Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.

• Keywords
• Bacteriophage, Biosensing Techniques, CRISPR-Cas10, Herelleviridae, Poly histidine Tag, Staphylococcus aureus,
• MeSH
• Biosensing Techniques * methods   MeSH
• CRISPR-Cas Systems * genetics   MeSH
• Gene Editing methods   MeSH
• Staphylococcus Phages * genetics   MeSH
• Staphylococcus virology   MeSH
• Publication type
• Journal Article MeSH

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.

• Keywords
• CRISPR CAS, Nagoya Protocol, antibiotic resistance, bacteriophage therapy, bacteriophages,
• Publication type
• Journal Article MeSH

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.

• Keywords
• Kayvirus, MALDI-MS, Staphylococcus, Viral proteins, bacteriophages, phage therapy,
• MeSH
• Biological Products isolation & purification   MeSH
• Chemical Fractionation methods   MeSH
• Humans MeSH
• Virus Replication MeSH
• Cluster Analysis MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   MeSH
• Staphylococcus Phages classification   metabolism   MeSH
• Staphylococcus aureus virology   MeSH
• Viral Proteins analysis   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biological Products MeSH
• viron MeSH Browser
• Viral Proteins MeSH

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

• Keywords
• Endolysin, Endopeptidases, Enzybiotics, Src homology domains, Staphylococcal infections, Staphylococcus bacteriophage,
• MeSH
• Endopeptidases genetics   isolation & purification   metabolism   MeSH
• Host Specificity * MeSH
• Mutant Proteins genetics   isolation & purification   metabolism   MeSH
• Myoviridae enzymology   genetics   physiology   MeSH
• Peptidoglycan metabolism   MeSH
• Protein Domains MeSH
• Sequence Deletion * MeSH
• Staphylococcus virology   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• endolysin MeSH Browser
• Endopeptidases MeSH
• Mutant Proteins MeSH
• Peptidoglycan MeSH