Chitinase from the Latex of Ficus benjamina L. Displays Antifungal Activity by Inducing ROS Generation and Structural Damage to the Fungal Cell Wall and Plasma Membrane.

BACKGROUND: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. OBJECTIVES: This study aimed to purify a chitinase from the latex of Ficus benjamina. METHODS: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. RESULTS: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. CONCLUSION: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-ß-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

Antidermatophytic activity of synthetic peptides: Action mechanisms and clinical application as adjuvants to enhance the activity and decrease the toxicity of Griseofulvin.

BACKGROUND: Dermatophytes belonging to the Trichophyton genus are important human pathogens, but they have developed resistance to griseofulvin, the most common antifungal drug used to treat dermatophytosis. OBJECTIVE: This study was aimed to evaluate the antidermatophytic activity of synthetic peptides, as well as mechanisms of action and synergistic effect with griseofulvin. METHODS: Scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FM) were employed to understand the activity and the mechanism of action of peptides. RESULTS: Here we report that synthetic peptides at 50 µg/mL, a concentration 20-fold lower than griseofulvin, reduced the microconidia viability of T. mentagrophytes and T. rubrum by 100%, whereas griseofulvin decreased their viability by only 50% and 0%, respectively. The action mechanism of peptides involved cell wall damage, membrane pore formation and loss of cytoplasmic content. Peptides also induced overproduction of reactive oxygen species (ROS) and enhanced the activity of griseofulvin 10-fold against both fungi, suggesting synergistic effects, and eliminated the toxicity of this drug to human erythrocytes. Docking analysis revealed ionic and hydrophobic interactions between peptides and griseofulvin, which may explain the decline of griseofulvin toxicity when mixed with peptides. CONCLUSION: Therefore, our results strongly suggest six peptides with high potential to be employed alone as new drugs or as adjuvants to enhance the activity and decrease the toxicity of griseofulvin.

Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge.

KEY MESSAGE: Cowpea miRNAs and Argonaute genes showed differential expression patterns in response to CPSMV challenge Several biotic stresses affect cowpea production and yield. CPSMV stands out for causing severe negative impacts on cowpea. Plants have two main induced immune systems. In the basal system (PTI, PAMP-triggered immunity), plants recognize and respond to conserved molecular patterns associated with pathogens (PAMPs). The second type (ETI, Effector-triggered immunity) is induced after plant recognition of specific factors from pathogens. RNA silencing is another important defense mechanism in plants. Our research group has been using biochemical and proteomic approaches to learn which proteins and pathways are involved and could explain why some cowpea genotypes are resistant whereas others are susceptible to CPSMV. This current study was conducted to determine the role of cowpea miRNA in the interaction between a resistant cowpea genotype (BRS-Marataoã) and CPSMV. Previously identified and deposited plant microRNA sequences were used to find out all possible microRNAs in the cowpea genome. This search detected 617 mature microRNAs, which were distributed in 89 microRNA families. Next, 4 out of these 617 miRNAs and their possible target genes that encode the proteins Kat-p80, DEAD-Box, GST, and SPB9, all involved in the defense response of cowpea to CPSMV, had their expression compared between cowpea leaves uninoculated and inoculated with CPSMV. Additionally, the differential expression of genes that encode the Argonaute (AGO) proteins 1, 2, 4, 6, and 10 is reported. In summary, the studied miRNAs and AGO 2 and AGO4 associated genes showed differential expression patterns in response to CPSMV challenge, which indicate their role in cowpea defense.

Trypsin inhibitor from Enterolobium contortisiliquum seeds impairs Aedes aegypti development and enhances the activity of Bacillus thuringiensis toxins.

BACKGROUND: Disease vector insects are barriers for human development. The use of synthetic chemicals to control these vectors has caused damage to the environment and contributed to the arising of resistant insect populations. This has led to an increased search for plant-derived molecules with insecticidal activity or that show synergistic effects with known insecticidal substances, such as protease inhibitors. Thus, we aimed to evaluate the effect of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on Aedes aegypti development as well as its effect on insecticidal activity of Bacillus thuringiensis toxins. RESULTS: EcTI showed an apparent molecular mass about of 20 kDa in SDS-PAGE and was able to inhibit in vitro the activity of trypsin and proteases from midgut of Ae. aegypti larvae. EcTI was not able to cause acute toxicity on mosquito larvae even at 1000 µg mL-1 , however it promoted a delay in larval development after prolonged exposure. The zymogram results for EcTI-treated larvae (from 50 to 200 µg mL-1 ) showed an increase of midgut proteases activity as a larvae defense mechanism, however no changes in the enzyme profile was observed. These same concentrations were able to enhance up to three fold the insecticidal activity of B. thuringiensis toxins without causing toxicity to Artemia sp. nauplii, a non-target organism. CONCLUSIONS: The results offer a novel approach by combining EcTI and B. thuringiensis toxins for combating Ae. aegypti larvae. © 2020 Society of Chemical Industry.

Gene expression during development and overexpression after Cercospora kikuchii and salicylic acid challenging indicate defensive roles of the soybean toxin.

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Anticandidal activity of synthetic peptides: Mechanism of action revealed by scanning electron and fluorescence microscopies and synergism effect with nystatin.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Scanning electron microscopy reveals deleterious effects of Moringa oleifera seed exuded proteins on root-knot nematode Meloidogyne incognita eggs.

Plant seeds can exudate active molecules with inhibitory effects against several soil pathogens, including nematodes. This study aimed to characterize and evaluate the nematicidal properties against Meloidogyne incognita of exuded proteins from Moringa oleifera seeds. M. oleifera seeds were soaked in distilled water, and exudates were harvested and analyzed for the presence of defense proteins and anthelmintic activity. Enzymatic assays revealed the existence of PR-proteins such as ß-1,3-glucanases (0.18 ± 0.003 nkatal mg-1 of protein), chitinases (0.22 ± 0.004 nkatal mg-1 of protein), proteases (261.30 ± 6.405 AU mg-1 of protein min-1), serine (190.30 ± 5.574 IA mg-1 of protein) and cysteine (231.70 ± 0.923 IA mg-1 of protein), protease inhibitors. The exuded proteins presented ovicidal activity and caused 100% mortality of second-stage juveniles (J2s). Scanning electron microscopy (SEM) revealed deleterious effects on M. incognita eggs, such as invaginations, cracks, scratched surface, and loss of internal content. These findings confirm the presence of anthelmintic proteins in M. oleifera seed exudate, possibly involved in plant defense during seed germination. Besides this, the exuded proteins exhibited strong biotechnological potential for use in the biocontrol of M. incognita infections, which are responsible for millions of dollars in staple crop losses every year.

RcAlb-PepII, a synthetic small peptide bioinspired in the 2S albumin from the seed cake of Ricinus communis, is a potent antimicrobial agent against Klebsiella pneumoniae and Candida parapsilosis.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

ClTI, a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds, exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis.

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.

Mo-CBP3, a 2S albumin from Moringa oleifera, is a complex mixture of isoforms that arise from different post-translational modifications.

Mo-CBP3 is a chitin-binding 2S albumin from Moringa oleifera. This seed storage protein is resistant to thermal denaturation and shows biological activities that might be of practical use, such as antifungal properties against Candida sp., a pathogen that causes candidiasis, and against Fusarium solani, a soil fungus that can cause diseases in plants and humans. Previous work has demonstrated that Mo-CBP3 is a mixture of isoforms encoded by members of a small multigene family. Mature Mo-CBP3 is a small protein (∼14 kDa), constituted by a small chain of approximately 4 kDa and a large chain of 8 kDa, which are held together by disulfide bridges. However, a more comprehensive picture on the spectrum of Mo-CBP3 isoforms which are found in mature seeds, is still lacking. In this work, genomic DNA fragments were obtained from M. oleifera leaves, cloned and completely sequenced, thus revealing new genes encoding Mo-CBP3. Moreover, mass spectrometry analysis showed that the mature protein is a complex mixture of isoforms with a remarkable number of molecular mass variants. Using computational predictions and calculations, most (∼86%) of the experimentally determined masses were assigned to amino acid sequences deduced from DNA fragments. The results suggested that the complex mixture of Mo-CBP3 isoforms originates from proteins encoded by closely related genes, whose products undergo different combinations of distinct post-translational modifications, including cleavage at the N- and C-terminal ends of both subunits, cyclization of N-terminal Gln, as well as Pro hydroxylation, Ser/Thr phosphorylation, and Met oxidation.

ClCPI, a cysteine protease inhibitor purified from Cassia leiandra seeds has antifungal activity against Candida tropicalis by inducing disruption of the cell surface.

Infections caused by Candida tropicalis have increased significantly worldwide in parallel with resistance to antifungal drugs. To overcome resistance novel drugs have to be discovered. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against C. tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of ß-mercaptoethanol and ESI-MS were 16.63 kDa and 18.362 kDa, respectively. ClCPI was stable in the pH range of 7.0-9.0 and thermostable up to 60 °C for 20 min. ClCPI inhibited cysteine proteases, but not trypsin, chymotrypsin neither alpha-amylase. Inhibition of papain was uncompetitive with a Ki of 4.1 × 10-7 M and IC50 of 8.5 × 10-7 M. ClCPI at 2.6 × 10-6 M reduced 50% C. tropicalis growth. ClCPI induced damages and morphological alterations in C. tropicalis cell surface, which led to death. These results suggest that ClCPI have great potential for the development of an antifungal drug against C. tropicalis.

Gene expression and spatiotemporal localization of antifungal chitin-binding proteins during Moringa oleifera seed development and germination.

MAIN CONCLUSION: Chitin-binding proteins behave as storage and antifungal proteins in the seeds of Moringa oleifera. Moringa oleifera is a tropical multipurpose tree. Its seed constituents possess coagulant, bactericidal, fungicidal, and insecticidal properties. Some of these properties are attributed to a group of polypeptides denominated M. oleifera chitin-binding proteins (in short, Mo-CBPs). Within this group, Mo-CBP2, Mo-CBP3, and Mo-CBP4 were previously purified to homogeneity. They showed high amino acid similarity with the 2S albumin storage proteins. These proteins also presented antimicrobial activity against human pathogenic yeast and phytopathogenic fungi. In the present study, the localization and expression of genes that encode Mo-CBPs and the biosynthesis and degradation of the corresponding proteins during morphogenesis and maturation of M. oleifera seeds at 15, 30, 60, and 90 days after anthesis (DAA) and germination, respectively, were assessed. The Mo-CBP transcripts and corresponding proteins were not detected at 15 and 30 days after anthesis (DAA). However, they accumulated at the latter stages of seed maturation (60 and 90 DAA), reaching the maximum level at 60 DAA. The degradation kinetics of Mo-CBPs during seed germination by in situ immunolocalization revealed a reduction in the protein content 48 h after sowing (HAS). Moreover, Mo-CBPs isolated from seeds at 60 and 90 DAA prevented the spore germination of Fusarium spp. Taken together, these results suggest that Mo-CBPs play a dual role as storage and defense proteins in the seeds of M. oleifera.

The expression of the genes involved in redox metabolism and hydrogen peroxide balance is associated with the resistance of cowpea [Vigna unguiculata (L.) Walp.] to the hemibiotrophic fungus Colletotrichum gloeosporioides.

Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.

Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII are synthetic antimicrobial peptides active against human pathogens by stimulating ROS generation and increasing plasma membrane permeability.

The efficiency of current antimicrobial drugs is noticeably decreasing and thus the development of new treatments is necessary. Natural and synthetic antimicrobial peptides (AMPs) have attracted great attention as promising candidates. Inspired on Mo-CBP3, an antimicrobial protein from Moringa oleifera seeds, we designed and synthesized three AMPs named Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII. All these three peptides inhibited the growth of Candida species and pathogenic bacteria, penetrate into microbial cells, but none is hemolytic or toxic to human cells. Mo-CBP3-PepIII, particularly, showed the strongest antimicrobial activity against Staphylococcus aureus and Candida species, important human pathogens. Additionally, Mo-CBP3-PepIII did not exhibit hemolytic or toxic activity to mammalian cells, but increased Staphylococcus aureus plasma membrane permeabilization. In Candida parapsilosis, Mo-CBP3-PepIII induced pore formation in the plasma membrane and overproduction of reactive oxygen species. Bioinformatics analysis suggested that Mo-CBP3-PepIII is resistant to pepsin digestion and other proteolytic enzymes present in the intestinal environment, which opens the possibility of oral delivery in future treatments. Together, these results suggest that Mo-CBP3-PepIII has great potential as an antimicrobial agent against the bacterium S. aureus and the fungi C. parapsilosis.

A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor ( LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin-trypsin inhibitor purified from Lonchocarpus sericeus seeds.

BACKGROUND: Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. RESULTS: LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). CONCLUSIONS: LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry.

A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production.

Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45-37.90 and 155.84-260.29 µM, respectively. In addition, Mo-CBP2 (18.90 µM) increased the cell membrane permeabilization and reactive oxygen species production in C. albicans and promoted degradation of circular plasmid DNA (pUC18) from Escherichia coli. The data presented in this study highlight the potential use of Mo-CBP2 as an anticandidal agent, based on its ability to inhibit Candida spp. growth with apparently low toxicity on mammalian cells.

Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV).

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. BIOLOGICAL SIGNIFICANCE: This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV.

cDNA cloning, molecular modeling and docking calculations of L-type lectins from Swartzia simplex var. grandiflora (Leguminosae, Papilionoideae), a member of the tribe Swartzieae.

The genus Swartzia is a member of the tribe Swartzieae, whose genera constitute the living descendants of one of the early branches of the papilionoid legumes. Legume lectins comprise one of the main families of structurally and evolutionarily related carbohydrate-binding proteins of plant origin. However, these proteins have been poorly investigated in Swartzia and to date, only the lectin from S. laevicarpa seeds (SLL) has been purified. Moreover, no sequence information is known from lectins of any member of the tribe Swartzieae. In the present study, partial cDNA sequences encoding L-type lectins were obtained from developing seeds of S. simplex var. grandiflora. The amino acid sequences of the S. simplex grandiflora lectins (SSGLs) were only averagely related to the known primary structures of legume lectins, with sequence identities not greater than 50-52%. The SSGL sequences were more related to amino acid sequences of papilionoid lectins from members of the tribes Sophoreae and Dalbergieae and from the Cladratis and Vataireoid clades, which constitute with other taxa, the first branching lineages of the subfamily Papilionoideae. The three-dimensional structures of 2 representative SSGLs (SSGL-A and SSGL-E) were predicted by homology modeling using templates that exhibit the characteristic ß-sandwich fold of the L-type lectins. Molecular docking calculations predicted that SSGL-A is able to interact with D-galactose, N-acetyl-D-galactosamine and α-lactose, whereas SSGL-E is probably a non-functional lectin due to 2 mutations in the carbohydrate-binding site. Using molecular dynamics simulations followed by density functional theory calculations, the binding free energies of the interaction of SSGL-A with GalNAc and α-lactose were estimated as -31.7 and -47.5 kcal/mol, respectively. These findings gave insights about the carbohydrate-binding specificity of SLL, which binds to immobilized lactose but is not retained in a matrix containing D-GalNAc as ligand.

Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.