Martin Benešík

Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate

Significance Resistance to antibiotics is widespread among pathogenic bacteria, including Staphylococcus aureus, which cause serious human diseases. Bacteriophages from the Twort-like genus of the family Myoviridae infect and kill pathogenic bacteria, and therefore are used to treat bacterial diseases. Detailed knowledge of the interactions of phages with bacterial cells is a prerequisite for the effective and safe use of phages for medical purposes. However, the molecular details of the processes regulating infections by these phages are not well understood. We used cryo-electron microscopy and tomography to describe the series of structural changes of a bacteriophage phi812 virion required to deliver its genome into the S. aureus cell. Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of the tail sheath enables the tail tube to penetrate through the bacterial cell wall and serve as a channel for the transport of the phage genome into the cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with “double-layered” baseplates were unknown. We used cryo-electron microscopy to show that the binding of the Twort-like phage phi812 to the Staphylococcus aureus cell wall requires a 210° rotation of the heterohexameric receptor-binding and tripod protein complexes within its baseplate about an axis perpendicular to the sixfold axis of the tail. This rotation reorients the receptor-binding proteins to point away from the phage head, and also results in disruption of the interaction of the tripod proteins with the tail sheath, hence triggering its contraction. However, the tail sheath contraction of Myoviridae phages is not sufficient to induce genome ejection. We show that the end of the phi812 double-stranded DNA genome is bound to one protein subunit from a connector complex that also forms an interface between the phage head and tail. The tail sheath contraction induces conformational changes of the neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. A subsequent structural change of the tail tube induced by its interaction with the S. aureus cell is required for the genome’s release.

Genetically modified bacteriophages in applied microbiology

Bacteriophages represent a simple viral model of basic research with many possibilities for practical application. Due to their ability to infect and kill bacteria, their potential in the treatment of bacterial infection has been examined since their discovery. With advances in molecular biology and gene engineering, the phage application spectrum has been expanded to various medical and biotechnological fields. The construction of bacteriophages with an extended host range or longer viability in the mammalian bloodstream enhances their potential as an alternative to conventional antibiotic treatment. Insertion of active depolymerase genes to their genomes can enforce the biofilm disposal. They can also be engineered to transfer various compounds to the eukaryotic organisms and the bacterial culture, applicable for the vaccine, drug or gene delivery. Phage recombinant lytic enzymes can be applied as enzybiotics in medicine as well as in biotechnology for pathogen detection or programmed cell death in bacterial expression strains. Besides, modified bacteriophages with high specificity can be applied as bioprobes in detection tools to estimate the presence of pathogens in food industry, or utilized in the control of food‐borne pathogens as part of the constructed phage‐based biosorbents.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity

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.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

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.

Antimicrobial effect of commercial phage preparation Stafal® on biofilm and planktonic forms of methicillin-resistant Staphylococcus aureus

Staphylococcus aureus may be a highly virulent human pathogen, especially when it is able to form a biofilm, and it is resistant to antibiotic. Infections caused by these bacteria significantly affect morbidity and mortality, primarily in hospitalized patients. Treatment becomes more expensive, more toxic, and prolonged. This is the reason why research on alternative therapies should be one of the main priorities of medicine and biotechnology. A promising alternative treatment approach is bacteriophage therapy. The effect of the anti-staphylococcal bacteriophage preparation Stafal® on biofilm reduction was assessed on nine S. aureus strains using both sonication with subsequent quantification of surviving cells on the catheter surface and evaluation of biofilm reduction in microtiter plates. It was demonstrated that the bacteriophages destroy planktonic cells very effectively. However, to destroy cells embedded in the biofilm effectively requires a concentration at least ten times higher than that provided by the commercial preparation. The catheter disc method (CDM) allowed easier comparison of the effect on planktonic cells and cells in a biofilm than the microtiter plate (MTP) method.