A68-11 A Corynebacterium Pseudodiphtheriticum Secreted Protease Inhibits Staphylococcus Aureus Virulence

Introduction Invasive Staphylococcus aureus infection is a highly morbid disease and a leading cause of hospital acquired infection including healthcare-associated pneumonia. S. aureus colonizes human nares and uses an arsenal of secreted toxins to break down tissue barriers and establish invasive infection. Identifying new ways to prevent and treat S. aureus infection is of critical importance. The upper airway microbiome impacts S. aureus colonization, and clarifying the mechanisms by which commensal bacteria influence S. aureus colonization and virulence could lead to novel tools to prevent invasive infection. Studies in humans and mice have shown that the presence of Corynebacterium pseudodiphtheriticum (a commensal bacteria found in the nasal cavity) is associated with reduced S. aureus infection risk. In recent work, we found that C. pseudodiphtheriticum inhibits S. aureus virulence through secreted factors which directly interfere with S. aureus toxin activity. We hypothesized that at least one of these secreted factors is a protease that directly attenuates S. aureus virulence. Methods Cell free culture media (CFCM), which contains molecules secreted by bacteria, was isolated from cultures of S. aureus (MRSA, USA300 strain) and C. pseudodiphtheriticum (DSM44287 strain). CFCM from S. aureus contains pore forming toxins, with toxin activity measured by hemolysis of human red blood cells in vitro. C. pseudodiphtheriticum CFCM was added to determine the impact on S. aureus hemolysis, with and without addition of protease inhibitors. Fast protein liquid chromatography (FPLC) size exclusion chromatography was used to separate C. pseudodiphtheriticum CFCM to identify which fractions contained proteins which blocked S. aureus hemolysis. Active fractions were analyzed by mass spectrometry to identify candidate proteins. Results C. pseudodiphtheriticum CFCM reduced S. aureus-mediated hemolysis of human red blood cells. This effect was reversed by addition of a cocktail of protease inhibitors. The serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) specifically inhibited C. pseudodiphtheriticum CFCM’s protective effect while other classes of protease inhibitors did not. Mass spectrometry of active fractions from C. pseudodiphtheriticum CFCM revealed four unique serine proteases with predicted size of 32-37 kD. Conclusions These data indicate that C. pseudodiphtheriticum secretes one or more serine proteases that directly block the activity of S. aureus pore forming toxins, an important indicator of S. aureus virulence. This work sheds light on the mechanism by which C. pseudodiphtheriticum, a common commensal bacteria in the human nasal cavity, protects against S. aureus infection and identifies a novel potential therapeutic intervention for S. aureus infection. This abstract is funded by: Boettcher Foundation (Boettcher)