gpwac of the T4-type bacteriophages: structure, function, and evolution of a segmented coiled-coil protein that controls viral infectivity

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the approximately 50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.     

Diversity and dynamics of bacteriophages in horse feces

The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5–11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (10³-10⁷ plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain.     

Effect of O-acetylation of O antigen of <Emphasis Type="Italic">Escherichia coli</Emphasis> lipopolysaccharide on the nonspecific barrier function of the outer membrane

Comparison of the methods for determination of permeability of the outer membrane of Escherichia coli strain 4s and its mutants was carried out. The studied isogenic strains E. coli 4s were obtained by selection of spontaneous mutants according to their sensitivity to bacteriophages recognizing the surface O antigen of the outer membrane lipopolysaccharide as a primary receptor. The variants differed in the presence and (de)acetylation of the lipopolysaccharide O antigen. A peptide antibiotic polymyxin, plasmid DNA, and lysozyme were used as probes. The role of acetylation of the O antigen of the lipopolysaccaride of E. coli outer membrane in modification of its permeability (correlating with bacteriophage sensitivity of the cells) was confirmed. Kinetic analysis using lysozyme was shown to be the optimal method for determination of the barrier function of E. coli outer membrane.     

A Simple Method for Extraction of the Horse Feces Virome DNA,Suitable for Oxford Nanopore Sequencing

Microbiology - Metagenomics of the viromes associated with symbiotic microbial communities is a rapidly expanding field of research. Most of the relevant studies deal with the viral communities of...     

Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.     

Prospects of the use of bacteriophage-based virus-like particles in the creation of anthrax vaccines

The profitability of vaccine production is less than that of other pharmaceutical goods worldwide. Thus, the cost of the vaccine substance determines the range of vaccines available for use. This is of particular importance for veterinary vaccines. In this review, we have surveyed the published data on exploited vaccines and concluded that the immunogenicity of antigen substances based on whole virions is higher than that of soluble antigens. The physiological basis of this phenomenon remains unknown; however, it may explain why most of the described recombinant vaccines have not yet been put into practice. All practically implemented antiviral vaccines (except that for hepatitis B) are based on viral substances produced by conventional cultural technologies. In light of this observation, an approach to the development of a universal platform for recombinant vaccines produced in the form of virus-like particles is suggested. To this end, a technique of designing fused bifunctional derivatives of bacteriophage proteins containing antigens of interest should be involved. The approach is depicted with the use of the protective anthrax antigen, a conventional vaccine antigen.     

Diversity and dynamics of bacteriophages in horse feces

The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5-11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (10(3)-10(7) plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain.