Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the<jats:named-content content-type="genus-species">Staphylococcus aureus</jats:named-content>bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live<jats:named-content content-type="genus-species">S. aureus</jats:named-content>cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed<jats:named-content content-type="genus-species">S. aureus</jats:named-content>cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human<jats:named-content content-type="genus-species">S. aureus</jats:named-content>strains, the methicillin-resistant<jats:named-content content-type="genus-species">S. aureus</jats:named-content>(MRSA) strain N315, and human<jats:named-content content-type="genus-species">Staphylococcus epidermidis</jats:named-content>strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5<jats:italic>in vitro</jats:italic>, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.</jats:p>

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…These enzyme pairs together were more efficient at degrading staphylococcal peptidoglycan than individually. 104 Similarly, this domain swapping approach has also proven to be useful for the improvement of lytic activity of the putative VAPGH within phage K open reading frame 56. 105 N-terminus deletions of orf56 defined the coding region responsible for a small PGH (Lys16) that retained staphylolytic activity.…”

Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus

“…However, no cell wall binding (CWB) domain could be identified in the amino acid sequence of HydH5. In a subsequent study, Rodríguez-Rubio et al (16) demonstrated that the activity of the protein HydH5 could be improved by fusing its CHAP domain to the CWB domain SH3b of lysostaphin. The resulting chimeric protein was designated CHAPSH3b and exhibited specific activity against staphylococci.…”

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

“…Therefore, although TG1 lytic activity was moderate against staphylococci, further investigation is worthwhile. We previously reported that the fusion of a staphylococcal SH3b cell wall-binding domain to the HydH5 CHAP domain resulted in a 4.8-fold increase of its lytic activity (15). Furthermore, it is plausible to swap different catalytic domains by protein engineering, generating proteins with improved lytic activity (4,15).…”

“…E. coli BL21(DE3)/pLysS was used in protein expression assays (19). Purification of TG1 (30.1 kDa) was performed by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (15). Peptidoglycan cleavage site determination was carried out by reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS) (2).…”