ABSTRACTHelicobacter pylorihas developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pyloritherapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C12K-2β12, that shows potentin vitrobactericidal activity againstH. pylori. Herein, we define the mechanism of action and evaluate thein vivoefficacy of C12K-2β12againstH. pyloriafter experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C12K-2β12rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C12K-2β12can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define thein vivoefficacy of C12K-2β12,H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C12K-2β121 day or 1 week postinfection. The efficacy of C12K-2β12was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P< 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C12K-2β12. Overall, our results demonstrate a dual mode of action of C12K-2β12against theH. pylorimembrane and cytoplasmic components. Moreover, and consistent with the previously reportedin vitroefficacy, C12K-2β12shows significantin vivoefficacy againstH. pyloriwhen used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment ofH. pyloriinfection.