300 words) 2 0Our laboratory recently reported that the EP 4 receptor antagonist, L-161,982, had 2 1 direct growth-inhibitory effects on Staphylococcus aureus in vitro and in vivo, 2 2 reducing microbial burden and providing significant protection against lethality in 2 3 models of S. aureus monomicrobial and polymicrobial intra-abdominal infection. 4This antimicrobial activity was observed with both methicillin-sensitive and 2 5 methicillin-resistant S. aureus (MRSA), as well as other Gram-positive bacteria. 6The antimicrobial activity of L-161,982 was independent of EP 4 receptor 2 7inhibitory activity. In this study, we investigated the mechanism of action (MOA) 2 8 of L-161,982, which contains a sulfonamide functional group. However, results 2 9 demonstrate L-161,982 does not affect folate synthesis (sulfonamide MOA), 3 0 oxidative stress, or membrane permeability. Instead, our results suggest that the 3 1 inhibitor works via effects on inhibition of the electron transport chain (ETC).3 2 Similar to other ETC inhibitors, L-161,982 exposure results in a small colony size 3 3 variant phenotype and inhibition of pigmentation, as well as significantly reduced 3 4 hemolytic activity, and ATP production. In addition, L-161,982 potentiated the 3 5 antimicrobial activity of another ETC inhibitor and inhibition was partially rescued 3 6by supplementation with nutrients required for ETC auxotrophs. Taken together, 3 7 these findings demonstrate that L-161,982 exerts antimicrobial activity against 3 8