title: Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88.
abstract: BACKGROUND
  Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG) hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria.
  
  
  RESULTS
  S. aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain (135 amino acid residues), and a C-terminal LYZ2 (lysozyme subfamily 2) domain (147 amino acid residues). These domains were also found when a predicted three-dimensional structure of HydH5 was made which provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and CFU reduction analysis. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45°C and in the absence of divalent cations (Ca2+, Mg2+, Mn2+). Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C.
  
  
  CONCLUSIONS
  The virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti-staphylococcal therapy.
authors.affiliation: Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Apdo, Asturias, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Rodríguez
authors.forename: Beatriz
authors.initials: B
authors.lastname: Martínez
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ana
authors.initials: A
authors.lastname: Rodríguez
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.forename: Pilar
authors.initials: P
authors.lastname: García
mesh_terms: D001433:Bacteriolysis; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017385:Sequence Homology; D013204:Staphylococcus Phages / Q000201:enzymology* / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000821:virology; D013696:Temperature; D014771:Virion / Q000201:enzymology / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase