To combat the emergence of antibiotic resistant pathogens, new strategies are being explored. In this context, the effectiveness of phage-encoded endolysins to eliminate certain infections has been well documented in mouse models [[36–38]]. The main advantage of these proteins is their ability to kill bacteria with near-species specificity and the reported low incidence of resistance development . Similarly, other phage lytic proteins that also hydrolyze essential PG bonds such as structural PG hydrolases, may also contribute to the supply of new antimicrobials.
Preliminary sequence analyses of the virion-associated PG hydrolase HydH5 revealed two putative lytic domains, namely, N-terminal CHAP domain and LYZ2 domain at the C-terminus. This protein organization resembles that of other phage muralytic enzymes which, similar to endolysins, appear to be modular enzymes containing separate catalytic domains. It has been proposed that the evolution of endolysins, and probably also structural PG hydrolases, has likely occurred through domain swapping and that phage lytic enzymes have co-evolved with host autolysins . In fact, the predicted 3D structure of HydH5 identified another central domain with remote homology to the AmiE catalytic domain of the autolysins AtlE and AtlA, the major S. epidermidis and S. aureus autolysins, respectively. However, key residue changes seem to have been selected in the active site of HydH5 despite the maintenance of the amidase-like fold, likely rendering a reduced activity amidase domain . Whether or not these mutations have catalytically inactivated the AmiE domain remains to be determined.
It should be noted that LYZ2 domains have been rarely studied in phages, being the phage phiMR11 the only example reported so far . However, it has been predicted that this lysozyme subfamily 2 catalytic domain (SMART accession number: SM00047) is widely distributed in Staphylococcus phage, Staphylococcus bacteria and other related bacteria.
In this work, we have demonstrated the staphylolytic activity of full-length HydH5 and each of its two catalytic domains by both zymogram analysis and CFU reduction analysis. Having two active catalytic domains decreases the likelihood of resistance development to this antimicrobial in that the pathogen would potentially need two simultaneous mutations in the same cell to become resistant. This is a very attractive trait for potential antimicrobials. Further biochemical analyses are required to definitively assign the endopeptidase and lysozyme activities to these domains and confirm to what extent both contribute to the lytic activity identified in our assays. It has been previously shown that some individual endolysin catalytic domains can lyse S. aureus cells in the absence of the complete protein. For example, phi11 and LysK endolysins have active CHAP domain constructs without either the amidase or SH3b domains required [[19, 30, 32]].
Our results also indicated that the lytic activity of HydH5 and the two catalytic domains does not require a CBD. Bioinformatic analysis of HydH5 failed to detect a known CBD. It has been speculated that some endolysins possess catalytic domains operating as cell wall-binding domains that direct the protein to target epitopes on the surface of susceptible bacteria [17, 40]. There are also numerous reports of C-terminally deleted lysins where the N-terminal lytic domain maintains their staphylococcal-  or streptococcal-specificity [41, 42] in the absence of their CBD. More surprising are recent studies showing that the lytic activity of the B30 (11) and PlyGBS  lysins were maintained or even enhanced, approximately 25-fold, respectively, in engineered lysins in which the SH3 domain has been removed. However, it is not entirely clear which part of the protein determines the specificity. Based on the results that showed binding of the catalytic domains to cells, we hypothesized that substrate recognition in HydH5 might be somehow mediated by its catalytic domains. However, further analyses are required to demonstrate the specificity of this binding for S. aureus cells. In this regard, preliminary results about the HydH5 lytic spectrum indicated that most of tested staphylococcal strains were susceptible to this protein (our unpublished results). It should be kept in mind that, in contrast to endolysins, phage structural PG hydrolases might not require a CBD because they are delivered to the PG substrate by the virion particle structure .
The proposed function of phage structural PG hydrolases during the first steps of the phage life cycle also implies that their hydrolytic activity should only damage the cell wall slightly in order to avoid premature lysis of the host cell. For this reason, it is not surprising that the lytic activity of HydH5 and both truncated versions were not detected in turbidity reduction assays but were capable of killing S. aureus Sa9 cells in the CFU reduction analysis. The variable quantitative behaviour of PG hydrolases activities in different lytic assays has also been observed by other authors [44, 45]. The killing activity of HydH5 was inhibited by some cations and sodium chloride. Although most of the endolysins described so far has not been tested for the effect of cations, there are some which lytic activity is dependent on or enhanced in the presence of calcium in the assay buffer [[32, 35, 46]].
The highest protein activity was detected against actively dividing log phase growth staphylococcal cells, possibly due to a different conformation of the PG. In fact, the degree of peptidoglycan cross-linking is significantly increased in stationary phase cells of species such as E. coli and Bacillus spp. [47, 48]. (An according result) A similar result was observed with the bacteriophage T7 gp16 structural transglycosylase which facilitated infection of E. coli cells growing to high cell densities or low temperatures. In these conditions the murein is more highly cross-linked and the internalization of the phage genome is significantly delayed in absence of gp16 protein . Different susceptibility as a function of growth stage was also observed in the Ply700 endolysin , which is more active against early and mid-exponential Streptococcus uberis cells.
Another feature that is characteristic of HydH5 and other phage structural hydrolases is their thermostability, most likely related to a high refolding capability. HydH5 retained 72% of its activity after a 5-min treatment at 100°C. Likewise, the structural lysozyme from phage phiKMV infecting Ps. aeruginosa is also a highly thermostable protein, retaining 26% of its activity after 2 h at 100°C and 21% after autoclaving . By contrast, the lytic activity of most phage endolysins is destroyed by heat treatment [35, 41]. This makes structural PG hydrolases attractive antimicrobials to be used in combination with other hygienic procedures based on high temperature such as those applied in food preservation and as structural models for highly thermostable enzymes.