Synthetic lipopeptides that interact with lipopolysaccharides are potent bactericidal compounds against Xylella fastidiosa

Abstract

Novel strategies to control diseases caused by Xylella fastidiosa that affect important crops like grapevine, olive, almond, and citrus are necessary to prevent its establishment and spread in several countries. Target-oriented functional peptides toward the pathogen are potential new compounds, and peptides directed to lipopolysaccharides (LPS) are suitable candidates. In the present work, 36 peptides, including previously reported sequences and newly synthesized lipopeptides, were studied for their bactericidal and LPS neutralization activities, as well as for their hemolytic and phytotoxic activities. In a first stage, a simple and rapid method for detecting peptide-LPS interactions based on a chromogenic endotoxin assay was developed. A linear relationship was observed between LPS neutralization and the bactericidal activity. A strong LPS interaction was related to a high bactericidal activity, whereas peptides with slight or low interaction with LPS had low bactericidal activity. A selected group of lipopeptides that strongly interacted with the LPS was highly bactericidal with moderate-to-low hemolytic and phytotoxic activity (BP389, BP473, and BP475). These lipopeptides incorporate a butanoyl group at the side chain of a lysine residue and an L- or D-phenylalanine at position 4. The interaction and cell lytic effect between BP473 and X. fastidiosa cells was confirmed by confocal microscopy experiments. These lipopeptides that target key components of the outer bacterial cell membrane are good candidates to mitigate infections by X. fastidiosa in plant hosts.IMPORTANCEXylella fastidiosa is a gram-negative bacterium that affects crops of economic importance such as grapevine, olive, almond, and citrus. The lack of efficient methods to control the diseases caused by this pathogen prompts the research of novel strategies. Since lipopolysaccharides (LPS) are a major virulence factor of X. fastidiosa, the development of new compounds that target LPS constitutes a promising approach. We identified cationic lipopeptides with a strong LPS interaction, high bactericidal activity, and low toxicity. These lipopeptides can be considered good candidates to control X. fastidiosa infections.

Keywords: LPS neutralization; antimicrobial peptides; lipopeptides; lytic effect; plant pathogens.

Fig 1

Electrophoresis gel showing extracts of LPS from X. fastidiosa subsp. fastidiosa. Direct LPS extract (lane 1), a 1/10 dilution (lane 2), and the molecular marker (lane 3). White arrows correspond to expected bands with LPS weights according to the extraction kit.

Fig 2

Effect of selected peptides at 150 µM on LPS neutralization. Bars correspond to assays using lyophilized LPS from E. coli 0111:B4 (black bars) or X. fastidiosa subsp. fastidiosa IVIA 5387.2 (gray bars) both at 0.5 EU/mL. Values are the means of three replicates, and error bars represent the confidence interval α = 0.05. Significant differences between techniques within each treatment according to a two-way ANOVA are indicated by asterisks (P < 0.05).

Fig 3

LPS neutralization and bactericidal activity of the 36 peptides screened.

Fig 4

Dose-response relationship between the reduction in viability of X. fastidiosa subsp. fastidiosa IVIA 5387.2 and peptide concentration after 3-hour contact with selected peptides. N₀ is 10⁷ cfu. Values are the means of three replicates, and error bars represent confidence interval (α = 0.05).

Fig 5

Relationship between bactericidal activity of peptides against X. fastidiosa and LPS neutralization. The regression equation is y = −1.63X + 2.26; R² = 0.582 and P-value = 1.57 × 10⁻⁷. BP178 has been excluded from the regression as an outlier.

Fig 6

Biological activity of lipopeptides. Bactericidal activity against X. fastidiosa, LPS neutralization, hemolysis, and effect on tobacco leaves. Values are the means of three replicates, and error bars represent the confidence interval (α = 0.05). REF indicates reference compounds: BP178 at 25 µM for bactericidal activity, YW12D for LPS neutralization, and melittin for hemolysis and effect on tobacco leaves. Horizontal lines represent arbitrarily defined thresholds used to select peptides based on their biological activity profiles. Black bars highlight peptides with high bactericidal activity, high LPS neutralization, and low toxicity.

Fig 7

Microscopy images after a contact test with CF or BP473-K(CF) and X. fastidiosa. (A) On the right, confocal fluorescence microscopy channel (excitation at 492 nm and emission at 517 nm), and on the left, a merge of confocal and interference contrast channels. Image at 30 s contact test with CF and X. fastidiosa. Treatments were performed at 3.1 µM. (B) Merge of confocal and interference contrast channels. Images after different contact times with BP473-K(CF). Treatments were performed at 5 µM.

Fig 8

General sequence of lipopeptides with the best biological activity profile studied in this work: BP389 (Phe⁴, Lys⁶, and Lys¹⁰[COC₃H₇]), BP473 (D-Phe⁴, Lys⁶[COC₃H₇], and Tyr¹⁰), and BP475 (D-Phe⁴, Lys⁶, and Lys¹⁰[COC₃H₇]). Lowercase letters correspond to a D-amino acid. Asterisk corresponds to a lysine incorporating a butanoyl group. Ac stands for acetyl.