Manipulating Interactions between T4 Phage Long Tail Fibers and Escherichia coli Receptors.

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K-12 strain, but not of the O157 strain, for adsorption, even though OmpCs from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in loop 4 of the K-12 OmpC as essential for T4 phage adsorption in the copresence of loops 1 and 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the distal tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K-12 strain. These results shed light on the mechanism underlying the phage host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein present in the distal tip (DT) region of the long tail fibers of T4 bacteriophages largely determines their host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which are likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

Bursting out: linking changes in nanotopography and biomechanical properties of biofilm-forming Escherichia coli to the T4 lytic cycle.

The bacteriophage infection cycle has been extensively studied, yet little is known about the nanostructure and mechanical changes that lead to bacterial lysis. Here, atomic force microscopy was used to study in real time and in situ the impact of the canonical phage T4 on the nanotopography and biomechanics of irreversibly attached, biofilm-forming E. coli cells. The results show that in contrast to the lytic cycle in planktonic cells, which ends explosively, anchored cells that are in the process of forming a biofilm undergo a more gradual lysis, developing distinct nanoscale lesions (~300 nm in diameter) within the cell envelope. Furthermore, it is shown that the envelope rigidity and cell elasticity decrease (>50% and >40%, respectively) following T4 infection, a process likely linked to changes in the nanostructure of infected cells. These insights show that the well-established lytic pathway of planktonic cells may be significantly different from that of biofilm-forming cells. Elucidating the lysis paradigm of these cells may advance biofilm removal and phage therapeutics.

Polycaprolactone film functionalized with bacteriophage T4 promotes antibacterial activity of food packaging toward Escherichia coli.

Bacteriophages (phages) have been extensively utilized as antibacterial agents in the food industry because of their host-specificity. However, their application in polymer films has been limited because of the lack of a strong attachment method for phage to the surface. We developed an antibacterial film by covalently immobilizing Escherichia coli (E. coli)-specific phage T4 on a polycaprolactone (PCL) film. The chemical bond formation was confirmed by XPS analysis, and the covalent attachment of phage T4 effectively inhibited E. coli growth even after external stimulation of the film by sonication. When applied as a packaging film for raw beef inoculated with E. coli O157:H7, the chemically functionalized PCL film showed approximately 30-fold higher bacterial inhibitory effects than the film with physically adsorbed phage T4. These results indicate the promising application potential of chemically functionalized PCL film with phage T4 as an antibacterial food packaging material against the foodborne pathogen E. coli.

Surface texture limits transfer of S. aureus, T4 bacteriophage, influenza B virus and human coronavirus.

Spread of pathogens on contaminated surfaces plays a key role in disease transmission. Surface technologies that control pathogen transfer can help control fomite transmission and are of great interest to public health. Here, we report a novel bead transfer method for evaluating fomite transmission in common laboratory settings. We show that this method meets several important criteria for quantitative test methods, including reasonableness, relevancy, resemblance, responsiveness, and repeatability, and therefore may be adaptable for standardization. In addition, this method can be applied to a wide variety of pathogens including bacteria, phage, and human viruses. Using the bead transfer method, we demonstrate that an engineered micropattern limits transfer of Staphylococcus aureus by 97.8% and T4 bacteriophage by 93.0% on silicone surfaces. Furthermore, the micropattern significantly reduces transfer of influenza B virus and human coronavirus on silicone and polypropylene surfaces. Our results highlight the potential of using surface texture as a valuable new strategy in combating infectious diseases.

Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses.

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

Modification of T2 phage infectivity toward Escherichia coli O157:H7 via using CRISPR/Cas9.

Phage therapy is getting considerable attention as a method for prophylaxis of food poisoning caused by Escherichia coli O157:H7, an important pathogen causing life-threatening bloody diarrhea. Despite previous studies have shown the feasibility of phage therapy to E. coli O157:H7, promising results have not been obtained in vivo yet. A major drawback of phage therapy is that bacteriophages have high specificity and cannot infect all the sub-strains of a particular pathogenic strain. To overcome this hurdle, we thought to establish a way to artificially expand the host-range of E. coli O157:H7-specific phages. To develop a proof-of-concept for this method, we focused on T2 phage, which cannot infect E. coli O157:H7 strains, and PP01 phage, which displays broad infectivity to them, and attempted to make T2 phage able to infect E. coli O157:H7 as efficiently as PP01. We report the trials of T2 genome editing using the CRISPR/Cas9 system and the modification of both long and short tail fibers of this phage based on comparison with PP01. The resultant recombinant showed the adsorption rate comparable to PP01. Thus, we provided the evidence that the short tail fiber of PP01 plays an important role in adsorption to E. coli O157:H7.

From Host to Phage Metabolism: Hot Tales of Phage T4's Takeover of E. coli.

The mechanisms by which bacteriophage T4 converts the metabolism of its E. coli host to one dedicated to progeny phage production was the subject of decades of intense research in many labs from the 1950s through the 1980s. Presently, a wide range of phages are starting to be used therapeutically and in many other applications, and also the range of phage sequence data available is skyrocketing. It is thus important to re-explore the extensive available data about the intricacies of the T4 infection process as summarized here, expand it to looking much more broadly at other genera of phages, and explore phage infections using newly-available modern techniques and a range of appropriate environmental conditions.

In Vivo Studies on the Influence of Bacteriophage Preparations on the Autoimmune Inflammatory Process.

Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.

Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease.

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

Protein determinants of phage T4 lysis inhibition.

Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.

Cationic phenylene ethynylene polymers and oligomers exhibit efficient antiviral activity.

The antiviral activities of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPE) and oligo-phenylene ethynylenes (OPE) were investigated using two model viruses, the T4 and MS2 bacteriophages. Under UV/visible light irradiation, significant antiviral activity was observed for all of the CPEs and OPEs; without irradiation, most of these compounds exhibited high inactivation activity against the MS2 phage and moderate inactivation ability against the T4 phage. Transmission electron microscopy (TEM) and SDS polyacrylamide gel electrophoresis (SDS-PAGE) reveal that the CPEs and OPEs exert their antiviral activity by partial disassembly of the phage particle structure in the dark and photochemical damage of the phage capsid protein under UV/visible light irradiation.

Unrestricted migration favours virulent pathogens in experimental metapopulations: evolutionary genetics of a rapacious life history.

Understanding pathogen infectivity and virulence requires combining insights from epidemiology, ecology, evolution and genetics. Although theoretical work in these fields has identified population structure as important for pathogen life-history evolution, experimental tests are scarce. Here, we explore the impact of population structure on life-history evolution in phage T4, a viral pathogen of Escherichia coli. The host-pathogen system is propagated as a metapopulation in which migration between subpopulations is either spatially restricted or unrestricted. Restricted migration favours pathogens with low infectivity and low virulence. Unrestricted migration favours pathogens that enter and exit their hosts quickly, although they are less productive owing to rapid extirpation of the host population. The rise of such 'rapacious' phage produces a 'tragedy of the commons', in which better competitors lower productivity. We have now identified a genetic basis for a rapacious life history. Mutations at a single locus (rI) cause increased virulence and are sufficient to account for a negative relationship between phage competitive ability and productivity. A higher frequency of rI mutants under unrestricted migration signifies the evolution of rapaciousness in this treatment. Conversely, spatially restricted migration favours a more 'prudent' pathogen strategy, in which the tragedy of the commons is averted. As our results illustrate, profound epidemiological and ecological consequences of life-history evolution in a pathogen can have a simple genetic cause.

Effects of temperature on the fitness cost of resistance to bacteriophage T4 in Escherichia coli.

Resistance to predation, herbivory, or disease often comes at a cost such that resistant genotypes are competitively inferior to their sensitive counterparts in the absence of predators, herbivores, or pathogens. The effects of this trade-off on natural populations depend on its sensitivity to environmental changes. We used Escherichia coli and bacteriophage T4 as a model predator/prey system to study the effects of temperature on the cost of resistance. An array of independent T4-resistant mutants, derived from a single ancestral strain of E. coli B, had a mean reduction in competitive fitness that depended strongly on environmental temperature; the cost of resistance generally increased with temperature. Genetic variance for fitness among phage-resistant mutants also depended on temperature; however, genetic variance increased at high and low thermal extremes. These results suggest that temperature is likely to be an important determinant of the consequences of predation in natural communities. We also discuss the underlying mechanistic basis for the cost of resistance in this system and its interaction with temperature.

Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid.

Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.

Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection.

This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich humic acid (AHA) added and coagulated with alum to form floc before filtration. The turbidity of the influent flocculated water was 6.4+/-1.5 NTU. Influent and filter effluent turbidity and particle counts were measured as well as headloss across the filter media. Filter effluent samples were collected for phage enumeration during three filter cycle stages: (i) filter ripening; (ii) stable operation; and (iii) end of filter cycle. Stable filter operation was defined according to a filter effluent turbidity goal of <0.3 NTU. Influent and filter effluent samples were subsequently exposed to UV light (254 nm) at 40 mJ/cm(2) using a low pressure UV collimated beam. The study found statistically significant differences (alpha=0.05) in the quantity of particle-associated phage present in the filter effluent during the three stages of filtration. There was reduced UV disinfection efficiency due to the presence of particle-associated phage in the filter effluent in trials with bacteriophage MS2 and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation of phage relative to particle-free control conditions for both phages. Trends in filter effluent turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The results therefore suggest that maintenance of optimum filtration conditions upstream of UV disinfection is a critical barrier to particle-associated viruses.

Application of microbial risk assessment on a residentially-operated bio-toilet.

The Sustainable Sanitation System is a new wastewater treatment system that incorporates a non-flushing toilet (Bio-toilet) that converts excreta into a reusable resource (as fertilizer or humus for organic agriculture) and reduces the pollution load to environments of the rivers, the lakes, and the sea. However, the risk of exposure to pathogens should be considered, because excrement is stored in the Bio-toilet. The aim of the present work is to analyze the health risk of dealing with the matrix (excreta and urine mixed with sawdust) of the Bio-toilet. Therefore, the fate of pathogenic viruses was investigated using coliphages as a virus index, and the modeling of the die-off rate in matrix was introduced. Then the microbial risk assessment was applied to a Bio-toilet that was actually used in a residential house; the infection risks of rotavirus and enterovirus as reference pathogens were calculated. According to the lab-scale experiment using coliphages for investing the die-off rate of viruses in the Bio-toilet, Qbeta had a higher die-off, which was greatly influenced by the water content and temperature. On the other hand, T4 showed a lower rate and was independent of water content. Therefore, these two phages' data were used as critical examples, such as viruses having high or low possibilities of remaining in the Bio-toilet during the risk assessment analysis. As the result of the risk assessment, the storage time required for an acceptable infectious risk level has wide variations in both rotavirus and enterovirus cases depending on the phage that was used. These were 0-260 days' and 0-160 days' difference, respectively.

Association and dissociation of the cell puncturing complex of bacteriophage T4 is controlled by both pH and temperature.

The tail lysozyme, gp5, of bacteriophage T4 is a trimeric protein and all the subunits are nicked between Ser351 and Ala352 during assembly through processing. When subsequently heated, the resulting (gp5*)(3) (gp5C)(3) (the asterisk "*" denotes that the intact pre-gp5 trimer has been nicked) dissociates into three gp5* (three independent N-terminal monomeric peptides, that carry lysozyme moieties at the C-termini of gp5*), and a C-terminal trimeric beta-helical structure (gp5C)(3). The interaction between gp27 and gp5* during infection is sundered by reducing pH. This dissociation would be physiologically relevant because the lysozyme moieties should be free in the periplasm (where the pH is low) and would digest the peptidoglycan layer, thereby enabling the tail tube to contact the inner membrane, and probably help to form a pore for DNA injection.

Local migration promotes competitive restraint in a host-pathogen 'tragedy of the commons'.

Fragmented populations possess an intriguing duplicity: even if subpopulations are reliably extinction-prone, asynchrony in local extinctions and recolonizations makes global persistence possible. Migration is a double-edged sword in such cases: too little migration prevents recolonization of extinct patches, whereas too much synchronizes subpopulations, raising the likelihood of global extinction. Both edges of this proverbial sword have been explored by manipulating the rate of migration within experimental populations. However, few experiments have examined how the evolutionary ecology of fragmented populations depends on the pattern of migration. Here, we show that the migration pattern affects both coexistence and evolution within a community of bacterial hosts (Escherichia coli) and viral pathogens (T4 coliphage) distributed across a large network of subpopulations. In particular, different patterns of migration select for distinct pathogen strategies, which we term 'rapacious' and 'prudent'. These strategies define a 'tragedy of the commons': rapacious phage displace prudent variants for shared host resources, but prudent phage are more productive when alone. We find that prudent phage dominate when migration is spatially restricted, while rapacious phage evolve under unrestricted migration. Thus, migration pattern alone can determine whether a de novo tragedy of the commons is resolved in favour of restraint.

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome.

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.

Inactivation of particle-associated viral surrogates by ultraviolet light.

This study investigated whether colloid-sized particles can enmesh and protect viruses from 254-nm ultraviolet (UV) light and sought to determine the particle characteristics (e.g. size, chemical composition) that are most relevant in causing a protective effect. Two viral surrogates (MS2 coliphage and bacteriophage T4), three types of particles (kaolin clay, humic acid powder, and activated sludge), two coagulants (alum and ferric chloride), two filtration conditions (none and 0.45 microm), and two UV doses (40 and 80 mJ/cm2 for MS2 coliphage; 2 and 7 mJ/cm2 for bacteriophage T4) were considered in a series of bench-scale UV collimated beam experiments. Transmission electron microscopy was used to qualitatively confirm the phage particle-association after coagulation. Humic acid and activated sludge floc particles shielded both viral surrogates to a statistically significant degree (with >99% confidence) relative to particle-free control conditions, while the kaolin clay particles provided no significant protection. The results of the study suggest that particles <2 microm in diameter are large enough to protect viruses from UV light and that particulate chemical composition (e.g. UV-absorbing organic content) may be a critical factor in the survival of particle-associated viruses during UV disinfection.