Controlled release of metal phenolic network protected phage for treating bacterial infection

Phage is a promising therapeutic agent for treating antibiotic resistant bacteria. However, in the process of treatment, phage may be cleared by the immune system and cleaved by protease, which could affect the efficacy of phage. In order to solve the above problems, phage encapsulation is usually adopted. In this study, we employed metal phenolic network (MPN) for efficient phage encapsulation which could protect phage from the cleavage of protease, and keep cytotoxicity weak. In the model of skin wound infection, the encapsulated phage could be released in response to pH change to achieve good antibacterial effect. Furthermore, the MPN encapsulation could prolong the T4 phage residence time at the wound. Our findings suggest that MPN can be a promising material for phage encapsulation.

Bacterial Consumption of T4 Phages

The bacterial consumption of viruses not been reported on as of yet even though bacteria feed on almost anything. Viruses are widely distributed but have no acknowledged active biocontrol. Viral biomass undoubtedly reintegrates trophic cycles; however, the mechanisms of this phase still remain unknown. 13C-labelled T4 phages monitor the increase of the density of the bacterial DNA concomitant with the decrease of plaque forming units. We used 12C T4 phages as a control. T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole the source of carbon, nitrogen, and phosphorus. Bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as in the gut or in water.

Immune Response to Therapeutic Staphylococcal Bacteriophages in Mammals: Kinetics of Induction, Immunogenic Structural Proteins, Natural and Induced Antibodies

Bacteriophages are able to affect the human immune system. Phage-specific antibodies are considered as major factors shaping phage pharmacokinetics and bioavailability. So far, general knowledge of phage antigenicity nevertheless remains extremely limited. Here we present comparative studies of immunogenicity in two therapeutic bacteriophages, A3R and 676Z, active against Staphylococcus aureus, routinely applied in patients at the Phage Therapy Unit, Poland. Comparison of the overall ability of whole phages to induce specific antibodies in a murine model revealed typical kinetics of IgM and IgG induction by these two phages. In further studies we identified the location of four phage proteins in the virions, with the focus on the external capsid head (Mcp) or tail sheath (TmpH) or an unidentified precise location (ORF059 and ORF096), and we confirmed their role as structural proteins of these viruses. Next, we compared the immune response elicited by these proteins after phage administration in mice. Similar to that in T4 phage, Mcp was the major element of the capsid that induced specific antibodies. Studies of protein-specific sera revealed that antibodies specific to ORF096 were able to neutralize antibacterial activity of the phages. In humans (population level), none of the studied proteins plays a particular role in the induction of specific antibodies; thus none potentially affects in a particular way the effectiveness of A3R and 676Z. Also in patients subjected to phage therapy, we did not observe increased specific immune responses to the investigated proteins.

Bacterial Predation on T4 Phages

Background: Bacterial consumption of viruses has never yet been reported, even though bacteria feed on almost anything. Viruses are omnipresent predators for all organisms, but have no acknowledged active biocontrol. The viral biomass undoubtedly reintegrates the trophic cycles, however the mechanisms of this phase still remain unknown.Methods: Here, we used stable isotope probing with 13C labelled T4 phages to monitor the increase of density of the bacterial DNA concomitant with the decrease of plaque forming units. We used 12C T4 phages as control.Results: T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole source of carbon, nitrogen and phosphorus.Conclusions: bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as gut or water.

Bacterial predation on T4 phages

Bacterial consumption of viruses has never yet been reported, even though bacteria feed on almost anything. Viruses are omnipresent predators for all organisms, but have no acknowledged active biocontrol. The viral biomass undoubtedly reintegrates the trophic cycles, however the mechanisms of this phase still remain unknown. Here, we show that an E. coli infecting virus - the coliphage T4- was actively consumed by bacteria. Using stable isotope probing with 13C labelled T4 phages, T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole source of carbon, nitrogen and phosphorus. Results demonstrate how bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as gut or water.

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 K12 strain, but not of the O157 strain, for adsorption, even though OmpC from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in Loop 4 of the K12 OmpC as essential for T4 phage adsorption in the copresence of Loop 1 and Loop 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 digital 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 K12 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 composing the digital tip (DT) region of the long tail fibers of bacteriophage T4 largely determines its 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 is 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.

Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research

Hydrophobicity is one of the most critical factors governing the adsorption of molecules and objects, such as virions, on surfaces. Even moderate change of wetting angle of plastic surfaces causes a drastic decrease ranging from 2 to 5 logs of the viruses (e.g., T4 phage) in the suspension due to adsorption on polymer vials' walls. The effect varies immensely in seemingly identical containers but purchased from different vendors. Comparison of glass, polyethylene, polypropylene, and polystyrene containers revealed a threshold in the wetting angle of around 95°: virions adsorb on the surface of more hydrophobic containers, while in more hydrophilic vials, phage suspensions are stable. The polypropylene surface of the Eppendorf-type and Falcon-type can accommodate from around 108 PFU/ml to around 1010 PFU/ml from the suspension. The adsorption onto the container’s wall might result in complete scavenging of virions from the bulk. We developed two methods to overcome this issue. The addition of surfactant Tween20 and/or plasma treatment provides a remedy by modulating surface wettability and inhibiting virions' adsorption. Plastic containers are essential consumables in the daily use of many bio-laboratories. Thus, this is important not only for phage-related research (e.g., the use of phage therapies as an alternative for antibiotics) but also for data comparison and reproducibility in the field of biochemistry and virology.

Structural basis of superinfection exclusion by bacteriophage T4 Spackle

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages – a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

Optimization of T4 phage engineering via CRISPR/Cas9

A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03–3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.