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The PlySs9 endolysin contains unique catalytic domains and is a potential therapeutic against Streptococcus suis

Increasing resistance to antibiotics amongst livestock has forced the discovery of alternative techniques to continue treating bacterial infections successfully. Bacteriophage-encoded peptidoglycan hydrolases, also referred to as endolysins, are able to lyse the bacterial cell wall and offer possible applications in food safety, human health and veterinary science. The treatment of Streptococcus suis infections in pigs specifically involves the latter three. Its zoonotic nature is a potential human health threat and the economic loss of $100 million per year is devastating on the swine industry. Preventing on-farm disease outbreaks is extremely difficult and current approaches to eradicate S. suis from herds are often ineffective. Therefore, a pressing need to identify and evaluate S. suis-specific endolysins arises. A bioinformatic approach was conducted to identify proteins in bacteriophage genomes with similar homology to known endolysin catalytic domains. We chose five candidates for synthesis, expression, purification and characterization upon discovery of lytic activity assayed by turbidity reduction. Binding capacity was evaluated by fluorescent microscopy. PlySs9 represents our lead candidate and is predicted to contain an N-terminal amidase catalytic domain, a central LysM-based cell wall binding domain, and a C-terminal CHAP catalytic domain. We have determined the optimal conditions for the lytic activity of PlySs9, characterized its broad activity spectrum, and investigated its ability to disrupt biofilms. Active-site residues were detected through site-directed mutagenesis. We also assessed the contribution of each individual domain to activity or binding. Lastly, a triple-acting enzyme of PlySs9 was engineered using three unique, potentially synergistic lytic domains to reduce the risk of resistance development. These results indicate that the broad lytic spectrum of PlySs9 and its derivatives have the potential to be used as therapeutic agents against S. suis infections.