Antimicrobial resistance is a global crisis that is fueled by an ever-growing demand for conventional antibiotics. Our goal isto determine if knottin proteins can be developed as a new and expanded class of antimicrobials. Knottins are small, robustproteins that are produced by everything from microbes to plants and animals. A subset of these proteins have antimicrobialproperties. We initiated an collaborative project with scientists at the Fred Hutchinson Cancer Research Center (Seattle, WA) to identify knottin proteins (called optides) that may have antimicrobialproperties. Initial screening successfully identified three such proteins (out of 44 "randomly" selected optides). Importantly, not only are theseoptides antimicrobial alone, but they can be combined with conventional antibiotics to enhance their efficacy. We propose to screen a new panel of additional optides (n=40) that share homology withknown antimicrobial knottins. We will screen these proteins against five species of drug-resistant pathogenic bacteria, assesstoxicity using a hemolysis assay, and identify optides that damage bacterial membranes. Data from this project will be usedto leverage extramural support forexpanded screening, characterization (including structural analysis) anddevelopment of a machine learning algorithm to mine public databases for additional candidate antimicrobial knottins.