RESUMO
Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.
Assuntos
Anuros , Leishmania mexicana , Plasmodium falciparum , Pele , Trypanosoma cruzi , Animais , Trypanosoma cruzi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Humanos , Leishmania mexicana/efeitos dos fármacos , Pele/parasitologia , Pele/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Equador , Doença de Chagas/tratamento farmacológicoRESUMO
Listeria monocytogenes is a human opportunistic foodborne pathogen that produces life-threatening infections with a high mortality rate. The control of Listeria in the food production environment and effective clinical management of human listeriosis are challenging due to the emergence of antibiotic resistance. Hence we evaluate the in vitro anti-Listeria activity of two synthetic cruzioseptins reproducing their natural sequences CZS-9, and CZS-12, and one engineered sequence based on CZS-1, named [K4K15]CZS-1. The assessment of the in vitro potential of cruzioseptins, highlighted the promising antibacterial effect of [K4K15]CZS-1 in very low concentrations (0.91 µM) and its thermal stability at high-temperature conditions, is compatible with the food industry. Microscopic and metabolomic analyses suggest cruzioseptin induces anti-Listeria bioactivity through membrane disruption and changes in the intracellular metabolome. We also report that [K4K15]CZS-1 is not resistant to peptidases/proteases emphasizing a key advantage for their use as a food preservative. However, there is a need for further structural and functional optimisations for the potential clinical application as an antibiotic. In conclusion, [K4K15]CZS-1 stand out as membrane-active peptides with the ability to induce shifts in the bacteria metabolome and inspire the development of strategies for the prevention of L. monocytogenes emergence and dissemination.
Assuntos
Antibacterianos , Listeria monocytogenes , Metabolômica , Listeria monocytogenes/efeitos dos fármacos , Antibacterianos/farmacologia , Humanos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genéticaRESUMO
Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.
RESUMO
Salmonella genus is a leading cause of food-borne infections with strong public health impact and economic ramifications. The development of antimicrobial resistance added complexity to this scenario and turned the antibiotic drug discovery into a highly important challenge. The screening of peptides has served as a successful discovery platform to design new antibiotic candidates. Motivated by this, the antimicrobial and cytotoxic properties of three cruzioseptins against Salmonella Typhimurium and RAW 264.7 murine macrophage cells, respectively, were investigated. [K4K15]CZS-1 was the most potent antimicrobial peptide identified in the screening step with a minimum inhibitory concentration (MIC) of 16 µg/mL (7.26 µM) and moderate cytotoxicity. From a structural point of view, in vitro and in silico techniques evidenced that [K4K15]CZS-1 is a α-helical cationic antimicrobial peptide. In order to capture mechanistic details and fully decipher their antibacterial action, we adopted a multidimensional approach, including spectroscopy, electron microscopy and omics analysis. In general lines, [K4K15]CZS-1 caused membrane damage, intracellular alterations in Salmonella and modulated metabolic pathways, such as the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and lipid metabolism. Overall, these findings provide deeper insights into the antibacterial properties and multidimensional mode of action of [K4K15]CZS-1 against Salmonella Typhimurium. In summary, this study represents a first step toward the screening of membrane-acting and intracellular-targeting peptides as potential bio-preservatives to prevent foodborne outbreaks caused by Salmonella.
RESUMO
Peptide engineering has gained attraction as a source of new cationicity-enhanced analogues with high potential for the design of next-generation antibiotics. In this context, cruzioseptin-1 (CZS-1), a peptide identified from Cruziohyla calcarifer, is recognized for its antimicrobial potency. However, this amidated-peptide is moderately hemolytic. In order to reduce toxicity and increase antimicrobial potency, 3 peptide analogues based on cruzioseptin-1 were designed and evaluated. [K4K15]CZS-1, an analogue with increased cationicity and reduced hydrophobicity, showed antibacterial, antifungal and antiproliferative properties. In addition, [K4K15]CZS-1 is less hemolytic than CZS-1. The in silico and scanning electron microscopy analysis reveal that [K4K15]CZS-1 induces a membranolytic effect on bacteria. Overall, these results confirm the potential of CZS-1 as source of inspiration for design new selective antimicrobial analogues useful for development of new therapeutic agents.