Rational Engineering of a Floral Nectar Peptide Generates Enhanced Antimicrobial Analogs with Different Modes of...

Antibiotic resistance is a critical challenge for global public health, stimulating the search for effective therapeutic alternatives. In this context, antimicrobial peptides (AMPs) stand out as promising candidates due to their structural diversity and multiple mechanisms of action. In this study, AMPs present in the floral nectar of Nicotiana sp. were evaluated for antibacterial activity against Staphylococcus epidermidis, S. aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Nicotianin-I (NI) was identified as the most active natural peptide, although with limited efficacy. Therefore, it was selected as a model for rational engineering. From its sequence, two bioinspired analogs, NI-A and NI-B, were developed and showed significantly increased antibacterial activity in a strain-dependent manner, as demonstrated by planktonic growth inhibition and cell viability assays. Mechanistic analyses indicated that NI-A acts predominantly by destabilizing bacterial membranes, while NI-B induces oxidative stress. Structural characterization revealed improved amphipathicity and environment-dependent folding behavior. Hemolysis assays demonstrated that only NI-B maintains low toxicity against human erythrocytes. Overall, these results highlight rational peptide engineering as a powerful strategy to transform low-activity natural AMPs into optimized antimicrobial candidates with defined mechanisms and therapeutic potential.