Development of antimicrobial protein-based polymers for biomedical applications

Inspired in naturally occurring fibrous proteins and composed of amino acid building blocks commonly found in structural proteins, protein-based polymers (PBPs) are a group of materials with unique chemical, physical and biological properties. Coventional recombinant DNA technology allows the biological synthesis of recombinant protein-based polymers (rPBPs) with precise control over its size and composition and the incorporation of functional bioactive domains such as antimicrobial peptides (AMPs). Owing to the unique balance between their mechanical properties, biocompatibility, biodegradability and thermostability, elastin-like recombinamers (ELRs) and silk-elastin like proteins (SELPs) are two of the most remarkable families of rPBP for biotechnological applications. Here, we describe the functionalization of a SELP and an ELR with different antimicrobial peptides that showed promising results against several Gram-positive and Gram-negative bacterial strains. This will provide the basis for the development of advanced biomaterials processed into different types of structures (e.g. hydrogels, films, fibers, particles) suitable for biomedical applications

Protein-engineered polymers functionalized with antimicrobial peptides for the development of active surfaces

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.This work was supported by the “Contrato-Programa” UIDB/04050/2020, project FunBioPlas (ERA-IB-2-6/0004/2014) and project FUN2CYT (POCI-01-0145-FEDER-030568) funded by Portugal national funds through the Fundação para a Ciência e a Tecnologia (FCT I.P.). A.M.P. acknowledges the Doctoral Programme in Applied and Environmental Microbiology (DP_AEM) and FCT I.P. for the PD/BD/113811/2015 grant. R.M. acknowledges FCT I.P. for funding in the scope of the Scientific Employment Stimulus instrument (CEECIND/00526/2018)

Profile of pterostilbene-induced redox homeostasis modulation in cardiac myoblasts and heart tissue

This study was designed to investigate the effect of pterostilbene (PTS) on cardiac oxidative stress in vitro, as this is a simple and promising methodology to study cardiac disease. Cardiac myoblasts (H9c2 cells) and homogenised cardiac tissue were incubated with the PTS and cyclodextrin (PTS ? HPbCD) complex for 1 and 24 h, respectively, at concentrations of 50 lM for the cells and 25 and 50 lM for cardiac tissue. The PTS ? HPbCD complex was used to increase the solubility of PTS in water. After the pretreatment period, cardiomyoblasts were challenged with hydrogen peroxide (6.67 lM) for 10 min, while cardiac tissue was submitted to a hydroxyl radical generator system (30 min). Cellular viability, oxidative stress biomarkers (e.g. total reactive oxygen species (ROS), carbonyl assay and lipoperoxidation) and the antioxidant response (e.g. sulfhydryl and the antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase) were evaluated. In cardiomyoblasts, the PTS ? HPbCD complex (50 lM) increased cellular viability. Moreover, the PTS ? HPbCD complex also significantly increased sulfhydryl levels in the cells submitted to an oxidative challenge. In cardiac tissue, lipid peroxidation, carbonyls and ROS levels were significantly increased in the groups submitted to oxidative damage, while the PTS ? HPbCD complex significantly reduced ROS levels in these groups. In addition, the PTS ? HPbCD complex also provoked increased catalase activity in both experimental protocols. These data suggest that the PTS ? HPbCD complex may play a cardioprotective role through a reduction of ROS levels associated with an improved antioxidant response

Shedding some light over the floral metabolism by Arum Lily (Zantedeschia aethiopica) Spathe de novo transcriptome assembly

Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) – encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information

Structural and Functional Analyses of Cone Snail Toxins

Cone snails are marine gastropod mollusks with one of the most powerful venoms in nature. The toxins, named conotoxins, must act quickly on the cone snails´ prey due to the fact that snails are extremely slow, reducing their hunting capability. Therefore, the characteristics of conotoxins have become the object of investigation, and as a result medicines have been developed or are in the trialing process. Conotoxins interact with transmembrane proteins, showing specificity and potency. They target ion channels and ionotropic receptors with greater regularity, and when interaction occurs, there is immediate physiological decompensation. In this review we aimed to evaluate the structural features of conotoxins and the relationship with their target types

Production and purification of two bioactive antimicrobial peptides using a two-step approach involving an elastin-like fusion tag

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.This work was supported by the “Contrato-Programa” UIDB/04050/2020, project FunBioPlas (ERA-IB-2-6/0004/2014) and project FUN2CYT (POCI-01-0145-FEDER-030568) funded by Portugal national funds through the Fundação para a Ciência e a Tecnologia (FCT I.P.)

Identification of four novel members of Kunitz-like a-amylase inhibitors family from delonix regia with activity toward coleopteran insects

Crop improvement generally focuses on yield, seed quality and nutritional characteristics, rather than resistance to biotic and abiotic stresses. A clear consequence of this approach is the absence of natural anti-feedant toxins in some improved seed materials, allowing predation of commercial crops by insect herbivores. Cowpea (Vigna unguiculata), commonly cultivated by small farmers, is particularly affected by insect-pests that reproduce and develop inside stored seeds. One alternative to conventional pesticides for pest control is the use of biotechnological tools, such as the digestive enzyme inhibitors, that could be introduced in transgenic crops to enhance resistance. In this study, it was verified that the in vivo bioassays using artificial seeds containing 0.5%, 1.0% and 1.5% (w/w) of Delonix regia rich fraction, containing a-amylase inhibitors with effectiveness toward insect a-amylases and other sources, caused remarkable reduction in development and increased mortality of Callosobruchus maculatus cowpea weevil and to cotton boll weevil Anthonomus grandis. Therefore, attempts were made to isolated those inhibitors by SPSepharose ion exchange chromatography followed by high performance liquid chromatography on a Vydac C18-TP analytical column. Four inhibitor peaks were obtained with molecular masses of 6.0, 20 and 24 kDa. Their N-termini showed high sequence similarities with Kunitz-like inhibitor family members. These results provide evidence that D. regia synthesizes a multiple family of Kunitz-like a-amylase inhibitors, with different molecular masses and a wide biotechnological potential to control insect-pests

Magnesium oxide prepared via metal?chitosan complexation method : application as catalyst for transesterification of soybean oil and catalyst deactivation studies.

A simple method to prepare magnesium oxide catalysts for biodiesel production by transesterification reaction of soybean oil with ethanol is proposed. The method was developed using a metal?chitosan complex. Compared to the commercial oxide, the proposed catalysts displayed higher surface area and basicity values, leading to higher yield in terms of fatty acid ethyl esters (biodiesel). The deactivation of the catalyst due to contact with CO2 and H2O present in the ambient air was verified. It was confirmed that the active catalytic site is a hydrogenocarbonate adsorption site

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to a-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian a-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil