Long Tail Fibers Research Articles

Biological properties of Staphylococcus virus ΦSA012 for phage therapy

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

RB49-like Bacteriophages Recognize O Antigens as One of the Alternative Primary Receptors.

The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer.

Molecular anatomy of the receptor binding module of a bacteriophage long tail fiber.

Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.

Modified Bacteriophage Tail Fiber Proteins for Labeling, Immobilization, Capture, and Detection of Bacteria.

A critical component of bacterial detection assays is choosing a suitable affinity molecule that retains sensitivity and specificity for the target pathogen over a wide range of in situ applications. Bacteriophages (phages) are bacterial viruses that bind and infect their host cells with unmatched specificity. Phage host range is often determined by their long tail fibers (LTFs) that mediate adsorption of the virus particle to potential bacterial host cells, by binding to specific cell surface receptors. The inherent specificity of the LTFs for distinct bacterial species makes them ideal candidates for development into recombinant affinity molecules. In this chapter, we describe the development of the Salmonella phage S16 LTF (S16 LTF) into an affinity molecule as part of a novel assay to detect Salmonella cells. The enzyme-linked long tail fiber assay (ELLTA) involves two steps: (1) Immobilization and separation of Salmonella cells using S16 LTF-coated paramagnetic beads (LTF-MBs), and (2) Labeling of bead-captured Salmonella using horseradish peroxidase-conjugated S16 LTF (HRP-LTF). Rapid HRP-mediated conversion of a chromogenic substrate provides visual confirmation for the presence of Salmonella. Overall, the ELLTA assay requires as little as 2h to detect as few as 102cfu/ml Salmonella cells from liquid culture. The absorbance of the enzyme-generated color substrate is largely proportional to the present bacterial concentrations between 102 and 107cfu/ml, providing semiquantitative determination of Salmonella cell counts. The methodology described in this chapter can be adapted for other phage receptor-binding proteins, to develop ELLTAs for the detection of other relevant bacterial pathogens.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition

The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range. Here we present the crystal structure of an adhesin (gp38) attached to a trimeric β-helical tip (gp37) from the Salmonella phage S16 LTF. Gp38 contains rare polyglycine type II helices folded into a packed lattice, herein designated "PGII sandwich." Sequence variability within the domain is limited to surface-exposed helices and distal loops that form putative receptor-binding sites. In silico analyses revealed a prevalence of the adhesin architecture among T-even phages, excluding the archetypal T4 phage. Overall, S16 LTF provides a valuable model for understanding binding mechanisms of phage adhesins, and for engineering of phage adhesins with expandable or modulated host ranges.

A Novel Long-Tail Fiber Current Sensor Based on Fiber Loop Ring-Down Spectroscopy and Fabry-Perot Cavity Filled With Magnetic Fluid

A novel long-tail optical fiber current sensor based on fiber loop ring-down spectroscopy (FLRDS) and magnetic-tunable refractive index properties and magnetic-tunable transmission properties of magnetic fluid is proposed and demonstrated for the first time. In the sensing system, a Fabry-Perot (F-P) cavity filled with magnetic fluid is used as the sensing head, and the sensitivity of current-sensing has been enhanced significantly. Relations between magnetic field and refractive index of magnetic fluid are measured. Refractive index of magnetic fluid increases linearly as the magnetic field increases and according to the F-P interferometer principle, the best gap length of F-P cavity is obtained. In this paper, the FLRDS sensing system has been theoretically modeled, and a new mathematical model developed for optimizing the coupling ratio of couplers has been presented, and the best coupling ratio has been obtained. The new mathematical model could also be used in the similar ring-down systems. Finally, the performances of the current-sensing system are tested by applying different measured currents. The final results indicate that a sensitivity of 14.88 mA/ $\mu $ s is achieved.

Long tail fibres of the novel broad‐host‐range T‐even bacteriophage S16 specifically recognize SalmonellaOmpC

We report isolation and characterization of the novel T4-like Salmonella bacteriophage vB_SenM-S16. S16 features a T-even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full-length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects 'rough' Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.

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.

Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins.

The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively. Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long. Their assembly requires the viral chaperones gp57A and gp38. Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement. Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution. Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E. A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip. Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

Stoichiometry and Domainal Organization of the Long Tail-fiber of Bacteriophage T4: A Hinged Viral Adhesin

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each ∼70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36 (23 kDa) and gp35 (30 kDa). LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35. We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds. These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35=3:3:3:1. Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains. At the proximal end is a globular domain of ∼145 kDa that becomes incorporated into the baseplate. It is followed by a rod-like shaft (33 × 4 nm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long. The proximal half-fiber terminates in three globular domains. The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 × 2.5 nm; 31 kDa). The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers. The latter hinge occurs at a site of local non-equivalence (the “kneecap”) at which density, correlated with the presence of gp35, bulges asymmetrically out on one side. Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe. Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12). Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded β-helix.

The influence of bacteriophage T4D short tailfibers on long tailfiber extension

The short tailfibers (STF) of bacteriophage T4 are important in the process of phage infection, in which they attach irreversibly to the bacterial surface. In this study we determined the effect of the short tailfibers on the extension of the long tailfibers (LTF) of the bacteriophage, by means of electric birefringence measurements. The following conclusions could be drawn: (i) The products of gene 12 carry a negative charge, which manifests itself as a contribution of two elementary charges to the effective charge of the particle in solution. (ii) The STF are folded closely to the baseplate structure. (iii) LTF extension is influenced by the presence of the STF which indicates that the baseplate structure is important for the movement of the LTF. The pH at which the LTF begin to extend (the “locking pH”) is not affected by the presence of the STF. (iv) The difference in rotational diffusion coefficient between T4, T2, and T6 is not caused by the short tailfibers.