Modification of a Tumor-Targeting Bacteriophage for Potential Diagnostic Applications

Background: Tumor-targeting bacteriophages can be used as a versatile new platform for the delivery of diagnostic imaging agents and therapeutic cargo. This became possible due to the development of viral capsid modification method. Earlier in our laboratory and using phage display technology, phages to malignant breast cancer cells MDA-MB 231 were obtained. The goal of this study was the optimization of phage modification and the assessment of the effect of the latter on the efficiency of phage particle penetration into MDA-MB 231 cells. Methods: In this work, we used several methods, such as chemical phage modification using FAM-NHS ester, spectrophotometry, phage amplification, sequencing, phage titration, flow cytometry, and confocal microscopy. Results: We performed chemical phage modification using different concentrations of FAM-NHS dye (0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM). It was shown that with an increase of the modification degree, the phage titer decreases. The maximum modification coefficient of the phage envelope with the FAM–NHS dye was observed with 4 mM modifying agent and had approximately 804,2 FAM molecules per phage. Through the immunofluorescence staining and flow cytometry methods, it was shown that the modified bacteriophage retains the ability to internalize into MDA-MB-231 cells. The estimation of the number of phages that could have penetrated into one tumor cell was conducted. Conclusions: Optimizing the conditions for phage modification can be an effective strategy for producing tumor-targeting diagnostic and therapeutic agents, i.e., theranostic drugs.

Evolution of the complex treatment of purulent diseases of the hand including laser irradiation (a review)

Effects of 1270 nm laser light irradiation at phage particles of virulent klebsiellosis bacteriophage were studied. The medical klebsiellosis bacteriophage, manufactured industrially, was taken as the study object. Klebsiella pneumonia N 296, sensitive to the selected phage, was used as a test-culture. An experimental device manufactured by LTD «New surgical technologies» was used as a source of light. Semiconductor diodes generate light with wavelength 1270 nm (1268–1272 nm) in the continuous mode. The number of viable phage particles in the initial solution of klebsiellosis bacteriophage was 5×108. Irradiation of the phage with 1270 nm laser light decreased the number of viable phage particles to 105. Results did not practically depend on the exposure time, i. e. phage titers were equally reduced when exposed to laser light for 5 min, 10 min and 15 min. Irradiation of Klebsiella bacteriophage with 1270 nm laser light reduced the number of viable phage particles by 3 log orders (initial titer was 108; after irradiation – 105 negative phage colonies). It is indicative of their damage. Mechanisms of phage particle damage should be the object of further research so as to defi ne if laser irradiation with the above mentioned wavelengths can be used in medical practice.

Swarm-mediated phage transport disrupts a biofilm inherently protected from phage penetration

Phage therapy is the treatment of chronic bacterial infections by virus that kill bacteria and it has shown promise in combating antimicrobial resistance (AMR). A typical phage particle is around 100 times bigger than a typical antibiotic molecule. Due to larger size, a phage particle diffuses slower than an antibiotic molecule, and can get trapped in the polymeric mesh of biofilm matrix. We report that a swarm of Capnocytophaga gingivalis, a bacterium abundant in the human oral microbiota, can actively transport phages over long distances. By tracking fluorescently labeled lambda phage particles that do not infect C. gingivalis, we demonstrate active predator transport by a C. gingivalis swarm. As a result, the rate of disruption of the prey i.e., an Escherichia coli colony increases 10 times. Production of curli fiber by a mature E. coli biofilm blocks the intercellular space and is known to inhibit the diffusion of phages within a biofilm. We find that C. gingivalis forms tunnels within the prey biofilm. When phages are actively delivered, curli fiber containing E. coli biofilms are no longer protected against phage infection. Our results demonstrate that active delivery of the predator by a self-propelled swarm might improve the pharmacokinetics of phage therapy. This can lead to the development of a tool to combat chronic AMR biofilms.

A Cell-free Infection System to Study Translation, Replication and Phage-particle Production During Infection of E. coli by Bacteriophage Qβ

Background Viruses infect all kingdoms of life, and new species are continuously being discovered. The single-stranded (+)-RNA viruses comprise the largest group of viruses, which includes pathogens such as Dengue virus, Corona virus and West Nile virus. Also, the simple bacteriophage Qβ belongs to this group of viruses. Studies of the mechanism of Qβ infection can increase our general understanding of the single-stranded (+)-RNA viruses, which can be exploited in the pursuit to treat and prevent of diseases caused by pathogenic (+)-RNA viruses.MethodsIn this study, we have analysed the production of infectious Qβ phage particles in three different cell-free infection systems upon addition of the Qβ genome as a template. The cell-free infection systems were based on cell-free protein expression systems: two commercial systems and one custom-made system. We studied the course of infection by analysing the production of viral RNA, proteins and phage particles produced in the cell-free reactions. The replication of the viral RNA was determined by RT-PCR, while the translation of the viral proteins was examined by radiolabelling, and the production of infectious phage particles was evaluated by double-layered plaque assays. ResultsBacteriophage Qβ was found to replicate in two of the three tested cell-free infection systems. Specifically, the viral RNA was replicated, the viral proteins were translated, and infectious phage particles were produced in the cell-free infection systems. The pattern of translation regulation of the viral proteins appeared similar to in vivo infection. Infectious Qβ phage particles were produced at yields of 25.4 × 104 PFU/μL reaction and 2.5 × 103 PFU/μL reaction in the commercial and custom-made system, respectively. Importantly, intact Qβ phage particles were shown not to replicate in the cell-free infection systems under the tested conditions. ConclusionCell-free infection systems can support replication of viral RNA, translation of viral proteins and self-assembly of infectious Qβ phage particles. We provide opportunities for further optimisation of the phage particle yield. Cell-free infection systems can be used in the future to study newly discovered viruses, the development of antiviral and antibacterial drugs, and in biotechnology.

Structure and Function of the T4 Spackle Protein Gp61.3

The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity—e and 5. Gene e encodes the well-known T4 lysozyme (commonly called T4L) that functions to break the peptidoglycan layer late in the infection cycle, which is required for liberating newly assembled phage progeny. Gene product 5 (gp5) is the tail-associated lysozyme, a component of the phage particle. It forms a spike at the tip of the tail tube and functions to pierce the outer membrane of the Escherichia coli host cell after the phage has attached to the cell surface. Gp5 contains a T4L-like lysozyme domain that locally digests the peptidoglycan layer upon infection. The T4 Spackle protein (encoded by gene 61.3) has been thought to play a role in the inhibition of gp5 lysozyme activity and, as a consequence, in making cells infected by bacteriophage T4 resistant to later infection by T4 and closely related phages. Here we show that (1) gp61.3 is secreted into the periplasm where its N-terminal periplasm-targeting peptide is cleaved off; (2) gp61.3 forms a 1:1 complex with the lysozyme domain of gp5 (gp5Lys); (3) gp61.3 selectively inhibits the activity of gp5, but not that of T4L; (4) overexpression of gp5 causes cell lysis. We also report a crystal structure of the gp61.3-gp5Lys complex that demonstrates that unlike other known lysozyme inhibitors, gp61.3 does not interact with the active site cleft. Instead, it forms a “wall” that blocks access of an extended polysaccharide substrate to the cleft and, possibly, locks the enzyme in an “open-jaw”-like conformation making catalysis impossible.

Bioprospecting Staphylococcus Phages with Therapeutic and Bio-Control Potential

Emergence of antibiotic-resistant bacteria is a serious threat to the public health. This is also true for Staphylococcus aureus and other staphylococci. Staphylococcus phages Stab20, Stab21, Stab22, and Stab23, were isolated in Albania. Based on genomic and phylogenetic analysis, they were classified to genus Kayvirus of the subfamily Twortvirinae. In this work, we describe the in-depth characterization of the phages that electron microscopy confirmed to be myoviruses. These phages showed tolerance to pH range of 5.4 to 9.4, to maximum UV radiation energy of 25 µJ/cm2, to temperatures up to 45 °C, and to ethanol concentrations up to 25%, and complete resistance to chloroform. The adsorption rate constants of the phages ranged between 1.0 × 10−9 mL/min and 4.7 × 10−9 mL/min, and the burst size was from 42 to 130 plaque-forming units. The phages Stab20, 21, 22, and 23, originally isolated using Staphylococcus xylosus as a host, demonstrated varied host ranges among different Staphylococcus strains suggesting that they could be included in cocktail formulations for therapeutic or bio-control purpose. Phage particle proteomes, consisting on average of ca 60–70 gene products, revealed, in addition to straight-forward structural proteins, also the presence of enzymes such DNA polymerase, helicases, recombinases, exonucleases, and RNA ligase polymer. They are likely to be injected into the bacteria along with the genomic DNA to take over the host metabolism as soon as possible after infection.

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

Characterization of the Replication, Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12

ABSTRACT We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (φZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of φZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear φZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear φZL12. φZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of φZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage φZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

The Genome and Structural Proteome of YuA, a New Pseudomonas aeruginosa Phage Resembling M6

ABSTRACT Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor λ-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, φJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a σ54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host σ54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.