Estudio de sustancias antimicrobianas de interés biotecnológico y biomédico

"El incremento de bacterias multidrogo-resistentes (MDR) representa un problema de salud pública a nivel mundial. Actualmente, las terapias antimicrobianas disponibles no son suficientes para eliminar a patógenos MDR, por lo que resulta indispensable el estudio de antimicrobianos con nuevos mecanismos de acción que favorezcan el control de estos patógenos. El uso de péptidos antimicrobianos (PAMs) ha sido sugerido como alternativa en el tratamiento de enfermedades infecciosas pues poseen mecanismos de acción diferentes a los de los antibióticos de amplio espectro; desafortunadamente, son pocos los PAMs que han sido aprobados para su uso terapéutico. Los PAMs del veneno de alacrán son un ejemplo de sustancias bioactivas con potencial para su uso contra bacterias MDR.

Charting the lipopeptidome of nonpathogenic Pseudomonas

Pseudomonas species are prominent producers of lipopeptides that support proliferation in a multitude of environments and foster varied lifestyles. By genome mining of biosynthetic gene clusters (BGCs) with lipopeptide-specific organization, we mapped the global Pseudomonas lipopeptidome and linked its staggering diversity to taxonomy of the producers, belonging to different groups within the major Pseudomonas fluorescens lineage

The importance of antimicrobial compounds produced by beneficial bacteria on the biocontrol of phytopathogens

Bacteria produce antimicrobial compounds to compete for nutrients and space in a particular habitat. Antagonistic interactions can be evaluated by several methodologies including the double-layer agar and simultaneous inhibition assays. Among the well-known inhibitory substances produced by bacteria are the broad-spectrum antibiotics, organic acids, siderophores, antifungal, and bacteriocins. The most studied bacterial genera able to produce these inhibitory substances are Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia, and Burkholderia. Some beneficial bacteria can promote plant growth and degrade toxic compounds in the environment representing an attractive solution to diverse issues in agriculture and soil pollution, particularly in fields with damaged soils where pesticides and fertilizers have been indiscriminately used. Beneficial bacteria may increase plant health by inhibiting pathogenic microorganisms; some examples include Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens, and Burkholderia tropica. However, most studies showing the antagonistic potential of these bacteria have been performed in vitro, and just a few of them have been evaluated in association with plants. Several inhibitory substances involved in pathogen antagonism have not been elucidated yet; in fact, we know only 1 % of the bacterial diversity in a natural environment leading us to assume that many other inhibitory substances remain unexplored. In this review, we will describe the characteristics of some antimicrobial compounds produced by beneficial bacteria, the principal methodologies performed to evaluate their production, modes of action, and their importance for biotechnological purposes

Structural characterization of scorpion peptides and their bactericidal activity against clinical isolates of multidrug-resistant bacteria.

Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against β-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria

Versatile role of Pseudomonas fuscovaginae cyclic lipopeptides in plant and microbial interactions

Pseudomonas fuscovaginae is the most prominent bacterial sheath rot pathogen, causing sheath brown rot disease in rice. This disease occurs worldwide and it is characterized by typical necrotic lesions on the sheath, as well as a reduction in the number of emitted panicles and filled grains. P. fuscovaginae has been shown to produce syringotoxin and fuscopeptin cyclic lipopeptides (CLPs), which have been linked to pathogenicity. In this study, we investigated the role of P. fuscovaginae UPB0736 CLPs in plant pathogenicity, antifungal activity and swarming motility. To do so, we sequenced the strain to obtain a single-contig genome and we constructed deletion mutants in the biosynthetic gene clusters responsible for the synthesis of CLPs. We show that UPB0736 produces a third CLP of 13 amino acids, now named asplenin, and we link this CLP with the swarming activity of the strain. We could then show that syringotoxin is particularly active against Rhizoctonia solani in vitro. By testing the mutants in planta we investigated the role of both fuscopeptin and syringotoxin in causing sheath rot lesions. We proved that the presence of these two CLPs considerably affected the number of emitted panicles, although their number was still significantly affected in the mutants deficient in both fuscopeptin and syringotoxin. These results reveal the importance of CLPs in P. fuscovaginae pathogenicity, but also suggest that other pathogenicity factors may be involved

Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in <i>Pseudomonas</i> Lipopeptide Biosynthesis

In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either ‘on’ (active) or ‘off’ (inactive). Here, we report the unprecedented observation of an E/C-domain that acts ‘on and off’, giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants