The oligo-acyl lysyl antimicrobial peptide C₁₂K-2β₁₂ exhibits a dual mechanism of action and demonstrates strong in vivo efficacy against Helicobacter pylori.

Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C(12)K-2β(12), that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C(12)K-2β(12) against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C(12)K-2β(12) rapidly 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 C(12)K-2β(12) can 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 the in vivo efficacy of C(12)K-2β(12), H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C(12)K-2β(12) 1 day or 1 week postinfection. The efficacy of C(12)K-2β(12) was 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 C(12)K-2β(12). Overall, our results demonstrate a dual mode of action of C(12)K-2β(12) against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C(12)K-2β(12) shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.