Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on cysteine, histidine-dependent amidohydrolases/peptidases activity for lysis ‘from without’

Abstract

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS.

Development of Phage Lysin LysA2 for Use in Improved Purity Assays for Live Biotherapeutic Products

“…One example is that of McCullers et al , 2007 [ 22 ], who used purified Cpl-1 lysin to effectively prevent acute otitis media in mice, inhibiting secondary bacterial infection following influenza infection, with no apparent toxicity of illness. Similar results for use of lysins as antimicrobials are reported in studies for other infections with potential applications against Staphylococci [ 23 , 24 ] and Streptococci [ 19 , 20 ].…”

The Phage Lytic Proteins from the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 Display Multiple Active Catalytic Domains and Do Not Trigger Staphylococcal Resistance

“…This is not expected to be the case for HydH5 since the CHAP and LYZ2 domains, as single domains obtained via deletion analysis, have shown similar lytic activity on live cells [18], [19]. This similar activity of both domains contradicts the results previously reported by other authors since one of the two catalytic domains seems to be almost inactive in some modular endolysins [20]–[23]. Similarly, the lysostaphin endopeptidase domain is active in the triple-lytic domain fusion HydH5Lyso, as the specific activity of this construct on live cells is higher than that previously obtained for HydH5SH3b [19].…”

A review of current methods using bacteriophages in live animals, food and animal products intended for human consumption

“…It was reported that optimum endolysin activity was achieved at a concentration of between 3 to 10 mM Ca 2+ . However, it must also be noted that they reported that the concentration of Mg 2+ had a negligible effect on enzyme activity, whilst the presence of Mn 2+ ions demonstrated an inhibitory effect, in vitro (Schmelcher et al, 2012). These data suggest that the chemical composition of food must be evaluated, as the activity of the endolysins, and hence the efficacy of the bacteriophages themselves could be adversely affected if not taken in to consideration.…”

“…Research by Schmelcher et al (2012) revealed that bacteriophage endolysin activity can be modulated by the concentration of divalent cations within the test medium. It was reported that optimum endolysin activity was achieved at a concentration of between 3 to 10 mM Ca 2+ .…”