Cutaneous glands of the striped toad, Rhinella crucifer (Wied-Neuwied, 1821) (Amphibia: Bufonidae): Histological study and bioactivities of glandular secretions.

This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting.

Pomegranate sarcotesta lectin (PgTeL) inhibits planktonic growth and disrupts biofilm formed by Cryptococcus neoformans.

AIMS: We investigated the putative fungistatic and fungicidal activities of pomegranate sarcotesta lectin (PgTeL) against Cryptococcus neoformans B3501 (serotype D), specifically the ability of PgTeL to inhibit yeast capsule and biofilm formation in this strain. METHODS AND RESULTS: PgTeL showed a minimum inhibitory concentration of 172.0 µg ml-1, at which it did not exhibit a fungicidal effect. PgTeL concentrations of 4.0-256.0 µg ml-1 reduced biofilm biomass by 31.0%-64.0%. Furthermore, 32.0-256.0 µg ml-1 PgTeL decreased the metabolic activity of the biofilm by 32.0%-93.0%. Scanning electron microscopy images clearly revealed disruption of the biofilm matrix. Moreover, PgTeL disrupted preformed biofilms. At concentrations of 8.0-256.0 µg ml-1, PgTeL reduced metabolic activity in C. neoformans by 36.0%-92.0%. However, PgTeL did not inhibit the ability of B3501 cells to form capsules under stress conditions. CONCLUSIONS: PgTeL inhibited biofilm formation and disrupted preformed biofilms, demonstrating its potential for use as an anticryptococcal agent.

Purification, Characterization, and Assessment of Antimicrobial Activity and Toxicity of Portulaca elatior Leaf Lectin (PeLL).

Lectins are carbohydrate-binding proteins with several bioactivities, including antimicrobial properties. Portulaca elatior is a species found at Brazilian Caatinga and data on the biochemical composition of this plant are scarce. The present work describes the purification of P. elatior leaf lectin (PeLL) as well as the assessment of its antimicrobial activity and toxicity. PeLL, isolated by chromatography on a chitin column, had native liquid charge and subunit composition evaluated by electrophoresis. Hemagglutinating activity (HA) of PeLL was determined in the presence of carbohydrates or divalent cations, as well as after heating and incubation at different pH values. Changes in the lectin conformation were monitored by evaluating intrinsic tryptophan fluorescence and using the extrinsic probe bis-ANS. Antimicrobial activity was evaluated against Pectobacterium strains and Candida species. The minimal inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations were determined. Finally, PeLL was evaluated for in vitro hemolytic activity in human erythrocytes and in vivo acute toxicity in mice (5 and 10 mg/kg b.w. per os). PeLL (pI 5.4; 20 kDa) had its HA was inhibited by mannose, galactose, Ca2+, Mg2+, and Mn2+. PeLL HA was resistant to heating at 100 °C, although conformational changes were detected. PeLL was more active in the acidic pH range, in which no conformational changes were observed. The lectin presented MIC and MBC of 0.185 and 0.74 µg/mL for all Pectobacterium strains, respectively; MIC of 1.48 µg/mL for C. albicans, C. tropicalis, and C. krusei; MIC and MFC of 0.74 and 2.96 µg/mL for C. parapsilosis. No hemolytic activity or signs of acute toxicity were observed in the mice. In conclusion, a new, low-toxic, and thermostable lectin was isolated from P. elatior leaves, being the first plant compound to show antibacterial activity against Pectobacterium.

Quantum dots conjugated to lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL) as potential fluorescent nanotools for investigating Cryptococcus neoformans.

This study reports the development of conjugates based on quantum dots (QD)s and lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL). Cryptococcus neoformans cells were chosen to evaluate the efficiency of the conjugates. Lectins were conjugated to QDs via adsorption, and the optical parameters (emission and absorption) were monitored. Lectin stability in the conjugates towards denaturing agents was investigated via fluorometry. The conjugation was evaluated using fluorescence microplate (FMA) and hemagglutination (HA) assays. The labeling of the C. neoformans cell surface was quantified using flow cytometry and observed via fluorescence microscopy. The QDs-SteLL and QDs-PgTeL conjugates, obtained at pH 7.0 and 8.0, respectively, showed the maintenance of colloidal and optical properties. FMA confirmed the conjugation, and the HA assay indicated that the lectin carbohydrate-binding ability was preserved after conjugation. SteLL and PgTeL showed stability towards high urea concentrations and heating. Conjugates labeled over 90% of C. neoformans cells as observed via flow cytometry and confirmed through fluorescence microscopy. C. neoformans labeling by conjugates was inhibited by glycoproteins, suggesting specific interactions through the lectin carbohydrate-binding site. Thus, an effective protocol for the conjugation of SteLL or PgTeL with QDs was proposed, yielding new nanoprobes useful for glycobiological studies.

Degradation of the residual textile mixture cetyltrimethylammonium bromide/remazol yellow gold RNL-150%/reactive blue BF-5G: evaluation photo-peroxidation and photo-Fenton processes in LED and UV-C photoreactors.

This article presents a study on the degradation of a residual textile mixture composed of cationic surfactant cetyltrimethylammonium bromide (CTAB) and the remazol yellow gold RNL-150% and reactive blue BF-5G textile dyes. This was carried out by employing the photo-peroxidation and photo-Fenton processes in LED and UV-C photoreactors. The photo-Fenton process was the most efficient as regards the degradation of the CTAB and dye mixture, for both types of radiation. In the kinetic study, degradations of 99% were obtained in 180 min for the chromophore groups using both types of radiation. The degradation of the CTAB and aromatic groups was, meanwhile, an average of 25% when employing LED radiation. The behavior of the degradation reaction was pseudo-first-order. Toxicity tests indicated that the solutions were better able to grow seeds and bacteria after treatment with the photo-Fenton process, using both types of radiation. The photo-Fenton processes carried out by employing LED and UV-C photoreactors were able to degrade the CTAB and dye mixture, thus highlighting the efficiency of LED radiation when its power (three times smaller) is compared to that of UV-C radiation. This process, therefore, represents an alternative for use in textile wastewater treatment systems.

Punica granatum sarcotesta lectin (PgTeL) has antibacterial activity and synergistic effects with antibiotics against ß-lactamase-producing Escherichia coli.

The sarcotesta of Punica granatum fruit contains an antimicrobial lectin called PgTeL. In this work, we evaluated the antibacterial activity of PgTeL against five drug-resistant Escherichia coli isolates able to produce ß-lactamases. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined by broth dilution. Morphometric and viability analyses were performed by flow cytometry, and ultrastructural changes were evaluated by scanning electron microscopy. Potential synergistic effects of PgTeL with antibiotics and anti-biofilm effect were also evaluated. PgTeL showed antibacterial activity against all isolates with MIC and MBC values ranging from 12.5 to 50.0 µg/mL and from 25.0 to 100.0 µg/mL, respectively. For most isolates, PgTeL postponed the growth start by at least ten hours. At the MIC, the lectin caused alterations in size, shape and structure of bacterial cells. The combination PgTeL-ceftazidime showed a synergistic effect for all isolates. Synergy was also detected with ampicillin (one isolate), carbenicillin (one isolate), cefotaxime (one isolate), cephalexin (four isolates) and cefuroxime (three isolates). PgTeL exhibited anti-biofilm activity against all isolates, causing ≥50% inhibition of biofilms at or above 6.25 µg/mL. The antibacterial effect of PgTeL and its synergy with antibiotics indicate that this fruit-derived molecule may have potential for future treatment of multidrug-resistant infections.

Punica granatum sarcotesta lectin (PgTeL) impairs growth, structure, viability, aggregation, and biofilm formation ability of Staphylococcus aureus clinical isolates.

In this work, we evaluated the ability of Punica granatum sarcotesta lectin (PgTeL) to impair the growth and viability of the Staphylococcus aureus clinical isolates 8325-4 (non-resistant) and LAC USA300 (MRSA strain). The effects of this lectin on aggregating, hemolytic activity, biofilm-forming ability, and expression of virulence genes (hla, rnaIII, and spa) were also investigated. PgTeL showed antibacterial activity against 8325-4 and LAC USA300 strains by interfering with both the growth (MIC50 of 6.25 and 12.5 µg/mL, respectively) and survival (MBC values of 25.0 and 50.0 µg/mL, respectively). Culture growth started only at the ninth (8325-4) and tenth (LAC USA300) hour in the presence of PgTeL at MIC50, while growth was detected since the first hour in the control. The lectin caused markedly altered cell morphology in both the strains. Although, at the MIC50, PgTeL caused structural alterations, most cells were still viable, while at the MBC it promoted cell injury and death. PgTeL showed anti-aggregation effect and exhibited antibiofilm activity against both the isolates. However, the lectin did not interfere with the hemolytic activity of LAC USA300 and with the expression of hla, rnaIII, and spa genes. In conclusion, PgTeL is a lectin with multiple inhibitory effects on S. aureus clinical isolates.

Portulaca elatior root contains a trehalose-binding lectin with antibacterial and antifungal activities.

Lectins are carbohydrate-binding proteins broadly distributed in plants and have several biological functions, including antimicrobial action. Portulaca elatior is a Caatinga plant whose chemical composition and biotechnological potential have not been extensively studied. In this work, a lectin was isolated from P. elatior root extract and evaluated for antimicrobial activity. The P. elatior root lectin (PeRoL) showed native molecular mass of 33 kDa, pI 3.8 and is comprised of two subunits of 15 kDa linked by disulfide bonds. No sequence similarities with Viridiplantae proteins were observed. The PeRoL hemagglutinating activity (HA) was not affected by heating and was detected in a pH ranging from 4.0 to 8.0. Trehalose was identified as an endogenous inhibitor of PeRoL present in the roots. Bacteriostatic activity was detected against Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus (minimal inhibitory concentration of 8.1, 32.5 and 4.06 µg/mL, respectively). PeRoL induced the death of Candida albicans, Candida parapsilosis, Candida krusei, and Candida tropicalis cells, with a minimal fungicidal concentration of 16 µg/mL. The lectin (100 µg/mL) was not cytotoxic to human peripheral blood mononuclear cells (PBMCs) and did not show hemolytic activity. In conclusion, the roots of P. elatior contain a trehalose-binding, thermostable, and antimicrobial lectin.

PgTeL, the lectin found in Punica granatum juice, is an antifungal agent against Candida albicans and Candida krusei.

The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100µg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25µg/mL; MFC: 50µg/mL) and C. krusei (MIC and MFC of 12.5µg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and »MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39µg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells.

Purification and characterization of a protease from the visceral mass of Mytella charruana and its evaluation to obtain antimicrobial peptides.

This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1 kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40 °C and stability in a wide pH range (3.0-9.0). Km was 4.28 ±â€¯0.34 mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056 ±â€¯0.001 nmol min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0 µg mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.

CasuL: A new lectin isolated from Calliandra surinamensis leaf pinnulae with cytotoxicity to cancer cells, antimicrobial activity and antibiofilm effect.

This work describes the isolation of a lectin (CasuL) from the leaf pinnulae of Calliandra surinamensis and the evaluation of its cytotoxic, antimicrobial and antibiofilm properties. Proteins from pinnulae extract were precipitated with ammonium sulphate (60% saturation) and submitted to Sephadex G-75 chromatography, which yielded isolated CasuL (purification factor: 113). Native CasuL is an acidic protein (pI 5.82) with a relative molecular mass of 48kDa. This lectin is also an oligomeric protein composed of three subunits and mass spectrometry revealed similarities with a Sorghum bicolor protein. CasuL did not undergo unfolding when heated but changes in conformation and hemagglutinating activity were detected at basic pH. CasuL did not reduce the viability of human peripheral blood mononuclear cells but was toxic to leukemic K562 cells (IC50 67.04±5.78µg/mL) and breast cancer T47D cells (IC50: 58.75±2.5µg/mL). CasuL (6.25-800µg/mL) only showed bacteriostatic effect but was able to reduce biofilm formation by Staphylococcus saprophyticcus and Staphylococcus aureus (non-resistant and oxacillin-resistant isolates). CasuL showed antifungal activity against Candida krusei causing alterations in cell morphology and damage to cell wall. In conclusion, the pinnulae of C. surinamensis leaves contain a thermo-stable lectin with biotechnological potential as cytotoxic, antibiofilm, and antifungal agent.

Evaluation of antimicrobial, cytotoxic, and hemolytic activities from venom of the spider Lasiodora sp.

This study characterized the protein/peptide profile of venom isolated from the spider Lasiodora sp. (Mygalomorphae, Theraphosidae) found in northeastern Brazil and determined its antimicrobial activity, toxicity against human cells, and hemolytic activity. Protein concentration of the Lasiodora sp. venom was 4.53 ± 0.38 mg/mL. SDS-PAGE showed proteins with molecular masses up to 75 kDa, some of which contained disulfide bridges. RP-HPLC analysis separate at least 12 peaks that were identified by mass spectrometry as peptides U1-theraphotoxin-Lp1a (lasiotoxin-1), U1-theraphotoxin-Lp1c (lasiotoxin-3), U3-theraphotoxin-Lsp1a (LTx5), and ω-theraphotoxin-Asp3a as well as the proteins phospholipase A2 (PLA2) and hyaluronidase. The crude venom exhibited bactericidal effect against Aeromonas sp., Bacillus subtilis, and Micrococcus luteus and fungicidal effect against Candida parapsilosis and Candida albicans. In addition, the venom exerted bacteriostatic effect against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus and fungistatic effect against Candida tropicalis and Candida krusei. The minimum inhibitory (MIC), minimum bactericidal (MBC), and minimum fungicidal (MFC) concentrations ranged from 3.9 to 500 µg/mL. The Lasiodora sp. venom decreased the viability of human peripheral blood mononuclear cells (PBMCs) by 50%-90% at concentrations of 0.1, 1, 10, and 100 µg/mL, promoting apoptosis of these cells. On the other hand, the venom showed weak hemolytic activity against Mus musculus erythrocytes (EC50: 757 µg/mL). In conclusion, the Lasiodora sp. spider venom is a rich source of antimicrobial agents. Future studies will focus on identifying antimicrobial agents present in this venom and evaluating whether these agents contribute to its cytotoxic effects against PBMCs.