Mechanism of action of a bactericidal lipopeptide which targets lipopolysaccharides of the plant pathogen Xylella fastidiosa.

The mechanism of action of BP473, a BP100-derived undecalipopeptide, was characterized through the evaluation of its bactericidal activity against different Xylella fastidiosa subspecies and strains, the study of the relationship between antibacterial activity and lipopolysaccharides (LPS) interaction, the analysis of its binding to LPS by NMR spectroscopy, and the assessment of its antibiofilm activity. The effect of BP473 on pathogen cell ultrastructure and on plant defense responses was also determined. In addition, its capacity to inhibit X. fastidiosa infections was further assessed in Nicotiana benthamiana using three X. fastidiosa subspecies. BP473 displayed potent bactericidal activity against subsp. fastidiosa and multiplex, whereas lower activity was observed against subsp. pauca. In contrast, its antibiofilm activity was limited. The bactericidal activity of BP473 was significantly reduced in the presence of free LPS, while that of BP178 remained unaffected, demonstrating the interaction between BP473 and LPS. NMR studies confirmed that all BP473 residues contribute to LPS binding, with the acetyl group and the aromatic protons of Phe4 showing the strongest interactions. Additional key binding epitopes included the meta aromatic protons of Tyr10 and the δ-CH3 and γ-CH2 protons of Leu3 and Ile7, respectively. Ultrastructural analyses by SEM in STEM mode revealed that both BP473 and the fluorescently labelled derivative BP473-K(CF) caused severe membrane damage in bacterial cells, supporting a membranolytic mode of action. BP473 elicited a weak plant defense response after a two-day treatment, mainly activating early signaling components without inducing a strong salicylic acid-dependent response. Importantly, BP473 reduced X. fastidiosa infection in planta, although its efficacy varied among bacterial subspecies. Overall, these results indicate that BP473 acts primarily through membrane disruption and LPS binding, while providing strain-dependent protection against X. fastidiosa infection.