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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #273291
Title: Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

Author
item RODRIGUEZ, LORENA - Consejo Superior De Investigaciones Cientificas (CSIC)
item MARTINEZ, BEATRIZ - Consejo Superior De Investigaciones Cientificas (CSIC)
item RODRIGUEZ, ANA - Consejo Superior De Investigaciones Cientificas (CSIC)
item Donovan, David
item GARCIA, PILAR - Consejo Superior De Investigaciones Cientificas (CSIC)

Submitted to: Antimicrobial Agents and Chemotherapy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2012
Publication Date: 1/20/2012
Citation: Rodriguez, L., Martinez, B., Rodriguez, A., Donovan, D.M., Garcia, P. 2012. Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5. Antimicrobial Agents and Chemotherapy. 78(7):2241-8.

Interpretive Summary: Problem: Staphylococci are gram positive pathogens that have large negative impacts on both agriculture and human health. Staphylococcus and streptococcus are leading causes of bovine mastitis, a 2 billion dollar a year loss, accounting for more than 40% of all cases in one study. Traditional treatments for mastitis, most commonly antibiotics, have been shown to be only marginally effective. Bacteriophage endolysins and virion associated peptidoglycan hydrdolases are a potential source of narrow spectrum antibiotics for treatment of antibiotic resistant pathogens. Accomplishments: We have determined the active domains of the H5 phage viron associated hydrolase (HydH5) and generated triple domain fusion constructs which are highly active against multiple strains of S. aureus . These fusion proteins, should be refractory to resistance development with at least two lytic activities expressed simultaneously. Contribution of Accomplishment to Solving the Problem: The findings of this work identify the necessary components of the HydH5 phage virion associated hyrolase for its application as an antimicrobial, and additionally describes the methodology for generating multiple domain fusions. These findings will aid in the development and analysis of similar molecules as antimicrobials.

Technical Abstract: Virion-associated peptidoglycan hydrolases have a 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 Staphylococcus aureus bacteriophage vB_SauS-phi-IPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP domain exhibited high lytic activity against live S. aureus 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 S. aureus cells in zymograms, plate lysis and turbidity reduction assays. In plate lysis assays, the HydH5 and its derivative fusions lysed bovine and human S. aureus, S. aureus MRSA N315 strain, and human Staphylococcus epidermidis strains. Several non-staphylococcal bacteria were not affected. HydH5 and its derivative fusions proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro suggesting that the two enzymes have distinct cut sites and thus may be more efficient in combination for the elimination of staphylococcal infections.