Purified phage endolysins have been used as therapeutics (so-called enzybiotics) against Streptococci in mice [13, 14] and have been proven effective against other Gram-positive pathogens including Enterococcus faecalis and E. faecium [15], Clostridium perfringens [16], group B Streptococci [17], Bacillus anthracis [18] and S. aureus [[19–21]]. Previously, we reported the isolation of the S. aureus bacteriophage vB_SauS-phiIPLA88

(in short, phiIPLA88) belonging to the Siphoviridae family [22]. The complete genome sequence was determined (Accession number NC_011614) and zymogram analysis revealed the presence of a phiIPLA88 virion-associated muralytic enzyme [23]. In this study, we describe the structural component of phiIPLA88 particle, HydH5, which exhibits lytic activity against S. aureus cells. HydH5 contains a CHAP [24, 25] and a LYZ2 [7] domain and the contribution of each to cell lysis GDC-0068 molecular weight has been analysed. Finally, we have determined the optimal activity conditions and heat-labile stability in order to assess

HydH5′s potential as an anti-Staphylococcus agent. Results S. aureus bacteriophage phiIPLA88 contains a structural Olaparib concentration component with a putative cell wall- degrading activity The virions of phage phiIPLA88 possess a structural component with lytic activity as was previously shown by zymogram analysis [23]. This lytic activity corresponded in size to that expected for the protein product of orf58 (72.5 kDa), which is located in the morphogenetic module with most of the phage head and MRIP tail structural genes. Computer-based similarity

searches revealed that protein gp58, designated here as HydH5 (634 amino acids, Acc. Number ACJ64586), showed 91% similarity with putative PG hydrolases identified in S. aureus phi11, phiNM and phiMR25 phages (Acc. Number NP_803302.1, YP_874009.1, YP_001949862.1). A 60% similarity was detected between HydH5 and the recently characterized PG hydrolase gp61 of S. aureus phiMR11 phage [7]. A phylogeny tree was generated from alignment of the known staphylococcal PG hydrolases (Figure 1). The 25 different proteins were clustered into two major groups. No relation between these groups and the previous S. aureus phages classification based on their genome organization was observed [26]. Interestingly, PG hydrolases from phages infecting S. epidermidis strains (phage CNPH82 and phage PH15) were found to be very similar to those from S. aureus phages. Furthermore, conserved-domain analyses of HydH5 identified two typical catalytic domains found in cell wall hydrolases. At its N-terminal region (15 to 149 amino acids) a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain was detected [24, 25]. The C-terminal region (483 to 629 amino acids) showed a LYZ2 (lysozyme subfamily 2 or glucosaminidase) [7] conserved domain.