Structural insights and antimicrobial synergy of a proto-galectin from the marine sponge Aiolochroia crassa.

In this study, we isolated a novel lectin from the marine sponge Aiolochroia crassa, named AcrL. The lectin showed a preference for glycans containing sialic acid terminal residues, as indicated by the strongest inhibition with fetuin and bovine submaxillary mucin. Primary structure determination by mass spectrometry revealed that AcrL is a galectin with conserved amino acid residues typically involved in carbohydrate binding. Structural modeling indicated that AcrL adopts a typical galectin ß-sandwich motif, featuring two anti-parallel ß-sheets with five strands each. Docking calculations revealed a carbohydrate-binding site composed of a main site, capable of hosting galactopyranosides, and an extended site, facilitating the binding of complex carbohydrates. AcrL inhibited significant biofilm formation against Staphylococcus aureus, S. epidermidis, and Escherichia coli with concentrations ranging from 500 to 15.6 µg.mL-1 for S. aureus, 7.8 µg.mL-1 for S. epidermidis, and 500 µg.mL-1 for E. coli. Furthermore, when combined with different antibiotics, AcrL potentiated their effect against pathogenic bacteria. The antimicrobial mechanism of AcrL was investigated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The analysis indicates that AcrL induces damage to the bacterial membrane. These findings underscore the discovery of a novel galectin in a basal organism and the comprehensive biochemical characterization conducted in this research, highlighting the potential of AcrL as a novel antibacterial agent and emphasizing its importance in combating bacterial infections.

Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Glucose-Binding Dioclea bicolor Lectin (DBL): Purification, Characterization, Structural Analysis, and Antibacterial Properties.

In this study, we purified a lectin isolated from the seeds of Dioclea bicolor (DBL) via affinity purification. Electrophoresis analysis revealed that DBL had three bands, α, ß, and γ chains, with molecular masses of approximately 29, 14, and 12 kDa, respectively. Gel filtration chromatography revealed that the native form of DBL had a molecular mass of approximately 100 kDa, indicating that it is a tetramer. Interestingly, DBL-induced hemagglutination was inhibited by several glucosides, mannosides, ampicillin, and tetracycline with minimum inhibitory concentration (MIC) values of 1.56-50 mM. Analysis of the complete amino acid sequence of DBL revealed the presence of 237 amino acids with high similarity to other Diocleinae lectins. Circular dichroism showed the prominent ß-sheet secondary structure of DBL. Furthermore, DBL structure prediction revealed a Discrete Optimized Protein Energy (DOPE) score of -26,642.69141/Normalized DOPE score of -1.84041. The DBL monomer was found to consist a ß-sandwich based on its 3D structure. Molecular docking showed the interactions between DBL and α-D-glucose, N-acetyl-D-glucosamine, α-D-mannose, α-methyl-D-mannoside, ampicillin, and tetracycline. In addition, DBL showed antimicrobial activity with an MIC of 125 µg/mL and exerted synergistic effects in combination with ampicillin and tetracycline (fractional inhibitory concentration index ≤ 0.5). Additionally, DBL significantly inhibited biofilm formation and showed no toxicity in murine fibroblasts (p < 0.05). These results suggest that DBL exhibits antimicrobial activity and works synergistically with antibiotics.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation.

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM - 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Purification, biochemical characterization of a lectin from marine sponge Ircinia strobilina and its effect on the inhibition of bacterial biofilms.

A new lectin from marine sponge Ircinia strobilina, denominated IsL, was isolated by combination of affinity chromatography in Guar gum matrix followed by size exclusion chromatography. IsL was able to agglutinate native and enzymatically treated rabbit erythrocytes, being inhibited by galactosides, such as α-methyl-D-galactopyranoside, ß-methyl-D-galactopyranoside and α-lactose. IsL hemagglutinating activity was stable at neutral to alkaline pH, however the lectin loses its activity at 40° C. The molecular mass determinated by mass spectrometry was 13.655 ± 5 Da. Approximately 40% of the primary structure of IsL was determined by mass spectrometry, but no similarity was observed with any protein. The secondary structure of IsL consists of 28% α-helix, 26% ß-sheet, and 46% random region, as determined by dichroism circular. IsL was a calcium-dependent lectin, but no significant variations were observed by circular dichroism when IsL was incubated in presence of calcium and EDTA. IsL was not toxic against Artemia nauplii and did not have antimicrobial activity against bacterial cells. However, the IsL was able to significantly inhibit the biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis.

United we stand, divided we fall: in-depth proteomic evaluation of the synergistic effect of Mo-CBP3-PepI and Ciprofloxacin against Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms, a structure that protects bacterial cells, conferring more resistance to difficult treatment. Synthetic peptides surge as an alternative to overcome the biofilm of multidrug-resistant pathogens. Mo-CBP3-PepI, when combined with Ciprofloxacin, reduced preformed S. aureus biofilm by 50% at low concentrations (0.2 and 6.2 µg. mL-1, respectively). The goal of this study was to evaluate the proteomic profile of biofilms after treatment with the Mo-CBP3-PepI combined with ciprofloxacin. Here, proteomic analysis confirmed with more depth previously described mechanisms and revealed changes in the accumulation of proteins related to DNA and protein metabolism, cell wall biosynthesis, redox metabolism, quorum sensing, and biofilm formation. Some proteins related to DNA and protein metabolism were reduced, while other proteins, like redox system proteins, disappeared in Ciprofloxacin+Mo-CBP3-PepI treatment. Our results indicated a synergistic effect of these two molecules with several mechanisms against S. aureus biofilm and opened new doors for combined treatments with other drugs.

Structural characterization of a galectin from the marine sponge Aplysina lactuca (ALL) with synergistic effects when associated with antibiotics against bacteria.

Lectins presents the ability to interact with glycans and trigger varied responses, including the inhibition of the development of various pathogens. Structural studies of these proteins are essential to better understand their functions. In marine sponges, so far only a few lectins have their primary structures completely determined. Thus, the objective of this work was to structurally characterize and evaluate antibacterial potential, in association with different antibiotics, of the lectin isolated from the marine sponge Aplysina lactuta (ALL). ALL is a homotetramer of 60 kDa formed by four 15 kDa-subunits. The lectin showed affinity only for the glycoproteins fetuin, asialofetuin, mucin type III, and bovine submaxillary mucin type I. The complete amino acid sequences of two isoforms of ALL, named ALL-a and ALL-b, were determined by a combination of Edman degradation and overlapped peptides sequenced by tandem mass spectrometry. ALL-a and ALL-b have 144 amino acids with molecular masses of 15,736 Da and 15,985 Da, respectively. Both structures contain conserved residues typical of the galectin family. ALL is a protein with antibacterial potential, when in association with ampicillin and oxacillin the lectin potentiates its antibiotic effect, included Methicillin-resistant Staphylococcus strains. Thus, ALL shows to be a molecule with potential for the development of new antibacterial drugs.

Structural study and antimicrobial and wound healing effects of lectin from Solieria filiformis (Kützing) P.W.Gabrielson.

The SfL-1 isoform from the marine red algae Solieria filiformis was produced in recombinant form (rSfL-1) and showed hemagglutinating activity and inhibition similar to native SfL. The analysis of circular dichroism revealed the predominance of ß-strands structures with spectra of ßI-proteins for both lectins, which had Melting Temperature (Tm) between 41 °C and 53 °C. The three-dimensional structure of the rSfL-1 was determined by X-ray crystallography, revealing that it is composed of two ß-barrel domains formed by five antiparallel ß chains linked by a short peptide between the ß-barrels. SfL and rSfL-1 were able to agglutinate strains of Escherichia coli and Staphylococcus aureus and did not show antibacterial activity. However, SfL induced a reduction in E. coli biomass at concentrations from 250 to 125 µg mL-1, whereas rSfL-1 induced reduction in all concentrations tested. Additionally, rSfL-1 at concentrations from 250 to 62.5 µg mL-1, showed a statistically significant reduction in the number of colony-forming units, which was not noticed for SfL. Wound healing assay showed that the treatments with SfL and rSfL-1 act in reducing the inflammatory response and in the activation and proliferation of fibroblasts by a larger and fast deposition of collagen.

New lectin isolated from the tropical sponge Haliclona (Reniera) implexiformis (Hechtel, 1965) shows antibiofilm effect.

A lectin from the marine sponge Haliclona (Reniera) implexiformis (HiL) was isolated by affinity chromatography on Sepharose™ matrix. HiL showed specificity for galactose and its derivatives. The glycoproteins porcine stomach mucin (PSM) and bovine stomach mucin (BSM) were potent inhibitors. Hemagglutinating activity of the lectin was maximal between pH 5.0 and 9.0. The lectin remained active until 60°C. The presence of CaCl2 and EDTA did not affect the hemagglutinating activity. In SDS-PAGE, HiL showed a single band of 20 kDa under reduced conditions, whereas in the non-reducing conditions, it showed a band of 20 kDa and one additional band of 36 kDa. The average molecular mass determined by Electrospray Ionization Mass Spectrometry (ESI-MS) was 35.874 ± 2 Da in native and non-reducing conditions, whereas carboxyamidomethylated-lectin showed 18,111 Da. These data indicated that HiL consists in a dimer formed by identical subunits linked by disulfide bonds. Partial amino acid sequence of HiL was determined by mass spectrometry, and revealed that it is a new type of lectin, which showed no similarity with any protein. Secondary structure consisted of 6% α-helice, 31% ß-sheet, 18% ß-turn and 45% random coil. HiL showed significant reduction in the number of viable cells of Staphylococcus biofilms.

No Chance to Survive: Mo-CBP3-PepII Synthetic Peptide Acts on Cryptococcus neoformans by Multiple Mechanisms of Action.

Multidrug-resistant Cryptococcus neoformans is an encapsulated yeast causing a high mortality rate in immunocompromised patients. Recently, the synthetic peptide Mo-CBP3-PepII emerged as a potent anticryptococcal molecule with an MIC50 at low concentration. Here, the mechanisms of action of Mo-CBP3-PepII were deeply analyzed to provide new information about how it led C. neoformans cells to death. Light and fluorescence microscopies, analysis of enzymatic activities, and proteomic analysis were employed to understand the effect of Mo-CBP3-PepII on C. neoformans cells. Light and fluorescence microscopies revealed Mo-CBP3-PepII induced the accumulation of anion superoxide and hydrogen peroxide in C. neoformans cells, in addition to a reduction in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) in the cells treated with Mo-CBP3-PepII. In the presence of ascorbic acid (AsA), no reactive oxygen species (ROS) were detected, and Mo-CBP3-PepII lost the inhibitory activity against C. neoformans. However, Mo-CBP3-PepII inhibited the activity of lactate dehydrogenase (LDH) ergosterol biosynthesis and induced the decoupling of cytochrome c (Cyt c) from the mitochondrial membrane. Proteomic analysis revealed a reduction in the abundance of proteins related to energetic metabolism, DNA and RNA metabolism, pathogenicity, protein metabolism, cytoskeleton, and cell wall organization and division. Our findings indicated that Mo-CBP3-PepII might have multiple mechanisms of action against C. neoformans cells, mitigating the development of resistance and thus being a potent molecule to be employed in the production of new drugs against C. neoformans infections.

Trypsin/α-Amylase Inhibitors from Capsicum chinense Seeds: Characterization and Antifungal Activity against Fungi of Agronomic Importance.

BACKGROUND: Protease inhibitors (PIs) have attracted attention due to their important roles in plant defense. OBJECTIVE: The objective of this work was to characterize and evaluate the antimicrobial activity of the peptides of a family of serine PIs from Capsicum chinense Jacq. seeds. METHODS: Initially, PIs were extracted from the seeds and subjected to purification by chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Subsequently, the PEF3 was subjected to trypsin inhibition assays, α-amylase activity assays, antimicrobial activity assays on phytopathogenic fungi, and assays to determine the likely mechanisms of action. RESULTS: The PEF3 was composed of three protein bands with molecular masses ranging between 6 and 14 kDa. The amino acid residues of the ~6 kDa band showed high similarity with serine PIs. PEF3 inhibited the activity of the enzymes trypsin, human salivary α-amylase, and Tenebrio molitor larval α-amylase and inhibited the growth of phytopathogenic fungi, showing 83.7% loss of viability in Fusarium oxysporum. PEF3 induced reactive oxygen species in Colletotrichum lindemuthianum and F. oxysporum to dissipate their mitochondrial membrane potential and activated caspases in C. lindemuthianum. CONCLUSION: Our results reinforce the importance of PIs in plant defense mechanisms against phytopathogenic fungi as well as in their biotechnological applications for the control of plant pathogens.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Chia (Salvia hispanica L.) Seeds Contain a Highly Stable Trypsin Inhibitor with Potential for Bacterial Management Alone or in Drug Combination Therapy with Oxacillin.

The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was purified from chia seeds through crude extract heat treatment, followed by affinity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~ 11 kDa under nonreducing conditions, confirmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min) and a broad pH range (2-10; 30 min). Upon exposure to DTT (0.1 M; 120 min), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disulfide bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 × 10-8 M; IC50 = 1.74 × 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI:trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains. Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.

Anti-Candida Potential of Peptides from Immature and Ripe Fruits of Capsicum chinense Jacq.

The objective of this work was to purify and evaluate the antifungal potential of peptides present in immature and ripe fruits of Capsicum chinense Jacq. (accession UENF 1706) on the medical importance yeasts. Initially the proteins of these seedless fruits were extracted, precipitated with ammonium sulfate at 70% saturation, followed by heating at 80 °C. Subsequently, the peptide-rich extract was fractionated by DEAE-Sepharose anion exchange. The whole process was monitored by tricine-SDS-PAGE. The results revealed that the fraction retained in anion exchange column, called D2, of immature and ripe fruits significantly inhibit the growth of Candida albicans and C. tropicalis yeasts. Due to the higher yield, the D2 fraction of immature fruits was selected for further purification by reverse phase chromatography on HPLC, where sixteen different fractions (H1-H16) were obtained and these were subjected to antifungal assay at 50 µg mL-1. Although almost all fractions tested had significant growth inhibition, the HI9 fraction inhibit 99% of the two yeasts tested. The effect of treatment with HI3, HI8, HI9, and HI14 fractions on the viability of yeast cells was analyzed due to their strong growth inhibition. We observed that only 50 µg mL-1 of the HI9 fraction is the lethal dose for 100% of the cells of C. albicans and C. tropicalis in the original assay. Although the HI9 fraction had a fungicidal effect on both tested yeasts, we only observed membrane permeabilization for C. tropicalis cells treated with 50 µg mL-1 of this fraction. Through mass spectrometry, we identified that the 6 kDa peptide band of HI9 fraction showed similarity with antimicrobial peptides belonging to the plant defensin family.

New Insights into the Mechanism of Antibacterial Action of Synthetic Peptide Mo-CBP3-PepI against Klebsiella pneumoniae.

Klebsiella pneumoniae is a multidrug-resistant opportunistic human pathogen related to various infections. As such, synthetic peptides have emerged as potential alternative molecules. Mo-CBP3-PepI has presented great activity against K. pneumoniae by presenting an MIC50 at a very low concentration (31.25 µg mL-1). Here, fluorescence microscopy and proteomic analysis revealed the alteration in cell membrane permeability, ROS overproduction, and protein profile of K. pneumoniae cells treated with Mo-CBP3-PepI. Mo-CBP3-PepI led to ROS overaccumulation and membrane pore formation in K. pneumoniae cells. Furthermore, the proteomic analysis highlighted changes in essential metabolic pathways. For example, after treatment of K. pneumoniae cells with Mo-CBP3-PepI, a reduction in the abundance of protein related to DNA and protein metabolism, cytoskeleton and cell wall organization, redox metabolism, regulation factors, ribosomal proteins, and resistance to antibiotics was seen. The reduction in proteins involved in vital processes for cell life, such as DNA repair, cell wall turnover, and protein turnover, results in the accumulation of ROS, driving the cell to death. Our findings indicated that Mo-CBP3-PepI might have mechanisms of action against K. pneumoniae cells, mitigating the development of resistance and thus being a potent molecule to be employed in producing new drugs against K. pneumoniae infections.

A fibrinogen-related Lectin from Echinometra lucunter represents a new FReP family in Echinodermata phylum.

Fibrinogen-related proteins (FREPs) have been identified in several animals. They are involved in the body's defense, acting as mediators of phagocytosis. Ficolins and intelectins are some of the most studied Fibrinogen-related Domain (FReD)-containing lectins. In this work, we have isolated a singular FReD-containing lectin, which cannot be classified as ficolin or intelectin. ELL (Echinometra lucunter lectin) was isolated from coelomic plasma by affinity chromatography on xanthan gum. Primary structure was determined by tandem mass spectrometry. Moreover, antimicrobial activity of ELL was evaluated against planktonic cells and biofilm of Escherichia coli, Staphylococcus aureus and S. epidermidis. ELL showed hemagglutinating activity in Ca2+ presence, which was inhibited by glycoprotein mucin and thyroglobulin. Complete amino acid sequence consisted of 229 residues, including a FReD in the N-terminal. Searches for similarity found that ELL was very close to putative proteins from Strongylocentrotus purpuratus. ELL showed moderate similarity with uncharacterized sea stars proteins and protochordate intelectins. ELL was able to inhibit the planktonic growth of the Gram-positive bacteria and significantly reduce the biofilm formation of all bacteria tested. In conclusion, we identified a new type of FReP-containing lectin with some structural and functional conservation towards intelectins.

Luffa operculata seed proteins: Identification by LC-ESI-MS/MS and biotechnological potential against Candida albicans and C. krusei.

L: operculata is a plant commonly found in the North and Northeast of Brazil. Although the regional population knows its medicinal potential, there are few scientific studies about its antimicrobial potential. Thus, this study aimed to characterize the proteins from L. operculata seeds extracted using different solutions and evaluate their antimicrobial potentials. The protein extracts obtained with NaCl and sodium acetate buffer presented the best inhibitory activities against Candida albicans and C. krusei. The study of the mechanism of action revealed proteins from L. operculata seeds induced pore formation on the membrane and ROS overaccumulation. Scanning Electron Microscopy images also showed severe morphological changes in Candida albicans and C. krusei. Proteins from L.operculata seeds did not show antibacterial activity. The enzymatic assays revealed the presence of proteolytic enzymes, serine and cysteine protease inhibitors, and chitinases in both protein extracts. Proteomic analysis by LC-ESI-MS/MS identified 57 proteins related to many biological processes, such as defense to (a)biotic stress, energetic metabolism, protein folding, and nucleotide metabolism. In conclusion, the L. operculata seed proteins have biotechnological potential against the human pathogenic yeasts Candida albicans and C. krusei.

JcTI-PepI, a synthetic peptide bioinspired in the trypsin inhibitor from Jatropha curcas, presents potent inhibitory activity against C. krusei, a neglected pathogen.

Antimicrobial resistance has been increasing globally, posing a global public health risk. It has prompted the scientific community to look for alternatives to traditional drugs. Antimicrobial Peptides (AMPs) have stood out in this context because they have the potential to control infectious diseases while causing no or little harm to mammalian cells. In the present study, three peptides, JcTI-PepI, JcTI-PepII, and JcTI-PepIII, were designed and tested for antimicrobial activity based on the primary sequence of JcTI-I, a 2S albumin with trypsin inhibitory activity from Jatropha curcas. JcTI-PepI strongly inhibited C. krusei growth, and it caused severe disruptions in cellular processes and cell morphology. C. krusei cells treated with JcTI-PepI showed indicative of membrane permeabilization and overproduction of Reactive Oxygen Species. Moreover, the yeast's ability to acidify the medium was severely compromised. JcTI-PepI was also effective against pre-formed biofilm and did not harm human erythrocytes and Vero cells. Overall, these characteristics indicate that JcTI-PepI is both safe and effective against C. krusei, an intrinsically resistant strain that causes serious health problems and is frequently overlooked. It implies that this peptide has a high potential for use as a new antimicrobial agent in the future.

Codium isthmocladum lectin 1 (CiL-1): Interaction with N-glycans explains antinociceptive and anti-inflammatory activities in adult zebrafish (Danio rerio).

Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.