Kayviruses are polyvalent broad host range staphylococcal phages with a potential to combat staphylococcal infections. However, the implementation of rational phage therapy in medicine requires a thorough understanding of the interactions between bacteriophages and pathogens at omics level. To evaluate the effect of a phage used in therapy on its host bacterium, we performed differential transcriptomic analysis by RNA-Seq from bacteriophage K of genus Kayvirus infecting two Staphylococcus aureus strains, prophage-less strain SH1000 and quadruple lysogenic strain Newman. The temporal transcriptional profile of phage K was comparable in both strains except for a few loci encoding hypothetical proteins. Stranded sequencing revealed transcription of phage noncoding RNAs that may play a role in the regulation of phage and host gene expression. The transcriptional response of S. aureus to phage K infection resembles a general stress response with differential expression of genes involved in a DNA damage response. The host transcriptional changes involved upregulation of nucleotide, amino acid and energy synthesis and transporter genes and downregulation of host transcription factors. The interaction of phage K with variable genetic elements of the host showed slight upregulation of gene expression of prophage integrases and antirepressors. The virulence genes involved in adhesion and immune evasion were only marginally affected, making phage K suitable for therapy. IMPORTANCE Bacterium Staphylococcus aureus is a common human and veterinary pathogen that causes mild to life-threatening infections. As strains of S. aureus are becoming increasingly resistant to multiple antibiotics, the need to search for new therapeutics is urgent. A promising alternative to antibiotic treatment of staphylococcal infections is a phage therapy using lytic phages from the genus Kayvirus. Here, we present a comprehensive view on the phage-bacterium interactions on transcriptomic level that improves the knowledge of molecular mechanisms underlying the Kayvirus lytic action. The results will ensure safer usage of the phage therapeutics and may also serve as a basis for the development of new antibacterial strategies.

• Klíčová slova
• Kayvirus, RNA-Seq, Staphylococcus aureus, Staphylococcus phages, bacteriophage therapy, noncoding RNA, phage-host interactions, prophages, transcriptome, viral transcription,
• MeSH
• lidé MeSH
• profágy genetika   MeSH
• stafylokokové bakteriofágy genetika   MeSH
• stafylokokové infekce * mikrobiologie   terapie   MeSH
• Staphylococcus aureus * MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lytic bacteriophages are valuable therapeutic agents against bacterial infections. There is continual effort to obtain new phages to increase the effectivity of phage preparations against emerging phage-resistant strains. Here we described the genomic diversity of spontaneous host-range mutants of kayvirus 812. Five mutant phages were isolated as rare plaques on phage-resistant Staphylococcus aureus strains. The host range of phage 812-derived mutants was 42% higher than the wild type, determined on a set of 186 methicillin-resistant S. aureus strains representing the globally circulating human and livestock-associated clones. Comparative genomics revealed that single-nucleotide polymorphisms from the parental phage 812 population were fixed in next-step mutants, mostly in genes for tail and baseplate components, and the acquired point mutations led to diverse receptor binding proteins in the phage mutants. Numerous genome changes associated with rearrangements between direct repeat motifs or intron loss were found. Alterations occurred in host-takeover and terminal genomic regions or the endolysin gene of mutants that exhibited the highest lytic activity, which implied various mechanisms of overcoming bacterial resistance. The genomic data revealed that Kayvirus spontaneous mutants are free from undesirable genes and their lytic properties proved their suitability for rapidly updating phage therapeutics.

• MeSH
• bakteriální léková rezistence MeSH
• bakteriofágy genetika   MeSH
• délka genomu MeSH
• genom virový MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• methicilin farmakologie   MeSH
• mutace * MeSH
• Staphylococcus aureus růst a vývoj   virologie   MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methicilin 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.

• Klíčová slova
• Endolysin, Endopeptidases, Enzybiotics, Src homology domains, Staphylococcal infections, Staphylococcus bacteriophage,
• MeSH
• endopeptidasy genetika   izolace a purifikace   metabolismus   MeSH
• hostitelská specificita * MeSH
• mutantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• Myoviridae enzymologie   genetika   fyziologie   MeSH
• peptidoglykan metabolismus   MeSH
• proteinové domény MeSH
• sekvenční delece * MeSH
• Staphylococcus virologie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endolysin MeSH Prohlížeč
• endopeptidasy MeSH
• mutantní proteiny MeSH
• peptidoglykan MeSH