Rafael de Almeida Barros

Bio: Rafael de Almeida Barros is an academic researcher from Universidade Federal de Viçosa. The author has contributed to research in topics: Anticarsia gemmatalis & Protease. The author has an hindex of 4, co-authored 17 publications receiving 35 citations.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Abstract: Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis.

Abstract: Purification of active trypsin in the digestive process of insects is essential for the development of potent protease inhibitors (PIs) as an emerging pest control technology and research into insect adaptations to dietary PIs. An important aspect is the presence of proteolytic microorganisms, which contribute to host nutrition. Here, we purified trypsins produced by bacteria Bacillus cereus, Enterococcus mundtii, Enterococcus gallinarum, and Staphylococcus xylosus isolated from the midgut of Anticarsia gemmatalis. The trypsins had a molecular mass of approximately 25 kDa. The enzymes showed increased activity at 40°C, and they were active at pH values 7.5–10. Aprotinin, bis-benzamidine, and soybean Kunitz inhibitor (SKTI) significantly inhibited trypsin activity. The l-1-tosyl-amido-2-phenylethylchloromethyl ketone (TPCK), pepstatin A, E-64, ethylenediamine tetraacetic acid, and calcium ions did not affect the enzyme activity at the concentrations tested. We infer the purified trypsins do not require calcium ions, by which they differ from the trypsins of other microorganisms and the soluble and insoluble trypsins characterized from A. gemmatalis. These data suggest the existence of different isoforms of trypsin in the velvetbean caterpillar midguts.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Abstract: Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Small peptides inhibit gut trypsin‐like proteases and impair Anticarsia gemmatalis (Lepidoptera: Noctuidae) survival and development

Abstract: Background Anticarsia gemmatalis larvae are key defoliating pests of soybean plants. Inorganic insecticides, harmful to the environment and human health, are the main molecules used in the control of this pest. To apply more sustainable management methods, organic molecules with high specificities, such as proteinaceous protease inhibitors, have been sought. Thus, molecular docking studies, kinetics assays, and biological tests were performed to evaluate the inhibitory activity of two peptides (i.e., GORE1 and GORE2) rationally designed to inhibit trypsin-like enzymes, which are the main proteases of A. gemmatalis midgut. Results The molecular docking simulations revealed critical hydrogen bonding patterns of the peptides with key active site residues of trypsin-like proteases of A. gemmatalis and other Lepidopteran insects. The negative values of binding energy indicate that hydrogen bonds potentiate the tight binding of the peptides with trypsin-like proteases, predicting an effective inhibition. The inhibition's rate constants (𝐾𝑖) were 0.49 mM and 0.10 mM for GORE1 and GORE2, resulting in effective inhibition of the activity trypsin on the L-BApNA substrate in the in vitro tests, pointing at the peptide GORE2 has higher inhibitory capacity on the A. gemmatalis trypsins. In addition, the two peptides were determined to be reversible competitive inhibitors. The in vivo test demonstrated that the peptides harm the survival and development of A. gemmatalis larvae. Conclusion This results suggest that the peptides are potential candidates in the management of A. gemmatalis larvae and provide baseline information for the design of new trypsin-like inhibitors based on peptidomimetic tools. This article is protected by copyright. All rights reserved.

Active response of soybean to defoliator Anticarsia gemmatalis Hübner: strategies to overcome protease inhibitor production

Abstract: Los mecanismos de resistencia endogenos de las plantas contra insectos herbivoros pueden permitir el desarrollo de estrategias alternativas para el control de plagas. La activacion de la via de las lipoxigenasas con la produccion de acido jasmonico, hormona responsable por la activacion de los genes inhibidores de proteasa, es una de las principales formas de defensa directa de las plantas contra los insectos. En el presente estudio, cultivares de soya en estadio V3, susceptibles y resistentes a insectos herbivoros fueron utilizadas para investigar las alteraciones en la actividad de LOX y la concentracion de PIs, en respuesta al ataque de orugas en cuarto y quinto instar de Anticarsia gemmatalis (Lepidoptera: Noctuidae) por 24 y 48 horas. La actividad de LOX (0,00 a 0,007 nm s-1/mg) y la concentracion de inhibidores de proteasa (60 a 125 mg de inhibidor de tripsina/ mg de proteina y 90 a 120 mg de inhibidor de tripsina/mg de proteina despues de 24 y 48 horas del ataque respectivamente) aumenta en respuesta a los danos ocasionados por A. gemmatalis. La actividad proteolitica disminuyo aproximadamente en 50% en los cultivares con diferentes grados de resistencia. El aumento del perfil enzimatico del intestino medio de A. gemmatalis puede estar relacionado al aumento de las enzimas tripticas para compensar la inhibicion de proteasas por sustancias entomotoxicas de los cultivares de soya.

The winds of (evolutionary) change: Breathing new life into microbiology

Abstract: To date, over 1500 prokaryotes have been characterized by small subunit rRNA sequencing and molecular phylogeny has had an equally profound effect on our understanding of relationship among eukaryotic microorganisms. The universal phylogenetic tree readily shows however how artificial the strong distinction between the eukaryote and prokaryotes has become. The split between the Archaea and the Bacteria is now recognized as the primary phylogenetic division and that the Eucarya have branched from the same side of the tree as the Archaea. Both prokaryotic domains would seem to be of thermophilic origin suggesting that life arose in a very warm environment. Among the Archaea, all of the Crenarchaeota cultured to date are thermophiles, and the deepest euryarchaeal branchings are represented exclusively by thermophiles. Among the Bacteria, the deepest known branchings are again represented exclusively by thermophiles, and thermophilia is widely scattered throughout the domain. The Archaea comprise a small number of quite disparate phenotypes that grow in unusual niches. All are obligate or facultative anaerobes. All cultured crenarchaeotes are thermophilic, some even growing optimally above the normal boiling temperature of water. The Archaeoglobales are sulfate reducers growing at high temperatures. The extreme halophiles grow only in highly saline environments. The methanogens are confined to a variety of anaerobic niches, often thermophilic. The Bacteria, on the other hand, are notable as being the source of life`s photosynthetic capacity. Five kingdoms of bacteria contain photosynthetic species; and each of the five manifests a distinct type of (chlorophyll-based) photosynthesis.

Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision?

Abstract: The insect gut microbiota has been shown to contribute to the host’s digestion, detoxification, development, pathogen resistance, and physiology. However, there is poor information about the ranking of these roles. Most of these results were obtained with cultivable bacteria, whereas the bacterial physiology may be different between free-living and midgut-colonizing bacteria. In this study, we provided both proteomic and genomic evidence on the ranking of the roles of gut bacteria by investigating the anal droplets from a weevil, Cryptorhynchus lapathi. The gut lumen and the anal droplets showed qualitatively and quantitatively different subsets of bacterial communities. The results of 16S rRNA sequencing showed that the gut lumen is dominated by Proteobacteria and Bacteroidetes, whereas the anal droplets are dominated by Proteobacteria. From the anal droplets, enzymes involved in 31 basic roles that belong to 7 super roles were identified by Q-TOF MS. The cooperation between the weevil and its gut bacteria was determined by reconstructing community pathway maps, which are defined in this study. A score was used to rank the gut bacterial roles. The results from the proteomic data indicate that the most dominant role of gut bacteria is amino acid biosynthesis, followed by protein digestion, energy metabolism, vitamin biosynthesis, lipid digestion, plant secondary metabolite (PSM) degradation, and carbohydrate digestion, while the order from the genomic data is amino acid biosynthesis, vitamin biosynthesis, lipid digestion, energy metabolism, protein digestion, PSM degradation, and carbohydrate digestion. The PCA results showed that the gut bacteria form functional groups from the point of view of either the basic role or super role, and the MFA results showed that there are functional variations among gut bacteria. In addition, the variations between the proteomic and genomic data, analyzed with the HMFA method from the point of view of either the bacterial community or individual bacterial species, are presented. The most dominant role of gut bacteria is essential nutrient provisioning, followed by digestion and detoxification. The weevil plays a pioneering role in diet digestion and mainly digests macromolecules into smaller molecules which are then mainly digested by gut bacteria.

Genome of Tetranychus urticae reveals herbivorous pest adaptations

Abstract: The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant–herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm.

Abstract: Protein–peptide interactions play a fundamental role in a wide variety of biological processes, such as cell signaling, regulatory networks, immune responses, and enzyme inhibition. Peptides are characterized by low toxicity and small interface areas; therefore, they are good targets for therapeutic strategies, rational drug planning and protein inhibition. Approximately 10% of the ethical pharmaceutical market is protein/peptide-based. Furthermore, it is estimated that 40% of protein interactions are mediated by peptides. Despite the fast increase in the volume of biological data, particularly on sequences and structures, there remains a lack of broad and comprehensive protein–peptide databases and tools that allow the retrieval, characterization and understanding of protein–peptide recognition and consequently support peptide design. We introduce Propedia, a comprehensive and up-to-date database with a web interface that permits clustering, searching and visualizing of protein–peptide complexes according to varied criteria. Propedia comprises over 19,000 high-resolution structures from the Protein Data Bank including structural and sequence information from protein–peptide complexes. The main advantage of Propedia over other peptide databases is that it allows a more comprehensive analysis of similarity and redundancy. It was constructed based on a hybrid clustering algorithm that compares and groups peptides by sequences, interface structures and binding sites. Propedia is available through a graphical, user-friendly and functional interface where users can retrieve, and analyze complexes and download each search data set. We performed case studies and verified that the utility of Propedia scores to rank promissing interacting peptides. In a study involving predicting peptides to inhibit SARS-CoV-2 main protease, we showed that Propedia scores related to similarity between different peptide complexes with SARS-CoV-2 main protease are in agreement with molecular dynamics free energy calculation. Propedia is a database and tool to support structure-based rational design of peptides for special purposes. Protein–peptide interactions can be useful to predict, classifying and scoring complexes or for designing new molecules as well. Propedia is up-to-date as a ready-to-use webserver with a friendly and resourceful interface and is available at: https://bioinfo.dcc.ufmg.br/propedia

Broad range flavonoid profiling by LC/MS of soybean genotypes contrasting for resistance to Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Abstract: Attack by herbivores is a major biotic stress limiting the soybean crop production. Plant defenses against caterpillars include the production of secondary metabolites such as flavonoids, which constitute a diverse group of plant secondary metabolites. Thus, a more discriminate metabolic profiling between genotypes are important for a more comprehensive and reliable characterization of soybean resistance. Therefore, in this study a non-targeted LC/MS-based for analysis of flavonoid profiles of soybean genotypes contrasting to the resistance to A. gemmatalis was applied. Clustering analysis revealed profiles highly distinct between the susceptible UFV 105 AP and the resistant IAC 17 genotypes. This comparative approach enables to identify directly from leaf extract some new compounds related to resistance, some of which were present in higher abundance specifically in the IAC 17 genotype: four Quercetin conjugates, Rutin (Quercetin 3-O-Rutinoside), Quercetin-3,7-O- di-glucoside, Quercetin-3-O-rhamnosylglycoside-7-O-glucoside and Quercetin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside; two Genistein conjugates, Genistein-7-O-diglucoside-dimalonylated and Genistein-7-O-6-O-malonylglucoside; and one Daidzein conjugate, Daidzein-7-O-Glucoside-malonate. The most abundant flavonoid glycoconjugates in soybean leaves belongs to Quercetin and Kaempferol classes. However, only one from the identified compounds was classified as a Kaempferol. The Kaempferol-3-O-L-rhamnopyranosyl-glucopyranoside showed high abundance in the resistant genotype IAC 17. The metabolic profiles generated by LC/MS allowed the reconstruction of the flavonoid biosynthetic pathways, which revealed a constitutive character for herbivory resistance in the resistant genotype IAC-17 and a metabolic regulation for the rechanneling of Quercetin, Kaempferol and Genistein conjugates in soybean. Highest relative abundances were detected for glyconjugates, such as Rutin, Quercetin 3-O-rhamnosylglycoside-7-O-glucoside and Quercitin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside in the leaves of the resistant genotype.