Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations

PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Phage display as a tool for identifying HIV-1 broadly neutralizing antibodies

Combinatorial biology methods offer a good solution for targeting interactions of specific molecules by a high-throughput screening and are widely used for drug development, diagnostics, identification of novel monoclonal antibodies, search for linear peptide mimetics of discontinuous epitopes for the development of immunogens or vaccine components. Among all currently available techniques, phage display remains one of the most popular approaches. Despite being a fairly old method, phage display is still widely used for studying protein-protein, peptide-protein and DNA-protein interactions due to its relative simplicity and versatility. Phage display allows highly representative libraries of peptides, proteins or their fragments to be created. Each phage particle in a library displays peptides or proteins fused to its coat protein and simultaneously carries the DNA sequence encoding the displayed peptide/protein in its genome. The biopanning procedure allows isolation of specific clones for almost any target, and due to the physical link between the genotype and the phenotype of recombinant phage particles it is possible to determine the structure of selected molecules. Phage display technology continues to play an important role in HIV research. A major obstacle to the development of an effective HIV vaccine is an extensive genetic and antigenic variability of the virus. According to recent data, in order to provide protection against HIV infection, the so-called broadly neutralizing antibodies that are cross-reactive against multiple viral strains of HIV must be induced, which makes the identification of such antibodies a key area of HIV vaccinology. In this review, we discuss the use of phage display as a tool for identification of HIV-specific antibodies with broad neutralizing activity. We provide an outline of phage display technology, briefly describe the design of antibody phage libraries and the affinity selection procedure, and discuss the biology of HIV-1-specific broadly neutralizing antibodies. Finally, we summarize the studies aimed at identification of broadly neutralizing antibodies using various types of phage libraries.

Whole Genome Sequence Analysis of Phage-Resistant Listeria monocytogenes Serotype 1/2a Strains from Turkey Processing Plants

Listeria monocytogenes is a Gram-positive bacterial pathogen and the causative agent of listeriosis, a severe foodborne infection. L. monocytogenes is notorious for its ability to persist in food processing environments (FPEs) via a variety of adaptive traits. Even though traits such as cold tolerance, biofilm formation and sanitizer resistance have been extensively investigated for their roles in persistence of L. monocytogenes in FPEs, much less is known about resistance to bacteriophages. Previous studies explored phage resistance mechanisms in laboratory-created mutants but it is imperative to investigate phage resistance that is naturally exhibited in FPE-derived strains. Here, we integrated the analysis of whole genome sequence data from a panel of serotype 1/2a strains of sequence types 321 and 391 from turkey processing plants, with the determination of cell surface substituents required for phage adsorption and phage infection assays with the four wide-host-range phages A511, P100, 20422-1 and 805405-1. Using a specific set of recombinant phage protein probes, we discovered that phage-resistant strains lacked one or both of the serogroup 1/2-specific wall teichoic acid carbohydrate decorations, N-acetylglucosamine and rhamnose. Furthermore, these phage-resistant strains harbored substitutions in lmo1080, lmo1081, and lmo2550, which mediate carbohydrate decoration of the wall teichoic acids.

Characterization of Phage Obtained from Methicillin -Resistant Staphylococcus aureus (MRSA)

Objective: The emergence of Staphylococcus aureus strains resistant to all antimicrobials and failure to discover new antibiotics have led researchers to phage therapy, which lost popularity after the discovery of antibiotics. The development of recombinant technology introduced the idea of creating lysogenic recombinant phages that provide controlled bacterial death and this required small- sized phages that were easy to manipulate. Our aim is to identify small-sized lysogenic bacteriophages that can be used safely in therapy. Method: The gene and protein map of the phage was created by analysis of sequencing after extracting a phage from the MRSA strain that is known to contain a small phage. Results: The phage was classified in Caudovirales spp. as it contains genes encoding tail proteins, and in Podoviridae spp. due to its genomic size and arrangement. Conclusion: To date, there are only sixteen phages from Podoviridae family uploaded on NCBI, and the phage described in this study is the seventeenth one. Only 41.4% of the ORFs (Open Reading Frames) in the genome could be matched with proteins using the NCBI BLAST. Recent studies suggest that 50-75% of bacteriophage ORFs do not correspond to any organism in GenBank. For better understanding of bacteriophages and their utilization in phage therapy, it is essential to sequence greater number of phages, and to discover their genomes and corresponding proteins. Since the genes and proteins of a lysogenic phage that can be used safely in recombinant phage therapies have been identified in our study, it will contribute to the relevant literature.

LysSAP26, a New Recombinant Phage Endolysin with a Broad Spectrum Antibacterial Activity

Multidrug-resistant (MDR) bacteria are a major threat to public health. Bacteriophage endolysins (lysins) are a promising alternative treatment to traditional antibiotics. However, the lysins currently under development are still underestimated. Herein, we cloned the lysin from the SAP-26 bacteriophage genome. The recombinant LysSAP26 protein inhibited the growth of carbapenem-resistant Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, oxacillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with minimum inhibitory concentrations of 5~80 µg/mL. In animal experiments, mice infected with A. baumannii were protected by LysSAP26, with a 40% survival rate. Transmission electron microscopy analysis confirmed that LysSAP26 treatment resulted in the destruction of bacterial cell walls. LysSAP26 is a new endolysin that can be applied to treat MDR A. baumannii, E. faecium, S. aureus, K. pneumoniae, P. aeruginosa, and E. coli infections, targeting both Gram-positive and Gram-negative bacteria.

The strategy of editing and modification of the genomes of bacterial viruses

In recent decades, a major problem for health systems around the world is the wide spread of bacterial pathogens that are resistant to various antimicrobial agents. A possible approach to solving this problem is the use of bacteriophages, viruses that specifically infect bacterial cells, as well as enzymes and proteins encoded in their genomes. The development of genomic editing technologies, including those based on CRISPR-Cas editing, makes it possible to create genetically engineered or recombinant phage particles with desired properties that are important for further practical application. In this review, we consider issues related to the characterization of bacteriophages as biological objects and as promising candidates for controlling the spread of antibioticresistant bacterial strains. We discuss modern approaches and strategies for modifying the phage genomes using various methods of genetic engineering and molecular biology to solve a variety of practical and research problems. Keywords: bacteriophages, phage genome editing, CRISPR-Cas system

The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10–1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.