Tesis doctoral de Rafael Ferre Malagón
Plant diseases caused by pathogenic microorganisms are one of the major factors limiting worldwide crop production. Their control requires the continued use of pesticides, being mainly based on antibiotics and copper compounds. Although antibiotics are highly efficient in plant disease control, it has been reported the emergence of resistant strains and their use has been banned in some countries, difficulting the plant disease management. Therefore, the search for novel antimicrobial agents has gained importance over the last several years. Antimicrobial peptides (amps) are essential compounds of the innate immune system of virtually all organisms and are being considered as a good alternative to conventional antibiotics. Due to their optimal properties, a great deal of scientific effort has been invested in studying their application in human, veterinary and plant disease control. the present phd study focused on the development of new sustainable antimicrobial agents derived from the cecropin a-melittin hybrid antimicrobial peptide wklfkkilkvl-nh2 (pep3) for use in plant protection. In particular, this work was centered on developing pep3 analogues active against the economically important plant pathogenic bacteria erwinia amylovora, pseudomonas syringae pv. Syringae, xanthomonas axonopodis pv. Vesicatoria, and the phytopathogenic fungi fusarium oxysporum, aspergillus niger, rhizopus stolonifer, and penicillium expansum. Pep3 analogues with high activity against these pathogens while exhibiting low cytotoxicity were obtained through a rational design taking into account some fundamental parameters that have been described to modulate the activity of amps, such as the positive charge, the overall hydrophobicity and amphipathicity. The work followed the standard approach in drug discovery which is based on a «hit to lead» stage followed by a «lead optimization» stage. First, we prepared a set of several analogues with the general structure r-x1klfkkilkx10l-nh2, where x1 and x10 corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and r included different n-terminal derivatizations. Peptides with improved microbicidal activity and minimized cytotoxicity and sensitivity to protease degradation, compared to pep3, were identified. Next, we succesfully optimized the best analog using a combinatorial chemistry approach. A 125-member library of synthetic linear undecapeptides was prepared by including 5 variations in each r, x1 and x10 positions. Analogues with the best profile in terms of in vitro antimicrobial activity, cytotoxicity and stability to proteinase k degradation were tested ex vivo by evaluating their capacity to prevent infections of e. Amylovora and p. Expansum on flowers and fruits, respectively. The peptide kklfkkilkyl-nh2 (bp100) was highly active to prevent infections of e. Amylovora in flowers showing efficacies of 63-76% at 100 um, being only slightly less effective than the antibiotic streptomycin, which is currently used in fire blight control. On the other hand, the peptide ts-fklfkkilkvl-nh2 (bp22) was the most effective to prevent p. Expansum infection in apple fruits, with an average efficacy of 56% disease reduction, being not significantly different than that of a commercial formulation of the reference fungicide imazalil. Therefore, these peptides might be considered as potential agents for use in plant protection either as pesticide ingredients or in some cases expressed in transgenic plants. moreover, insights into the mode of action of bp100 were carried out to fully rationalize the biological properties of these peptides and to further improve them. Taking advantage of the intrinsic tyr fluorescence of bp100, we investigated its binding affinity and damaging effect on phospholipid bilayers modeling the bacterial and mammalian cytoplasmatic membranes using spectroscopic methodologies. Results showed a stronger selectivity of bp100 towards anionic bacterial membrane models as indicated by the high obtained partition constants, being one order of magnitude greater than that for the neutral mammalian membrane models. For the anionic systems, membrane saturation was observed at high peptide/lipid ratios and found to be related with bp100-induced vesicle permeabilization, membrane electroneutrality, and vesicle aggregation. Occurrence of bp100 translocation was unequivocally detected at both high and low peptide/lipid ratios. These findings unravel the relationship among the closely coupled processes of charge neutralization, permeabilization, and translocation in the mechanism of action of antimicrobial peptides. Moreover, it was deduced an equation that correlates the minimum inhibitory concentration (mic) of an amp with the partition constant and the threshold concentration in the membrane. This equation provides an easily prediction of in vivo antimicrobial activities from simple biophysical parameters.
Datos académicos de la tesis doctoral «Design and synthesis of short antimicrobial peptides for plant protection. study of their mode of action.«
- Título de la tesis: Design and synthesis of short antimicrobial peptides for plant protection. study of their mode of action.
- Autor: Rafael Ferre Malagón
- Universidad: Girona
- Fecha de lectura de la tesis: 23/07/2010
Dirección y tribunal
- Director de la tesis
- Marta Planas Grabuleda
- Tribunal
- Presidente del tribunal: jean Martinez
- María del rosario González muñiz (vocal)
- nuno Santos (vocal)
- María isabel Romero García (vocal)