Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.
BACKGROUND The most common milk‐clotting enzymes in the cheese industry are recombinant chymosins. Food naturalness is a factor underpinning consumers' food choice. For consumers who avoid food with ingredients from genetically modified organisms (GMOs), the use of vegetable‐based rennet substitute in the cheese formulation may be a suitable solution. Artichokes that deviate from optimal products, when allowed to bloom due to flower protease composition, are excellent as raw material for vegetable rennet preparation. As enzymatic milk clotting exerts a significant impact on the characteristics of the final product, this product should be studied carefully. RESULTS Mature flowers from unharvested artichokes (Cynara scolymus cv. Francés) that did not meet aesthetic standards for commercialization were collected and used to prepare a flower extract. This extract, as a coagulant preparation, enabled the manufacture of cheeses with distinctive characteristics compared with cheeses prepared with chymosin. Rennet substitution did not affect the actual yield but led to significant changes in dry matter yield, humidity, water activity, protein content, and color, and conferred antioxidant activity to the cheeses. The rennet substitution promoted significant modifications in springiness, and in the microstructure of the cheese, with a more porous protein matrix and an increment in the size of the fat globules. Both formulations showed a similar microbiota evolution pattern with excellent microbiological quality and good sensory acceptance. CONCLUSIONS The rennet substitute studied here produced a cheese adapted to specific market segments that demand more natural and healthier products made with a commitment to the environment but well accepted by a general cheese consumer. © 2020 Society of Chemical Industry
The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide’s mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells’ cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization.
Frente al ataque de un patógeno, las plantas pueden producir una gran diversidad de factores de defensa tales como metabolitos secundarios y una amplia gama de proteínas y péptidos antimicrobianos (AMPs), entre los que se encuentran las defensinas. Estas son pequeñas proteínas catiónicas ricas en cisteína cuya principal actividad reportada es la antifúngica, pero se han descripto también muchas otras acciones para estas proteínas. En vista su alto grado de conservación estructural, las diferencias en las especificidades y las múltiples actividades de estas proteínas pueden ser atribuidas a la variación en la secuencia aminoacídica y a la distribución de cargas de los loops expuestos al solvente que modulan su función biológica. En estos loops se localizan dos motivos importantes su actividad, el g-core, un motivo de relevancia para los AMPs en general, y el α-core que no ha sido estudiado en profundidad. Este trabajo de tesis describe la obtención y el estudio de nuevas proteínas de defensa de una especie vegetal silvestre de la flora argentina, que pueden resultar moléculas atractivas para su potencial aplicación agronómica o médica. Las especies silvestres constituyen una valiosa fuente de AMPs por su notable adaptación al medio ambiente, lo que resulta en una mayor resistencia a enfermedades en comparación con los cultivos agrícolas. A partir de ARNm de flores de Silybum marianum se clonaron dos defensinas (DefSm1D y DefSm2D), se realizó el análisis de las secuencias, la predicción de la estructura terciaria a través del modelado molecular por homología y su análisis funcional. Con el fin de profundizar el conocimiento de las relaciones estructura-función e identificar regiones relacionadas con la actividad antifúngica, a partir del estudio estructural de las defensinas se realizó el diseño racional péptidos derivados de las regiones g- y α-core. Se realizó la síntesis química de péptidos en fase sólida utilizando la estrategia Fmoc y se purificaron y analizaron por HPLC-RP, ESI-MS y CD. Se evaluó y caracterizó la acción de los péptidos sobre dos hongos fitopatógenos del género Fusarium. Los péptidos derivados de DefSm2D resultaron ser activos en concentraciones micromolares frente a Fusarium graminearum, un fitopatógeno que afecta al cultivo de trigo produciendo no sólo mermas significativas en el rendimiento sino también micotoxinas que permanecen en los granos. Se evaluó y caracterizó la actividad de los péptidos sobre conidios a través de distintas técnicas. Los péptidos derivados del α-core produjeron agregación de los conidios del hongo y un efecto diferencial sobre la morfología de la pared celular. Se estudió también la actividad de péptidos modificados, derivados de la región α-core. Nuestra investigación sugiere que las regiones α- y g-core estarían implicadas en la actividad antifúngica de la defensina de origen actuando mediante mecanismos diferenciales.
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