Employment of mastoparan-like peptides to prevent Staphylococcus aureus associated with bovine mastitis.

Bovine mastitis is a frequent infection in lactating cattle, causing great economic losses. Staphylococcus aureus represents the main etiological agent, which causes recurrent and persistent intramammary infections because conventional antibiotics are ineffective against it. Mastoparan-like peptides are multifunctional molecules with broad antimicrobial potential, constituting an attractive alternative. Nevertheless, their toxicity to host cells has hindered their therapeutic application. Previously, our group engineered three mastoparan-L analogs, namely mastoparan-MO, mastoparan-R1, and [I5, R8] MP, to improve cell selectivity and potential. Here, we were interested in comparing the antibacterial efficacy of mastoparan-L and its analogs against bovine mastitis isolates of S. aureus strains, making a correlation with the physicochemical properties and structural arrangement changes promoted by the sequence modifications. As a result, the analog's hemolytic and/or antimicrobial activity was balanced. All the peptides displayed α-helical folding in hydrophobic and membrane-mimetic environments, as determined by circular dichroism. The peptide [I5, R8] MP stood out for its enhanced selectivity and antibacterial features related to mastoparan-L and the other derivatives. Biophysical approaches revealed that [I5, R8] MP rapidly depolarizes the bacterial membrane of S. aureus, causing cell death by subsequent membrane disruption. Our results demonstrated that the [I5, R8] MP peptide could be a starting point for the development of peptide-based drugs for the treatment of bovine mastitis, with the advantage of no residue in milk, which would help reduce the use of classical antibiotics.IMPORTANCEStaphylococcus aureus is a leading cause of mastitis, the world's most important dairy cattle disease. The multidrug resistance and zoonotic potential of S. aureus, besides the likelihood of antibiotic residues in milk, are of critical concern to public and animal health. Antimicrobial peptides offer a novel antimicrobial strategy. Here, we demonstrate that [I5, R8] MP is a potent and selective peptide, which acts on S. aureus by targeting the bacterial membrane. Therefore, understanding the physicochemical determinants and the modes of action of this class of antimicrobials opens novel prospects for peptide development with enhanced activities in the bovine mastitis context.

Deciphering the structure and mechanism of action of computer-designed mastoparan peptides.

Mastoparans are cationic peptides with multifunctional pharmacological properties. Mastoparan-R1 and mastoparan-R4 were computationally designed based on native mastoparan-L from wasps and have improved therapeutic potential for the control of bacterial infections. Here, we evaluated whether these peptides maintain their activity against Escherichia coli strains under a range of salt concentrations. We found that mastoparan-R1 and mastoparan-R4 preserved their activity under the conditions tested, including having antibacterial activities at physiological salt concentrations. The overall structure of the peptides was investigated using circular dichroism spectroscopy in a range of solvents. No significant changes in secondary structure were observed (random coil in aqueous solutions and α-helix in hydrophobic and anionic environments). The three-dimensional structures of mastoparan-R1 and mastoparan-R4 were elucidated through nuclear magnetic resonance spectroscopy, revealing amphipathic α-helical segments for Leu3-Ile13 (mastoparan-R1) and Leu3-Ile14 (mastoparan-R4). Possible membrane-association mechanisms for mastoparan-R1 and mastoparan-R4 were investigated through surface plasmon resonance and leakage studies with synthetic POPC and POPC/POPG (4:1) lipid bilayers. Mastoparan-L had the highest affinity for both membrane systems, whereas the two analogs had weaker association, but improved selectivity for lysing anionic membranes. This finding was also supported by molecular dynamics simulations, in which mastoparan-R1 and mastoparan-R4 were found to have greater interactions with bacteria-like membranes compared with model mammalian membranes. Despite having a few differences in their functional and structural profiles, the mastoparan-R1 analog stood out with the highest activity, greater bacteriostatic potential, and selectivity for lysing anionic membranes. This study reinforces the potential of mastoparan-R1 as a drug candidate.

Geometric deep learning as a potential tool for antimicrobial peptide prediction.

Antimicrobial peptides (AMPs) are components of natural immunity against invading pathogens. They are polymers that fold into a variety of three-dimensional structures, enabling their function, with an underlying sequence that is best represented in a non-flat space. The structural data of AMPs exhibits non-Euclidean characteristics, which means that certain properties, e.g., differential manifolds, common system of coordinates, vector space structure, or translation-equivariance, along with basic operations like convolution, in non-Euclidean space are not distinctly established. Geometric deep learning (GDL) refers to a category of machine learning methods that utilize deep neural models to process and analyze data in non-Euclidean settings, such as graphs and manifolds. This emerging field seeks to expand the use of structured models to these domains. This review provides a detailed summary of the latest developments in designing and predicting AMPs utilizing GDL techniques and also discusses both current research gaps and future directions in the field.

Wound healing strategies based on nanoparticles incorporated in hydrogel wound patches.

The intricate, tightly controlled mechanism of wound healing that is a vital physiological mechanism is essential to maintaining the skin's natural barrier function. Numerous studies have focused on wound healing as it is a massive burden on the healthcare system. Wound repair is a complicated process with various cell types and microenvironment conditions. In wound healing studies, novel therapeutic approaches have been proposed to deliver an effective treatment. Nanoparticle-based materials are preferred due to their antibacterial activity, biocompatibility, and increased mechanical strength in wound healing. They can be divided into six main groups: metal NPs, ceramic NPs, polymer NPs, self-assembled NPs, composite NPs, and nanoparticle-loaded hydrogels. Each group shows several advantages and disadvantages, and which material will be used depends on the type, depth, and area of the wound. Better wound care/healing techniques are now possible, thanks to the development of wound healing strategies based on these materials, which mimic the extracellular matrix (ECM) microenvironment of the wound. Bearing this in mind, here we reviewed current studies on which NPs have been used in wound healing and how this strategy has become a key biotechnological procedure to treat skin infections and wounds.

Antimicrobial potential of a ponericin-like peptide isolated from Bombyx mori L. hemolymph in response to Pseudomonas aeruginosa infection.

The main effectors in the innate immune system of Bombyx mori L. are antimicrobial peptides (AMPs). Here, we infected B. mori with varied inoculum sizes of Pseudomonas aeruginosa ATCC 25668 cells to investigate changes in morpho-anatomical responses, physiological processes and AMP production. Ultraviolet-visible spectra revealed a sharp change in λmax from 278 to 285 nm (bathochromic shift) in the hemolymph of infected B. mori incubated for 24 h. Further, Fourier Transform InfraRed studies on the hemolymph extracted from the infected B. mori showed a peak at 1550 cm-1, indicating the presence of α-helical peptides. The peptide fraction was obtained through methanol, acetic acid and water mixture (90:1:9) extraction, followed by peptide purification using Reverse Phase High Performance Liquid Chromatography. The fraction exhibiting antibacterial properties was collected and characterized by Matrix-Assisted Laser Desorption/Ionization-Time of Flight. A linear α-helical peptide with flexible termini (LLKELWTKMKGAGKAVLGKIKGLL) was found, corresponding to a previously described peptide from ant venom and here denominated as Bm-ponericin-L1. The antibacterial activity of Bm-ponericin-L1 was determined against ESKAPE pathogens. Scanning electron microscopy confirmed the membrane disruption potential of Bm-ponericin-L1. Moreover, this peptide also showed promising antibiofilm activity. Finally, cell viability and hemolytic assays revealed that Bm-ponericin-L1 is non-toxic toward primary fibroblasts cell lines and red blood cells, respectively. This study opens up new perspectives toward an alternative approach to overcoming multiple-antibiotic-resistance by means of AMPs through invertebrates' infection with human pathogenic bacteria.

Antimicrobial peptide production in response to gut microbiota imbalance.

The gut microbiota presents essential functions in the immune response. The gut epithelium acts as a protective barrier and, therefore, can produce several antimicrobial peptides (AMPs) that can act against pathogenic microorganisms, including bacteria. Several factors cause a disturbance in gut microbiota, including the exacerbated and erroneous use of antibiotics. Antibiotic therapy has been closely related to bacterial resistance and is also correlated with undesired side-effects to the host, including the eradication of commensal bacteria. Consequently, this results in gut microbiota imbalance and inflammatory bowel diseases (IBD) development. In this context, AMPs in the gut epithelium play a restructuring role for gut microbiota. Some naturally occurring AMPs are selective for pathogenic bacteria, thus preserving the health microbiota. Therefore, AMPs produced by the host's epithelial cells represent effective molecules in treating gut bacterial infections. Bearing this in mind, this review focused on describing the importance of the host's AMPs in gut microbiota modulation and their role as anti-infective agents against pathogenic bacteria.

Noncompetitive tight-binding inhibition of Anticarsia gemmatalis trypsins by Adenanthera pavonina protease inhibitor affects larvae survival.

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant Ki and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å2 . The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.

Immobilization of antimicrobial trypsin inhibitors onto cashew gum polysaccharide/PVA films.

In this work, films produced by blending cashew gum polysaccharide (CGP) with PVA were used as support for immobilization of trypsin inhibitors with antimicrobial activity obtained from Platypodium elegans (PeTI) and Inga laurina (ILTI). The produced films had a homogeneous macroscopic surface with an absence of bubbles and cracks. SEM of CGP/PVA confirmed the porous structure of these materials, being observed a high incidence of pores with a diameter ranging from 0.4 to 7 µm after swelling in a solution with basic pH. CGP/PVA-F2 presented improved mechanical properties when compared with CGP/PVA-F1, showing higher values of tensile strength and elongation. Results from immobilization efficiency proved that both CGP/PVA formulations were able to entrap trypsin inhibitors. However, the inhibitory activity of the immobilized PeTI was two-fold higher than that observed for ILTI, independently of the film formulation. The release profile of PeTI was similar in both formulations, with an increase in the amount of released inhibitor as a function of time. For ILTI, the CGP/PVA-F2 presented higher release than that from CGP/PVA-F1, achieving a maximum release after 720 min. Also, the released inhibitors showed high stability after 24 h of storage. This study confirmed that CGP/PVA films are versatile and efficient materials to be used as support for immobilization of biomolecules.

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity.

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization⁻time-of-flight (MALDI⁻TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI.

Adaptive mechanisms of insect pests against plant protease inhibitors and future prospects related to crop protection: a review.

The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term.

Entomocidal effects of beech apricot, Labramia bojeri, seed extract on a soybean pest, the velvetbean moth, Anticarsia gemmatalis, and its enzymatic activity.

The effects of the beech apricot, Labramia bojeri A. de Candolle (Sapotales: Sapotaceae), seed aqueous extract on the larval development of the velvetbean moth, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was evaluated. The extract inhibited larval development, pupal weight, and survival and emergence of adults. Digestive proteolytic activity in larval midgut and feces extracts was determined. Larvae fed 10 g/L of the aqueous extract showed a significant reduction in trypsin activity (~64%), when compared with control larvae. Trypsin and chymotrypsin activities were also detected in fecal material in aqueous-extract-fed larvae, with about ~4.5 times more trypsin activity than the controls. The results from dietary utilization experiments with A. gemmatalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food and an increase in approximate digestibility and metabolic cost. The effect of the extract suggests the potential use of L. bojeri seeds to inhibit the development of A. gemmatalis via oral exposure. The L. bojeri extract can be an alternative to other methods of control.

Short and long-term antinutritional effect of the trypsin inhibitor ApTI for biological control of sugarcane borer.

Plant-derived trypsin inhibitors have been shown to have potent anti-insect effects and are a promising alternative for the biological control of pests. In this work, we tested the anti-insect activity of Adenanthera pavonina trypsin inhibitor (ApTI) against Diatraea saccharalis larvae, a major insect pest in sugarcane. The addition of 0.1% ApTI in short-term assays resulted in 87% and 63% decreased trypsin and chymotrypsin activities respectively. ApTI was not digested after 60h incubation with D. saccharalis midgut proteases. The chronic effects of ApTI on F0 and F1 generations of D. saccharalis were also analyzed. The larvae from the F0 generation showed 55% and 21% decreased larval and pupal viability, respectively. ApTI-fed larvae from the F1 generation showed a decrease of 33% in survival rate and 23% in the average larval weight. Moreover, ApTI treatment reduced trypsin and chymotrypsin activities in F1 larvae. Thus, the anti-insect effects of ApTI on consecutive generations (F0 and F1) of D. saccharalis larvae demonstrate its potential for long-term control of this pest.

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens.

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.

Inhibition of bacterial adherence to saliva-coated through plant lectins.

In the present study, we evaluated the ability of lectin from Talisia esculenta (TEL) and a protein from Labramia bojeri seeds (Labramin) to inhibit adherence of microorganisms and exert antimicrobial effects. The minimum inhibitory and bactericidal concentrations of these proteins were determined using 5 species of bacteria: Streptococcus mutans UA159, Streptococcus sobrinus 6715, Streptococcus sanguinis ATCC10556, Streptococcus mitis ATCC903 and Streptococcus oralis PB182. In addition, an adherence assay was performed using these 5 bacterial species and sterile polystyrene microtiter plates coated with human saliva. Filtered protein solutions (6.25 to 100 mug/ml) were added to saliva-coated plates, and the plates were then incubated for 1 h at 37 degrees C. After incubation, the plates were washed, and a bacterial suspension (10(6 )CFU/ml) was then transferred to each plate, followed by incubation at 37 degrees C for 1 h (10% CO(2)). Adherence of bacteria to the acquired pellicle was visualized by staining with crystal violet, and absorbance was measured using a plate reader at 575 nm. Neither Labramin nor TEL, at any of the concentrations used, inhibited growth of any of the microorganisms. However, Labramin inhibited adherence of S. mutans and S. sobrinus. The present results indicate that Labramin is potentially useful as a biofilm-inhibiting drug.