Abstract
The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88) and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso). We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture) was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections.
Highlights
Staphylococcus aureus is a dangerous pathogen responsible for a variety of infections ranging from skin abscesses to fatal sepsis, endocarditis, osteomyelitis, septicemia, pneumonia and meningitis [1]
In order to perform an in deep characterization of these phage derived proteins, we studied the catalytic activity of the domains included in LysH5, HydH5SH3b and HydH5Lyso by mass spectrometry analysis of the specific cleavage sites in the S. aureus PG
To determine the LysH5, HydH5SH3b and HydH5Lyso PG cut sites, Liquid Chromatography-Mass Spectrometry (LC-MS) was performed on proteins-digested S. aureus PG preparations
Summary
Staphylococcus aureus is a dangerous pathogen responsible for a variety of infections ranging from skin abscesses to fatal sepsis, endocarditis, osteomyelitis, septicemia, pneumonia and meningitis [1]. Two phage encoded peptidoglycan (PG) hydrolytic activities (endolysins and virion-associated PG hydrolases) showed antimicrobial activity against Gram-positive pathogens [5,6] This has boosted the study of these proteins to be used as therapeutic agents, in external applications [7,8]. Multiple studies demonstrate the control of both streptococcal (pneumonia, endocarditis, otitis media, meningitis) and Bacillus anthracis (intraperitoneal) infections in mice by phage lysins. These results support the application of endolysins to treat human and animal infections [7,8]. Similar results were obtained with LysGH15 since the intraperitoneal administration of the lytic enzyme 30 min after MRSA infection was sufficient to guarantee survival of the mice for up to 60 days after treatment [10]
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