From Mitochondrial Targeting to Microbial Killing: Twinkle-Derived Peptides as an Emerging Class of Potent AMPs

Abstract Mitochondrial targeting sequences are short N-terminal peptides that direct proteins to mitochondria. Notably, their amphipathic α-helical architecture closely resembles that of many potent antimicrobial peptides (AMPs). This structural convergence reflects the endosymbiotic origin of mitochondria and suggests that contemporary mitochondrial targeting sequences may retain latent antimicrobial properties. Here, this evolutionary link was exploited to investigate the mitochondrial-targeting sequences of a Twinkle helicase as a scaffold for the rational design of a new class of antimicrobials. Using an in silico strategy, two peptides, TAMP1 (Twinkle AntiMicrobial Peptide1) and its optimized derivative, TAMP2 (Twinkle AntiMicrobial Peptide2), were designed starting from the mitochondrial targeting sequences of the Twinkle helicase from the Antarctic fish Notothenia coriiceps. Both 18-residue peptides were structurally and functionally characterized as proof of concept for mitochondrial targeting sequence-derived antimicrobials. They adopted stable amphipathic α-helical conformations and exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Notably, TAMP2 demonstrated enhanced efficacy, effectively targeting an extended panel of bacteria, including spoilage, foodborne, and multidrug-resistant pathogens, with MBC values in the range of 0.5–32 μM. Microscale thermophoresis and molecular dynamics simulations further supported these observations, indicating strong interactions with bacterial membrane mimetics and providing mechanistic insight into peptide activity. These findings establish Twinkle-derived mitochondrial targeting sequence peptides as a promising, scalable platform for developing a novel class of antimicrobial agents inspired by mitochondrial evolution.