The C-Terminal Phenylalanine of the Thanatin-like Peptide Rip-2 Contributes to Its Enhanced LptA-Associated Antimicrobial Activity Compared with Thanatin

The proteins of the lipopolysaccharide transport (Lpt) complex have emerged as promising targets for thanatin-like antimicrobial peptides (AMPs) active against Gram-negative bacteria. Here, we investigated the molecular features associated with the enhanced antibacterial activity of the thanatin-like peptide Rip-2 from the bean bug Riptortus pedestris, compared with thanatin. Microscale thermophoresis showed that Rip-2 produced a concentration-dependent response with recombinant LptAm-sfGFP-His6 that differed from that for thanatin. A phenotypic counter-screening was performed to identify genetic changes associated with Rip-2-selective reduced susceptibility. Subsequent whole-genome sequencing identified the same LptA R76C substitution as a recurrent resistance-associated change in all selected clones. This substitution was associated with only a two-fold increase in the MIC of thanatin, whereas the MIC of Rip-2 was 16-fold higher. Structure-guided modeling suggested that the guanidinium group of LptA Arg76 may contribute to Rip-2 engagement by forming predicted contacts with both the terminal carboxylate and the aromatic side chain of the peptide C-terminal Phe18 residue. Consistent with this model, substitutions of this phenylalanine impaired the peptide antibacterial activity, with more tolerated aromatic replacements in comparison to nonaromatic ones. These findings support an important role for the Rip-2 C-terminal Phe18 aromatic residue in the enhanced activity of this peptide relative to thanatin and provide a framework for the rational design of more potent thanatin-derived antibiotics.