Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureusbacteriophage vB_SauS-phiIPLA88

Abstract

BackgroundStaphylococcus 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.ResultsS. 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.ConclusionsThe virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti-staphylococcal therapy.

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain

“…Nevertheless, the activity seems to be somewhat weaker than that shown by other staphylococcal virion-associated peptidoglycan hydrolases such phiIPLA88 HydH5 (14) and phiMR11 gp61 (13). This weaker specific activity (about 23-fold lower than that of HydH5) could have been due to the presence of only one catalytic domain, the lysozyme-like domain, in contrast to HydH5, which showed two active catalytic domains (14). Nevertheless, the peptidoglycan hydrolytic activity of TMP as part of phiIPLA35 virions might also be enhanced by the peptidase_M23 domain.…”

“…However, the presence in phages phiIPLA88 and phiMR11 of proteins with muralytic activities might indicate its involvement in local cell wall degradation, allowing the subsequent introduction of DNA into the host cytoplasm (13,14). In phiIPLA35, the presence of an active muramidase domain in the TMP could also indicate its contribution to the infection process, taking into account that no other virion-associated proteins with peptidoglycan hydrolase activity were identified in this phage (8).…”

“…Lysozyme activities catalyze the cleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetyl-D-glucosamine (GlcNAc). Lysozyme catalytic domains are often found in cell wall hydrolases from S. aureus phages (14). However, only phages 42E, phi47, phi12, phiSLT, tp310-2, and phi3A revealed a TMP with a lysozyme-like domain homolog to the phiIPLA35 TMP.…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…101 Likewise, the S. aureus phage FMR11 also encodes a putative VAPGH with a CHAP domain followed by a muramidase domain, which are independently staphylolytic, and lacks a CBD. 76 The S. aureus phage vB_SauS-phiIPLA88 VAPGH HydH5 is also a dual EAD enzyme, 102 In contrast, the S. aureus phage P68 has a VAPGH (protein P17) that has muralytic activity and a C-terminus region that acts as a substrate recognition and/or binding domain. 103 Since the removal of proteins and lipids from the staphylococcal cell wall does not affect binding, it was speculated that protein P17 binds to a carbohydrate component covalently attached to the peptidoglycan.…”

The Peptidoglycan Hydrolase of Staphylococcus aureus Bacteriophage ϕ11 Plays a Structural Role in the Viral Particle

“…The complete genome sequence and zymogram analysis of the S. aureus bacteriophage vB_SauS-phiIPLA88 revealed the presence of HydH5, a VAPGH able to lyse viable S. aureus cells (12). HydH5 (634 amino acids, accession no.…”