Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate.

Bacterial resistance is a threat to health worldwide, mainly due to reduced effective treatment. In this context, the search for strategies to control such infections and suppress antimicrobial resistance is necessary. One of the strategies that has been used is combination therapy. In the present work, we investigated the in vitro efficacy of the antimicrobials diminazene aceturate (DA), chloramphenicol (CHL), and streptomycin (STP) alone and in combination against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus clinical isolates. DA was capable of inhibiting all strains with MIC of 25-400 µg mL-1, while STP and CHL showed antibacterial activity with minimum inhibitory concentration (MICs) of ≤3.12-400 µg mL-1. The combination of aceturate with STP showed synergism toward almost all Gram-negative bacteria, with fractional inhibitory concentration index (FICIs) of 0.09-0.37. In addition, for CHL and aceturate, synergisms for Gram-negative and -positive strains were observed. A time-kill assay against E. coli revealed that the aceturate and STP combination can inhibit bacterial growth in a shorter time when compared with single antibiotics. In addition, antimicrobials did not show hemolytic activity even at the highest concentrations used. Therefore, the antimicrobial combinations presented in this work showed important results, demonstrating that combined therapy can be used as an alternative strategy for pathogen control.

Anti-Staphy Peptides Rationally Designed from Cry10Aa Bacterial Protein.

Bacterial infections pose a significant threat to human health, constituting a major challenge for healthcare systems. Antibiotic resistance is particularly concerning in the context of treating staphylococcal infections. In addressing this challenge, antimicrobial peptides (AMPs), characterized by their hydrophobic and cationic properties, unique mechanism of action, and remarkable bactericidal and immunomodulatory capabilities, emerge as promising alternatives to conventional antibiotics for tackling bacterial multidrug resistance. This study focuses on the Cry10Aa protein as a template for generating AMPs due to its membrane-penetrating ability. Leveraging the Joker algorithm, six peptide variants were derived from α-helix 3 of Cry10Aa, known for its interaction with lipid bilayers. In vitro, antimicrobial assays determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) required for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Acinetobacter baummanii, Enterobacter cloacae, Enterococcus facallis, Klebsiella pneumonia, and Pseudomonas aeruginosa. Time-kill kinetics were performed using the parental peptide AMPCry10Aa, as well as AMPCry10Aa_1 and AMPCry10Aa_5, against E. coli ATCC, S. aureus 111 and S. aureus ATCC strains showing that AMPCry10Aa_1 and AMPCry10Aa_5 peptides can completely reduce the initial bacterial load with less than 2 h of incubation. AMPCry10Aa_1 and AMPCry 10Aa_5 present stability in human serum and activity maintenance up to 37 °C. Cytotoxicity assays, conducted using the MTT method, revealed that all of the tested peptides exhibited cell viability >50% (IC50). The study also encompassed evaluations of the structure and physical-chemical properties. The three-dimensional structures of AMPCry10Aa and AMPCry10Aa_5 were determined through nuclear magnetic resonance (NMR) spectroscopy, indicating the adoption of α-helical segments. Electron paramagnetic resonance (EPR) spectroscopy elucidated the mechanism of action, demonstrating that AMPCry10Aa_5 enters the outer membranes of E. coli and S. aureus, causing substantial increases in lipid fluidity, while AMPCry10Aa slightly increases lipid fluidity in E. coli. In conclusion, the results obtained underscore the potential of Cry10Aa as a source for developing antimicrobial peptides as alternatives to conventional antibiotics, offering a promising avenue in the battle against antibiotic resistance.

Peptide Stapling Applied to Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are considered a promising therapeutic approach against multi-drug resistant microorganisms. Besides their advantages, there are limitations to be overcome so that these molecules can become market competitive. One of the biggest limitations is proteolytic susceptibility, which could be overcome by structural modifications such as cyclization, especially for helix-constraining strategies. Over the years, many helix stabilization techniques have arisen, such as lactam-bridging, triazole-based, N-alkylation and all-hydrocarbon stapling. All-hydrocarbon stapling takes advantage of modified amino acid residues and olefinic cross-linking to constrain peptide helices. Despite being a well-established strategy and presenting efficient stability results, there are different limitations especially related to toxicity. In this review, recent studies on stapled AMPs for antimicrobial usage are explored with the aim of understanding the future of these molecules as putative antimicrobial agents.

Nanoparticles in association with antimicrobial peptides (NanoAMPs) as a promising combination for agriculture development.

Antimicrobial peptides are small molecules, up to 10 kDa, present in all kingdoms of life, including in plants. Several studies report that these molecules have a broad spectrum of activity, including antibacterial, antifungal, antiviral, and insecticidal activity. Thus, they can be employed in agriculture as alternative tools for phytopathogen and pest control. However, the application of peptides in agriculture can present challenges, such as loss of activity due to degradation of these molecules, off-target effects, and others. In this context, nanotechnology can offer versatile structures, including metallic nanoparticles, liposomes, polymeric nanoparticles, nanofibers, and others, which might act both in protection and in release of AMPs. Several polymers and biomaterials can be employed for the development of nanostructures, such as inorganic metals, natural or synthetic lipids, synthetic and hybrid polymers, and others. This review addresses the versatility of NanoAMPs (Nanoparticles in association with antimicrobial peptides), and their potential applications in agribusiness, as an alternative for the control of phytopathogens in crops.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

Screening for cysteine-stabilized scaffolds for developing proteolytic-resistant AMPs.

Antimicrobial peptides (AMP) are present in all organisms and can present several activities and potential applications in human and animal health. Screening these molecules scaffolds represents a key point for discovering and developing novel biotechnological products, including antimicrobial, antiviral and anticancer drugs candidates and insecticidal molecules with potential applications in agriculture. Therefore, considering the amount of biological data currently deposited on public databases, computational approaches have been commonly used to predicted and identify novel cysteine-rich peptides scaffolds with known or unknown biological properties. Here, we describe a step-by-step in silico screening for cysteine-rich peptides employing molecular modeling (with a core focus on comparative modeling) and atomistic molecular dynamics simulations. Moreover, we also present the concept of additional tools aiming at the computer-aided screening of new Cs-AMPs based drug candidates. After the computational screening and peptide chemical synthesis, we also provide the reader with a step-by-step in vitro activity evaluation of these candidates, including antibacterial, antifungal, and antiviral assays.