HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.

The search for less harmful, ecologically efficient, more specific, and natural alternatives for the control of pathogens is essential. Bauhinia variegata lectin (BvL) is a protein that has numerous biological activities, including antifungal. The present study examines the potential in vitro of B. variegata lectin against the fungus Bipolaris oryzae, responsible for agricultural losses in southern Brazil, due to damage to rice fields during seed germination. Bioassays to assess the inhibition potential of BvL were performed, including fungal growth, spore formation, and germination, in concentrations of 0, 25, 50, and 100 µg mL-1. Only the concentration of 100 µg mL-1 successfully inhibited mycelial growth and spore germination, while in spore formation, all treatments inhibited sporulation. In addition, fluorescence microscopy analysis demonstrated the ability of lectin to bind to the fungus and the lack of detection in the presence of lactose, suggesting its interaction with the fungal cell wall structures. This study highlights the potential of B. variegata seed lectin to control mycelial growth, sporulation, and germination of the phytopathogenic fungus B. oryzae, posing as a new biotechnological possibility for biological control.

Glycan-binding specificity was studied for Jacalin, RCA 120, SBA, PHA-L, PHA-E, WGA, UEA, AAL, LTL, LEL, SNA, DSA, LCA, MAH and Con A, lectins widely used in histochemistry. Oligosaccharide- and polysaccharide-based glycan arrays were applied. Expected specificity was confirmed for only 6 of the 15 lectins and the glycan binding profiles of some lectins were dramatically broader than generally accepted. WGA, LEL and DSA known as chitooligosaccharide-specific, were unexpectedly polyreactive, binding to other glycans with the same affinity as to chitobiose, ABH antigens and oligolactosamines (unsubstituted and sialylated). SBA, in addition to expected binding to glycans with terminal GalNAcα, also had high affinity for the GM1 ganglioside. MAH demonstrated much higher affinity to a variety of sulfated glycans compared to Neu5Acα2-3Galβ1-3GalNAcα. Contrary to the common view, LCA demonstrated the maximum binding to (GlcNAcβ1-2Manα1)2-3,6-Manβ1-4GlcNAcβ1-4GlcNAc N-glycan, while it had no interaction with corresponding Gal or Neu5Ac terminated versions. This observed polyreactivity of some lectins casts doubt on their use in accurately determining the presence of a specific glycan structure by histochemical studies. However, comparisons of sera from healthy and diseased individuals with help of a lectin array can easily establish differences in glycosylation patterns and presumptive glycan identities, which can later be clarified using more accurate methods of structural analysis.

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.

Legume lectins are a diverse family of carbohydrate-binding proteins that share significant similarities in their primary, secondary, and tertiary structures, yet exhibit remarkable variability in their quaternary structures and carbohydrate-binding specificities. The tertiary structure of legume lectins, characterized by a conserved β-sandwich fold, provides the scaffold for the formation of a carbohydrate-recognition domain (CRD) responsible for ligand binding. The structural basis for the binding is similar between members of the family, with key residues interacting with the sugar through hydrogen bonds, hydrophobic interactions, and van der Waals forces. Variability in substructures and residues within the CRD are responsible for the large array of specificities and enable legume lectins to recognize diverse sugar structures, while maintaining a consistent structural fold. Therefore, legume lectins can be classified into several specificity groups based on their preferred ligands, including mannose/glucose-specific, N-acetyl-d-galactosamine/galactose-specific, N-acetyl-d-glucosamine-specific, l-fucose-specific, and α-2,3 sialic acid-specific lectins. In this context, this review examined the structural aspects and carbohydrate-binding properties of representative legume lectins and their specific ligands in detail. Understanding the structure/binding relationships of lectins continues to provide valuable insights into their biological roles, while also assisting in the potential applications of these proteins in glycobiology, diagnostics, and therapeutics.

Korean mistletoe (Viscum album L. var. coloratum) is renowned for its medicinal properties, including anti-cancer and immunoadjuvant effects. This study aimed to elucidate the mechanisms by which Korean mistletoe lectin (V. album L. var. coloratum agglutinin; VCA) modulates breast cancer cell apoptosis and macrophage polarization. The specific objectives were to (1) investigate the direct effects of VCA on MCF-7 breast cancer cells and THP-1-derived M1/M2 macrophages; (2) analyze the impact of VCA on the paracrine interactions between these cell types; and (3) compare the efficacy of VCA in 2D vs. 3D co-culture models to bridge the gap between in vitro and in vivo studies. We employed both 2D and 3D models, co-culturing human M1/M2 macrophages with human MCF-7 breast cancer cells in a Transwell system. Our research demonstrated that M1 and M2 macrophages significantly influenced the immune and apoptotic responses of breast cancer cells when exposed to VCA. M1 macrophages exhibited cytotoxic characteristics and enhanced VCA-induced apoptosis in both 2D and 3D co-culture models. Conversely, M2 macrophages initially displayed a protective effect by reducing apoptosis in breast cancer cells, but this protective effect was reversed upon exposure to VCA. Furthermore, our findings illustrate VCA\'s ability to modulate M1 and M2 polarization in breast cancer cells. Finally, the use of magnetic 3D cell cultures suggests their potential to yield results comparable to conventional 2D cultures, bridging the gap between in vitro and in vivo studies.

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50% by VEA purified from accessions #5 and #36. VEA antibiofilm variability between accessions was observed, likely due to co-purified small molecules rather than differences in VEA protein sequences. In conclusion, VEA seed extracts from the accessions with the highest antibiofilm activity could represent a valid approach for the development of an effective antibiofilm agent.

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja\'s binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.

Leishmaniases, caused by Leishmania parasites, are widespread and pose significant health risks globally. Visceral leishmaniasis (VL) is particularly prevalent in Brazil, with high morbidity and mortality rates. Traditional treatments, such as pentavalent antimonials, have limitations due to toxicity and resistance. Therefore, exploring new compounds like lectins is crucial. Concanavalin A (ConA) has shown promise in inhibiting Leishmania growth. This study aimed to evaluate its leishmanicidal effect on L. infantum promastigotes and understand its mechanism of action. In vitro tests demonstrated inhibition of promastigote growth when treated with ConA, with IC50 values ranging from 3 to 5 μM over 24-72 h. This study suggests that ConA interacts with L. infantum glycans. Additionally, ConA caused damage to the membrane integrity of parasites and induced ROS production, contributing to parasite death. Scanning electron microscopy confirmed morphological alterations in treated promastigotes. ConA combined with the amphotericin B (AmB) showed synergistic effects, reducing the required dose of AmB, and potentially mitigating its toxicity. ConA demonstrated no cytotoxic effects on macrophages, instead stimulating their proliferation. These findings reinforce that lectin exhibits promising leishmanicidal activity against L. infantum promastigotes, making ConA a potential candidate for leishmaniasis treatment.

Bacterial cellulose (BC), produced by bacterial fermentation, is a high-purity material. BC can be oxidized (BCOXI), providing aldehyde groups for covalent bonds with drugs. Frutalin (FTL) is a lectin capable of modulating cell proliferation and remodeling, which accelerates wound healing. This study aimed to develop an FTL-incorporated dressing based on BC, and to evaluate its physicochemical properties and biological activity in vitro. An experimental design was employed to maximize FTL loading yield onto the BC and BCOXI, where independent variables were FTL concentration, temperature and immobilization time. BCOXI-FTL 1 (44.96 % ± 1.34) had the highest incorporation yield (IY) at the experimental conditions: 6 h, 5 °C, 20 μg mL-1. The second highest yield was BCOXI-FTL 6 (23.28 % ± 1.43) using 24 h, 5 °C, 100 μg mL-1. Similarly, the same reaction parameters provided higher immobilization yields for native bacterial cellulose: BC-FTL 6 (16.91 % ± 1.05) and BC-FTL 1 (21.71 % ± 1.57). Purified FTL displayed no cytotoxicity to fibroblast cells (<50 μg mL-1 concentration) during 24 h. Furthermore, BCOXI-FTL and BC-FTL were non-cytotoxic during 24 h and stimulated fibroblast migration. BCOXI-FTL demonstrated neutrophil activation in vitro similar to FTL. These promising results indicate that the bacterial cellulose matrices containing FTL at low concentrations, could be used as an innovative biomaterial for developing wound dressings.