Lipidated SNAPP-Stars Target and Kill Multidrug-Resistant Bacteria within Minutes

The fast emergence of bacteria resistance has already threatened global health, and immediate action is required before the emergence of another global pandemic. Despite substantial progress in the chemical synthesis of novel antimicrobial compounds and advancements in understanding antimicrobial resistance, there has been only a handful of new antibiotics coming to the market. Structurally Nanoengineered Antimicrobial Peptide Polymers (SNAPP-stars) are a new class of antimicrobials. Here, we show that lipidation of lysine-valine 16-armed SNAPP-star, S16 (lipo-SNAPP-star) where the N-terminal arms are conjugated with different fatty acids (caproic, C6, lauric, C12, and stearic acid, C18) enhanced the antimicrobial activity toward <i>S. aureus</i> and MRSA. Lipidation enhanced activity by targeting the SNAPP-stars to the bacterial surface by binding to peptidoglycan, leading to greater inner membrane disruption and depolarization. Lipo-SNAPP-stars killed bacteria in under a minute, whereas vancomycin took >16 h. Lipo-SNAPP-stars were found to preferentially target and kill MRSA rather than <i>S. aureus</i> in a mixed bacteria model. Lipid chain length affected activity, with C6-S16 having greater activity compared to C12-S16 > C18-S16. Lauric and stearic acid enhanced SNAPP-star binding to the bacterial surface and membrane depolarization but impeded SNAPP-stars' ability to transit through the peptidoglycan layer to disrupt the inner membrane. Microbial flow cytometry showed that lipidation aided binding to bacteria via lipoteichoic acid and specifically to peptidoglycan. Further, lipid length enhanced bacterial binding with C18-S16 > C12-S16 > C6-S16 = S16, which contrasts the activity order of C6-S16 > S16 ≫ C12-S16 ≫ C18-S16. Our data demonstrate that lipidation enhances antimicrobial activity by targeting and binding an antimicrobial to peptidoglycan, but increasing lipid length reduces activity by retaining the antimicrobial in the outer layer. Lipidation of SNAPP-stars did not increase cytotoxicity, with C6-S16 having an improved therapeutic index compared to S16. Our data show how lipidation of SNAPP-stars enhances its antimicrobial activity, resulting in a highly biocompatible antimicrobial that targets and kills the "superbug" MRSA within minutes.