Peptide Conjugates Derived from flg15, Pep13, and PIP1 That Are Active against Plant-Pathogenic Bacteria and Trigger Plant Defense Responses.

Thirty peptide conjugates were designed by combining an antimicrobial peptide (BP16, BP100, BP143, KSL-W, BP387, or BP475) at the N- or C-terminus of a plant defense elicitor peptide (flg15, BP13, Pep13, or PIP1). These conjugates were highly active in vitro against six plant-pathogenic bacteria, especially against Xanthomonas arboricola pv. pruni, Xanthomonas fragariae and Xanthomonas axonopodis pv. vesicatoria. The most active peptides were those incorporating Pep13. The order of the conjugation influenced the antibacterial activity and the hemolysis. Regarding the former, peptide conjugates incorporating the elicitor peptide flg15 or Pep13 at the C-terminus were, in general, more active against Pseudomonas syringae pv. actinidiae and P. syringae pv. syringae, whereas those bearing these elicitor peptides at the N-terminus displayed higher activity against Erwinia. amylovora and the Xanthomonas species. The best peptide conjugates displayed MIC values between 0.8 and 12.5 µM against all the bacteria tested and also had low levels of hemolysis and low phytotoxicity. Analysis of the structural and physicochemical parameters revealed that a positive charge ranging from +5 to +7 and a moderate hydrophobic moment/amphipathic character is required for an optimal biological profile. Interestingly, flg15-BP475 exhibited a dual activity, causing the upregulation of the same genes as flg15 and reducing the severity of bacterial spot in tomato plants with a similar or even higher efficacy than copper oxychloride. Characterization by nuclear magnetic resonance (NMR) of the secondary structure of flg15-BP475 showed that residues 10 to 25 fold into an α-helix. This study establishes trends to design new bifunctional peptides useful against plant diseases caused by plant-pathogenic bacteria. IMPORTANCE The consequences of plant pathogens on crop production together with the lack of effective and environmentally friendly pesticides evidence the need of new agents to control plant diseases. Antimicrobial and plant defense elicitor peptides have emerged as good candidates to tackle this problem. This study focused on combining these two types of peptides into a single conjugate with the aim to potentiate the activity of the individual fragments. Differences in the biological activity of the resulting peptide conjugates were obtained depending on their charge, amphipathicity, and hydrophobicity, as well as on the order of the conjugation of the monomers. This work provided bifunctional peptide conjugates able to inhibit several plant-pathogenic bacteria, to stimulate plant defense responses, and to reduce the severity of bacterial spot in tomato plants. Thus, this study could serve as the basis for the development of new antibacterial/plant defense elicitor peptides to control bacterial plant pathogens.

D-Amino Acid-Containing Lipopeptides Derived from the Lead Peptide BP100 with Activity against Plant Pathogens.

From a previous collection of lipopeptides derived from BP100, we selected 18 sequences in order to improve their biological profile. In particular, analogues containing a D-amino acid at position 4 were designed, prepared, and tested against plant pathogenic bacteria and fungi. The biological activity of these sequences was compared with that of the corresponding parent lipopeptides with all L-amino acids. In addition, the influence of the length of the hydrophobic chain on the biological activity was evaluated. Interestingly, the incorporation of a D-amino acid into lipopeptides bearing a butanoyl or a hexanoyl chain led to less hemolytic sequences and, in general, that were as active or more active than the corresponding all L-lipopeptides. The best lipopeptides were BP475 and BP485, both incorporating a D-Phe at position 4 and a butanoyl group, with MIC values between 0.8 and 6.2 µM, low hemolysis (0 and 24% at 250 µM, respectively), and low phytotoxicity. Characterization by NMR of the secondary structure of BP475 revealed that the D-Phe at position 4 disrupts the α-helix and that residues 6 to 10 are able to fold in an α-helix. This secondary structure would be responsible for the high antimicrobial activity and low hemolysis of this lipopeptide.

A Bifunctional Peptide Conjugate That Controls Infections of Erwinia amylovora in Pear Plants.

In this paper, peptide conjugates were designed and synthesized by incorporating the antimicrobial undecapeptide BP16 at the C- or N-terminus of the plant defense elicitor peptide flg15, leading to BP358 and BP359, respectively. The evaluation of their in vitro activity against six plant pathogenic bacteria revealed that BP358 displayed MIC values between 1.6 and 12.5 µM, being more active than flg15, BP16, BP359, and an equimolar mixture of BP16 and flg15. Moreover, BP358 was neither hemolytic nor toxic to tobacco leaves. BP358 triggered the overexpression of 6 out of the 11 plant defense-related genes tested. Interestingly, BP358 inhibited Erwinia amylovora infections in pear plants, showing slightly higher efficacy than the mixture of BP16 and flg15, and both treatments were as effective as the antibiotic kasugamycin. Thus, the bifunctional peptide conjugate BP358 is a promising agent to control fire blight and possibly other plant bacterial diseases.

Solid-Phase Synthesis of Biaryl Cyclic Lipopeptides Derived from Arylomycins.

An efficient approach for the solid-phase synthesis of N-methylated tailed biaryl cyclic lipopeptides based on the structure of arylomycins was established. Each of these analogues incorporates an N-terminal linear lipopeptide attached to a biaryl cyclic tripeptide containing a Phe-Tyr, a Tyr-Tyr, or a His-Tyr linkage. This methodology first involved an intramolecular Suzuki-Miyaura arylation of a linear peptidyl resin incorporating the corresponding halogenated amino acid at the N-terminus and a boronotyrosine at the C-terminus. After N-methylation of the resulting biaryl cyclic peptidyl resin, the N-methylated lipopeptidyl tail was then assembled. The biaryl cyclic lipopeptides were purified and characterized.

Solid-phase synthesis of biaryl bicyclic peptides containing a 3-aryltyrosine or a 4-arylphenylalanine moiety.

A methodology for the solid-phase synthesis of biaryl bicyclic peptides containing a Phe-Phe, a Phe-Tyr or a Tyr-Tyr motif has been devised. This approach comprises two key steps. The first one involves the cyclization of a linear peptidyl resin containing the corresponding halo- and boronoamino acids via a microwave-assisted Suzuki-Miyaura cross coupling. This step is followed by the macrolactamization of the resulting biaryl monocyclic peptidyl resin leading to the formation of the expected biaryl bicyclic peptide. This study provides the first solid-phase synthesis of this type of bicyclic compounds being amenable to prepare a diversity of synthetic or natural biaryl bicyclic peptides.

Total Solid-Phase Synthesis of Dehydroxy Fengycin Derivatives.

A rapid and efficient solid-phase strategy for the synthesis of dehydroxy fengycins derivatives is described. This synthetic approach involved the linkage of a Tyr to a Wang resin via a Mitsunobu reaction and the elongation of the peptide sequence followed by subsequent acylation of the N-terminus of the resulting linear peptidyl resin, esterification of the phenol group of a Tyr with an Ile, and final macrolactamization. The amino acid composition as well as the presence of the N-terminal acyl group significantly influenced the stability of the macrolactone. Cyclic lipodepsipeptides with a l-Tyr3/d-Tyr9 configuration were more stable than those containing the Tyr residues with an opposite configuration. This work constitutes the first approach on the total solid-phase synthesis of dehydroxy fengycin derivatives.

Antimicrobial activity of linear lipopeptides derived from BP100 towards plant pathogens.

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria. These lipopeptides were synthesized on solid-phase and screened for their antimicrobial activity, toxicity and proteolytic stability. They incorporated a butanoyl, a hexanoyl or a lauroyl group at the N-terminus or at the side chain of a lysine residue placed at each position of the sequence. Their antimicrobial activity and hemolysis depended on the fatty acid length and its position. In particular, lipopeptides containing a butanoyl or a hexanoyl chain exhibited the best biological activity profile. In addition, we observed that the incorporation of the acyl group did not induce the overexpression of defense-related genes in tomato. Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested (minimum inhibitory concentration of 0.8 to 12.5 µM), low hemolytic, low phytotoxic and significantly stable to protease degradation. This family of lipopeptides might be promising functional peptides useful for plant protection.