Bifunctional Inhibitors from Capsicum chinense Seeds with Antimicrobial Activity and Specific Mechanism of Action Against Phytopathogenic Fungi.

BACKGROUND: Antimicrobial peptides (AMPs) are found in the defense system in virtually all life forms, being present in many, if not all, plant species. OBJECTIVE: The present work evaluated the antimicrobial, enzymatic activity and mechanism of action of the PEF2 fraction from Capsicum chinense Jack. seeds against phytopathogenic fungi. METHODS: Peptides were extracted from C. chinense seeds and subjected to reverse-phase chromatography on an HPLC system using a C18 column coupled to a C8 guard column, then the obtained PEF2 fraction was rechromatographed using a C2/C18 column. Two fractions, named PEF2A and PEF2B, were obtained. The fractions were tested for antimicrobial activity on Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and α-amylase activity assays were also performed. The mechanism of action by which PEF2 acts on filamentous fungi was studied through analysis of membrane permeability and production of reactive oxygen species (ROS). Additionally, we investigated mitochondrial functionality and caspase activation in fungal cells. RESULTS: It is possible to observe that PEF2 significantly inhibited trypsin activity and T. molitor larval α-amylase activity. The PEF2 fraction was able to inhibit the growth of C. gloeosporioides, C. lindemuthianum and F. oxysporum. PEF2A inhibited the growth of C. lindemuthianum (75%) and F. solani (43%). PEF2B inhibited C. lindemuthianum growth (66%) and F. solani (94%). PEF2 permeabilized F. solani cell membranes and induced ROS in F. oxysporum and F. solani. PEF2 could dissipate mitochondrial membrane potential but did not cause the activation of caspases in all studied fungi. CONCLUSION: The results may contribute to the biotechnological application of these AMPs in the control of pathogenic microorganisms in plants of agronomic importance.

Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi.

Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

The toxicity of a lipid transfer protein (Cc-LTP1) from Coffea canephora Seeds on the larval development of Callosobruchus maculatus (Coleoptera: Bruchidae).

In this work, we analyzed the effects of coffee seed proteins, especially Cc-LTP1 on the larval development of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a bruchid pest of beans and the most important insect pest of Vigna unguiculata (L.) Walp. Artificial seed assay, which incorporated the F/0-90 fraction from Coffea canephora seeds, resulted in the reduction of oviposition and caused an inhibition of C. maculatus larval development in a dose-dependent manner. The F/0-90 fraction used at a 4 % concentration resulted in the survival of no larvae. The purified Cc-LTP1, at a concentration of 0.5 %, also demonstrated effective inhibition of larval development, reducing both females oviposition and the weight and number of larvae. Cc-LTP1 was also able to inhibit the C. maculatus gut α-amylase activity, and immunolabeling by an anti-LTP serum was observed in the midgut tissues of the C. maculatus larvae. Cc-LTP1 has shown binding affinity towards microvillar cells, endoplasmic reticulum and mitochondria, as demonstrated by micrographic images taken by a transmission electron microscope. The results from this study indicate that Cc-LTP1 has insecticidal actions toward C. maculatus and exerts anti-nutritional effects with direct actions on intestinal tissues.

Alfa-amylase inhibitors of legume seeds and their involvement in the resistance to bruchid beetles

The presence of inhibitors of alfa-amylases from several sources (bacterial, plant, insect and mammallian) was investigated in seeds of several food legumes. No inhibitor of any of the tested enzymes was found in Phaseolus lunatus (Lima bean) seeds while the presence of inhibitors of insect (bruchid), porcine pancreas and human saliva alfa-amylases was confirmed in the seeds of P. vulgaris (common bean). Glycine max (soybean) seeds showed alfa-als for all the tested enzymes except for the porcine pancreatic amylase. Although we have found low levels of alfa-als in both bruchid-susceptible and resistant cowpea (Vigna unguiculata) seeds their presence does not correlate with the resistance shown by the seeds of some cultivars to the cowpea weevil Callosobruchus maculatus. The results reported here suggest that alfa-Als are not involved in the resistance of seeds of some cowpea lines to C. maculatus and that variant vicilins, the 7 S storage proteins involved in this resistance, do not show any inhibitory towards bruchi alfa-amylases