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.

Antimicrobial and Antibiofilm Activities of Helical Antimicrobial Peptide Sequences Incorporating Metal-Binding Motifs.

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. CM15 ATCUN variants, when combined with Meropenem, streptomycin, or chloramphenicol, showed synergistic effects against E. coli (KpC+ 1812446) biofilms. Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the possible use of size, charge, and hydrophobicity to fine-tune AMP biological function. Our data indicate that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties. These principles may be applied to augment the activities of other peptide sequences.

A short peptide with selective anti-biofilm activity against Pseudomonas aeruginosa and Klebsiella pneumoniae carbapenemase-producing bacteria.

Biofilm-related infections represent an enormous clinical challenge nowadays. In this context, diverse studies are underway to develop effective antimicrobial agents targeting bacterial biofilms. Here, we describe the antibacterial and anti-biofilm activities of a short, cationic peptide named R5F5, obtained from sliding-window analysis based on a peptide (PcDBS1R5) derived from Plasmodium chabaudi. Ten fragments were generated (R5F1 to F10) and submitted to initial antibacterial assays against Pseudomonas aeruginosa. As a result, R5F5 showed the highest antimicrobial activity. We therefore carried out further antibacterial and anti-biofilm assays against P. aeruginosa and Klebsiella pneumoniae carbapenemase-producing bacterial strains. R5F5 revealed selective anti-biofilm activity, as the peptide inhibited >60% biofilm formation in all cases from 8 to 64 µg·mL-1. Moreover, R5F5 was not hemolytic against mice erythrocytes at 640 µg mL-1. Cytotoxic effects on human lung fibroblast cells were not detected at 160 µg·mL-1. Structural studies revealed that R5F5 presents random coil conformations in water and 50% 2,2,2-trifluoroethanol (TFE)/water (v/v), whereas amphipathic, extended conformations were observed in contact with sodium dodecyl sulfate (SDS) micelles. Thus, here we report a novel peptide with selective anti-biofilm activity against susceptible and resistant bacterial strains, with no toxicity toward mammalian cells and that adopts a stable structure in anionic environment.

Non-Lytic Antibacterial Peptides That Translocate Through Bacterial Membranes to Act on Intracellular Targets.

The advent of multidrug resistance among pathogenic bacteria has attracted great attention worldwide. As a response to this growing challenge, diverse studies have focused on the development of novel anti-infective therapies, including antimicrobial peptides (AMPs). The biological properties of this class of antimicrobials have been thoroughly investigated, and membranolytic activities are the most reported mechanisms by which AMPs kill bacteria. Nevertheless, an increasing number of works have pointed to a different direction, in which AMPs are seen to be capable of displaying non-lytic modes of action by internalizing bacterial cells. In this context, this review focused on the description of the in vitro and in vivo antibacterial and antibiofilm activities of non-lytic AMPs, including indolicidin, buforin II PR-39, bactenecins, apidaecin, and drosocin, also shedding light on how AMPs interact with and further translocate through bacterial membranes to act on intracellular targets, including DNA, RNA, cell wall and protein synthesis.

Effect of Acrocomia aculeata Kernel Oil on Adiposity in Type 2 Diabetic Rats.

The macauba palm (Acrocomia aculeata) is native of tropical America and is found mostly in the Cerrados and Pantanal biomes. The fruits provide an oily pulp, rich in long chain fatty acids, and a kernel that encompass more than 50% of lipids rich in medium chain fatty acids (MCFA). Based on biochemical and nutritional evidences MCFA is readily catabolized and can reduce body fat accumulation. In this study, an animal model was employed to evaluate the effect of Acrocomia aculeata kernel oil (AKO) on the blood glucose level and the fatty acid deposit in the epididymal adipose tissue. The A. aculeata kernel oil obtained by cold pressing presented suitable quality as edible oil. Its fatty acid profile indicates high concentration of MCFA, mainly lauric, capric and caprilic. Type 2 diabetic rats fed with that kernel oil showed reduction of blood glucose level in comparison with the diabetic control group. Acrocomia aculeata kernel oil showed hypoglycemic effect. A small fraction of total dietary medium chain fatty acid was accumulated in the epididymal adipose tissue of rats fed with AKO at both low and high doses and caprilic acid did not deposit at all.

Antimicrobial peptides could antagonize uncontrolled inflammation via Toll-like 4 receptor.

Antimicrobial peptides are part of the organism's defense system. They are multifunctional molecules capable of modulating the host's immune system and recognizing molecules present in pathogens such as lipopolysaccharides (LPSs). LPSs are recognized by molecular patterns associated with pathogens known as Toll-like receptors (TLRs) that protect the organism from pathological microorganisms. TLR4 is responsible for LPS recognition, thus inducing an innate immune response. TLR4 hyperstimulation induces the uncontrolled inflammatory process that is observed in many illnesses, including neurodegenerative, autoimmune and psoriasis). Molecules that act on TLR4 can antagonize the exacerbated inflammatory process. In this context, antimicrobial peptides (AMPs) are promising molecules capable of mediating toll-like receptor signaling. Therefore, here we address the AMPs studied so far with the aim of inhibiting the intense inflammatory process. In addition, we aim to explore some of the interactions between exogenous AMPs and TLR4.

An N-capping asparagine-lysine-proline (NKP) motif contributes to a hybrid flexible/stable multifunctional peptide scaffold.

Structural diversity drives multiple biological activities and mechanisms of action in linear peptides. Here we describe an unusual N-capping asparagine-lysine-proline (NKP) motif that confers a hybrid multifunctional scaffold to a computationally designed peptide (PaDBS1R7). PaDBS1R7 has a shorter α-helix segment than other computationally designed peptides of similar sequence but with key residue substitutions. Although this motif acts as an α-helix breaker in PaDBS1R7, the Asn5 presents exclusive N-capping effects, forming a belt to establish hydrogen bonds for an amphipathic α-helix stabilization. The combination of these different structural profiles was described as a coil/N-cap/α-helix scaffold, which was also observed in diverse computational peptide mutants. Biological studies revealed that all peptides displayed antibacterial activities. However, only PaDBS1R7 displayed anticancer properties, eradicated Pseudomonas aeruginosa biofilms, decreased bacterial counts by 100-1000-fold in vivo, reduced lipopolysaccharide-induced macrophages stress, and stimulated fibroblast migration for wound healing. This study extends our understanding of an N-capping NKP motif to engineering hybrid multifunctional peptide drug candidates with potent anti-infective and immunomodulatory properties.

Antimicrobial Peptides and Cell-Penetrating Peptides for Treating Intracellular Bacterial Infections.

Bacterial infections caused by intracellular pathogens are difficult to control. Conventional antibiotic therapies are often ineffective, as high doses are needed to increase the number of antibiotics that will cross the host cell membrane to act on the intracellular bacterium. Moreover, higher doses of antibiotics may lead to elevated severe toxic effects against host cells. In this context, antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) have shown great potential to treat such infections by acting directly on the intracellular pathogenic bacterium or performing the delivery of cargos with antibacterial activities. Therefore, in this mini-review, we cover the main AMPs and CPPs described to date, aiming at intracellular bacterial infection treatment. Moreover, we discuss some of the proposed mechanisms of action for these peptide classes and their conjugation with other antimicrobials.

Antimicrobial peptides from Bombyx mori: a splendid immune defense response in silkworms.

Bombyx mori L., a primary producer of silk, is the main tool in the sericulture industry and provides the means of livelihood to a large number of people. Silk cocoon crop losses due to bacterial infection pose a major threat to the sericulture industry. Bombyx mori L., a silkworm of the mulberry type, has a sophisticated inherent innate immune mechanism to combat such invasive pathogens. Among all the components in this defense system, antimicrobial peptides (AMPs) are notable due to their specificity towards the invading pathogens without harming the normal host cells. Bombyx mori L. so far has had AMPs identified that belong to six different families, namely cecropin, defensin, moricin, gloverin, attacin and lebocin, which are produced by the Toll and immune deficiency (IMD) pathways. Their diverse modes of action depend on microbial pathogens and are still under investigation. This review examines the recent progress in understanding the immune defense mechanism of Bombyx mori based on AMPs.