study, we characterized two induced myoviruses from one marine photoheterotrophic bacterium Porphyrobacter sp. YT40 belonging to the Sphingomonadales family in Alphaproteobacteria. The genome sequence of prophage A is ∼36.9kb with an average GC content of 67.1%, and its core or functional genes are homologous to Mu or Mu-like phages. Furthermore, induced viral particles from prophage A show a knob-like neck structure, which is only found in bacteriophage Mu. The genome size of prophage B is ∼36.8kb with an average GC content of 65.3%. Prophage B contains a conserved gene cluster Q-P-O-N-M-L, which is unique in P2 phages. Induced viral particles from prophage B display an icosahedral head with a diameter of ∼55nm and a 130±5nm long contractile tail. To our knowledge, this is the first report that characterizes the induced P2-like phage in marine Alphaproteobacteria. Phylogeny analyses suggest that these two types of prophages are commonly found in sequenced bacteria of the Sphingomonadales family. This study sheds light on the ongoing interaction between marine bacteria and phages, and improves our understanding of bacterial genomic plasticity and evolution.

How pirate phage interferes with helper phage: Comparison of the two distinct strategies

Pirate phages use the structural proteins encoded by unrelated helper phages to propagate. The best-studied example is the pirate P4 and helper P2 of coliphages, and it has been known that the Staphylococcus aureus pathogenicity islands (SaPIs) that can encode virulence factors act as pirate phages, too. When alone in the host, the pirate phages act as a prophage, but when the helper phage gene is also in the same host cell, the pirate phage has ability to exploit the helper phages structural proteins to produce pirate phage particles and spread, interfering with the helper phage production. The known helper phages in these systems are temperate phages. Interestingly, the interference of the pirate phage to the helper phage occurs in a different manner between the SaPI-helper system and the P4-P2 system. SaPIs cannot lyse a helper lysogen upon infection, while when a helper phage lyse a SaPI lysogen, most of the phage particles produced are the SaPI particles. On the contrary, in the P4-P2 system, a pirate phage P4 can lyse a helper P2 lysogen to produce mostly the P4 particles, while when P2 phage lyses a P4 lysogen, most of the produced phages are the P2 particles. Here, the consequences of these different strategies in the pirate and helper phage spreading among uninfected host is analyzed by using mathematical models. It is found that SaPI’s strategy interferes with the helper phage spreading significantly more than the P4’s strategy, because SaPI interferes with the helper phage’s main reproduction step, while P4 interferes only by forcing the helper lysogens to lyse. However, the interference is found to be weaker in the spatially structured environment than in the well-mixed environment. This is because, in the spatial setting, the system tends to self-organize so that the helper phages take over the front of propagation due to the need of helper phage for the pirate phage spreading.

Expression and purification of codon-optimized cre recombinase in E. coli

The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

A diarylamine derived from anthranilic acid inhibits ZIKV replication

Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3Hel) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3Hel.

1952. Bacteriophage Treatment Improves Survival of Mice Infected with Carbapenem-Resistant Klebsiella pneumoniae

BackgroundBacteriophage (phage) therapy is being considered as a treatment option for patients with multi-drug-resistant bacterial infections. However, there is a dearth of controlled clinical data to support therapeutic phage efficacy. As a first step toward addressing this deficiency, we tested the ability of two well-characterized phages, alone and in combination, to kill carbapenem-resistant Klebsiella pneumoniae (ST258) in blood in vitro and rescue mice from lethal ST258 infection.MethodsWild-type C57BL/6J mice were infected with a lethal inoculum of ST258 by intra-peritoneal (IP) injection followed 1 hour later by IP administration of lytic phage P1, P2, or P1+P2 at a multiplicity of infection (MOI) estimated at 1. Survival of each group of mice was tracked for 10 days. In separate experiments, mice were sacrificed at 1 hour, 24 hours, and 48 hours post-phage treatment. Mouse blood and tissues were collected at each timepoint for enumeration of bacteria and phage, screening for phage resistance, and histopathology.ResultsST258 survival in mouse blood in vitro was significantly less after 1 hour of incubation with P1 or P1+P2 (MOI 1) compared with the control group (no phage). Consistent with the in vitro data, none of the mice (0/15) in the control group (no phage) survived to 10 days post-infection, whereas 12/15, 14/15, and 15/15 mice survived in the P2, P1, and P1+P2-treated groups, respectively (P < 0.0001).ConclusionPrompt, systemic administration of lytic bacteriophages rescued mice from lethal ST258 infection. These data support the potential of phage therapy to effectively treat infections caused by ST258. It will be important to assess whether, for other phage-bacteria combinations, in vitro lysis in blood correlates with in vivo treatment efficacy and therefore may have predictive utility.Disclosures All Authors: No reported Disclosures.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Enclosed in a 23 MDa Procapsid

Scaffolding proteins (SPs) are required for the capsid shell assembly of many tailed double-stranded DNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, only one high-resolution structure is available for SPs within procapsids. Here, we use the inherent size limit of NMR to identify mobile segments of the 303-residue phage P22 SP free in solution and when incorporated into a ∼23 MDa procapsid complex. Free SP gives NMR signals from its acidic N-terminus (residues 1–40) and basic C-terminus (residues 264–303), whereas NMR signals from the middle segment (residues 41–263) are missing because of intermediate conformational exchange on the NMR chemical shift timescale. When SP is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix domain disappear because of binding to the procapsid interior. Signals from the N-terminal domain persist, indicating that this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus, coupled with its high content of negative charges, is likely important for dissociation and release of SP during the double-stranded DNA genome packaging step accompanying phage maturation.

Productive infection and transduction by bacteriophage P1 in the species Salmonella bongori

Productive infection and transduction by bacteriophage P1 in the species Salmonella bongori

Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens.

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3'-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.

Production of bacteriophage-encoded endolysin, LysP11, in Nicotiana benthamiana and its activity as a potent antimicrobial agent against Erysipelothrix rhusiopathiae.

We produced a biologically active phage-encoded endolysin, LysP11, in N. benthamiana. Plant-produced LysP11 exhibited robust antimicrobial activity against E. rhusiopathiae, and C-terminal domain of LysP11 bound specifically to E. rhusiopathiae. Bacterial resistance to antibiotics, a serious issue in terms of global public health, is one of the leading causes of death today. Thus, new antimicrobial agents are needed to combat pathogens. Recent research suggests that bacteriophages and endolysins derived from bacteriophages are potential alternatives to traditional antibiotics. Here, we examined the antimicrobial activity of LysP11, which is encoded by Propionibacterium phage P1.1 and comprises an N-terminal amidase-2 domain and a C-terminal domain with no homology to other bacteriophage endolysins. LysP11 was produced in Nicotiana benthamiana (N. benthamiana) using an Agrobacterium-mediated transient expression strategy. LysP11 was purified on microcrystalline cellulose-binding resin after attachment of the Clostridium thermocellum-derived family 3 cellulose-binding domain as an affinity tag. The affinity tag was removed using the small ubiquitin-related modifier (SUMO) domain and SUMO-specific protease. Plant-produced LysP11 showed strong antimicrobial activity toward Erysipelothrix rhusiopathiae (E. rhusiopathiae), mediated via lysis of the cell wall. Lytic activity was optimal at pH 8.0-9.0 (37°C) and increased at higher concentrations of NaCl up to 400mM. Furthermore, the C-terminal domain of LysP11 bound specifically to the E. rhusiopathiae cell wall. Based on these results, we propose that LysP11 is a potential candidate antimicrobial agent against E. rhusiopathiae.