|LINDEN, SARAH - University Of Maryland|
|NELSON, DANIEL - University Of Maryland|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/2/2016
Publication Date: N/A
Interpretive Summary: N/A
Technical Abstract: The crisis of increasing resistance of pathogenic bacteria to classical antibiotics has driven research towards identification of other means to fight infectious disease. One particularly attractive option is the use of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These enzymes are able to lyse the bacterial cell wall upon direct contact when applied externally and lack the drawbacks of typical antimicrobials (1). Endolysins have already shown potential in the areas of food safety, human health, and veterinary science (2). One specific area that could benefit from endolysin application is the overwhelming problem of Streptococcus suis infections of pigs. While the economic loss of $100 million per year on the swine industry can be devastating in the event of an outbreak, it is the zoonotic nature of S. suis that is particularly alarming. There are currently no effective approaches to eradicate S. suis from pig herds and preventing disease outbreaks has proven extremely difficult. Therefore, there is a pressing need to identify and evaluate novel S. suis-specific endolysins for antibacterial activity. Utilizing the genomes of the S. suis bacteriophage SMP and 16 sequenced S. suis strains, many of which contain prophage elements, a bioinformatic approach was conducted to identify proteins with similar homology to known endolysin catalytic domains. Initially, 165 distinct proteins belonging to 9 different architectures were found. Four of these architectures lacked domains that we considered essential for lytic activity and thus proteins categorized as such were eliminated. One protein from each of the 5 remaining architectures was chosen for synthesis, expression, purification, and further characterization upon discovery of lytic activity. Preliminary data indicates that the protein we have named Ply1 is a lead candidate. Based on homology, this enzyme was predicted to contain an N-terminal amidase catalytic domain, a central LysM cell wall binding domain, and a C-terminal CHAP catalytic domain. Using turbidity reduction of stationary phase S. suis as a measure of activity, we have determined the optimal pH, salt concentration, contribution of reducing agents, divalent cations, or cofactors, and temperature for Ply1. In addition, we have characterized its host range (it maintains lytic activity against all strains of S. suis tested as well as other Gram-positive species) and its ability to disrupt biofilms. We have determined the active-site residues through site-directed mutagenesis. Further, we intend to engineer Ply1 (a dual-acting endolysin with two active lytic domains) and reduce the risk of resistance development via the engineering of a triple-acting enzyme with three unique, synergistic lytic domains. These initial results indicate that Ply1 has the potential to be used as a therapeutic agent against S. suis infections.