RESUMO
The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.
Assuntos
Proteínas de Anfíbios/farmacologia , Anfíbios/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/química , Vírus de DNA/efeitos dos fármacos , Vírus de RNA/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Lipídeos/química , SARS-CoV-2/efeitos dos fármacos , Células VeroRESUMO
BACKGROUND: Colistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa. OBJECTIVES: Identification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants. METHODS: The available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A. RESULTS: A putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially. CONCLUSIONS: This study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.
Assuntos
Colistina , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Translocador Nuclear Receptor Aril Hidrocarboneto , Colistina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pseudomonas aeruginosaRESUMO
When cultured in minimal growth medium, the B38 strain of Bacillus subtilis did not exhibit any antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate. Coculturing B38 strain with viable MRSA cells weakly increased antibacterial activity production (20 AU/ml). Addition of dead MRSA cells in a B38 culture, increased by 8-fold the B. subtilis strain antibacterial activity reaching 160 AU/ml against MRSA strain. This antibacterial activity recovered from cell-free supernatants was stimulated by an autoinducing compound which is sensitive to the action of proteinase K suggesting a proteinaceous nature. This compound was heat-stable till 80 °C and showed a molecular mass around 20 kDa as determined by SDS-PAGE. These results suggest that the production of antibacterial compounds by B38 strain is dependent on the amount of the autoinducing compound.
Assuntos
Antibacterianos/farmacologia , Antibiose , Bacillus subtilis/metabolismo , Proteínas de Bactérias/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacillus subtilis/química , Bacillus subtilis/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Técnicas de Cocultura , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade MicrobianaRESUMO
Bacillus subtilis B38, isolated from soil, showed antimicrobial activity against human pathogenic Candida albicans species. Specific PCR primers revealed the presence of the bamC gene, which is involved in the biosynthesis of bacillomycin D. Three anti-Candida compounds designated a(1) , a(2) and a(3) were purified from culture supernatant and identified using matrix-assisted laser desorption/ionization time-of-flight MS as analogues of bacillomycin D-like lipopeptides of 14, 15 and 16 carbon fatty acid long chains, respectively. The compound a(3) displayed the strongest fungicidal activity against pathogenic C. albicans strains. It was even more active than amphotericin B with a lethal concentration of 59.07 vs. 135.26 µM of the antimycotic drug against the pathogenic strain C. albicans sp. 311 isolated from finger nail. Only moderate or weak anti-Candida activity was recorded for a(1) and a(2) compounds. Furthermore, a(3) showed the highest hemolytic activity, reaching 50% hemolysis at 22.14 µM, whereas a(1) and a(2) displayed a limited hemolysis at 68.26 and 37.41 µM, respectively. These findings suggest that the acyl chain length of bacillomycin D-like lipopeptides plays a major role in hemolytic and antifungal activities.
Assuntos
Antifúngicos/farmacologia , Bacillus subtilis/metabolismo , Candida albicans/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptídeos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Peptídeos Catiônicos Antimicrobianos , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Hemolíticos/química , Hemolíticos/isolamento & purificação , Hemolíticos/metabolismo , Hemolíticos/farmacologia , Humanos , Lipopeptídeos/química , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Microbiologia do SoloRESUMO
An antibacterial compound, S07-2, was purified to homogeneity by hydrophobic interaction, anion exchange, C18 reverse-phase and HS PEG HPLC. The molecular mass of S07-2 was 905.6 Da as determined by MS. The S07-2 compound was resistant to high temperatures (up to 100 degrees C) and could withstand a wide range of pH from 3 to 10. In addition, its antibacterial activity was preserved after treatment with proteases. Biochemical characterization revealed its cyclic peptide structure. This compound showed a bactericidal effect against important food-spoilage bacteria and food-borne pathogens including Listeria monocytogenes and Enterococcus faecalis with lethal concentration values of 62.5 microg mL(-1) and against Salmonella enteritidis at a concentration of 31.25 microg mL(-1). However, no cytotoxic effect against human erythrocytes was recorded. Furthermore, the S07-2 compound displayed a remarkable Fe(2+)-chelating activity (EC(50)=9.76 microg mL(-1)) and 1-diphenyl-2-picrylhydrazyl-scavenging capacity (IC(50)=65 microg mL(-1)). All these chemical and biological features make S07-2 a useful compound in the food industry as a natural preservative.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Bacillus subtilis/metabolismo , Antibacterianos/química , Antibacterianos/toxicidade , Antioxidantes/química , Antioxidantes/toxicidade , Quelantes/química , Quelantes/isolamento & purificação , Quelantes/farmacologia , Quelantes/toxicidade , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Estabilidade de Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Salmonella enteritidis/efeitos dos fármacosRESUMO
An antimicrobial activity produced by Bacillus subtilis B38 was found to be effective against several bacteria, including pathogenic and spoilage microorganisms such as, Listeria monocytogenes, Salmonella enteridis, and clinical isolates of methicillin-resistant Staphylococcus species. Nutrients such as carbon, nitrogen sources, and inorganic salts enhanced the production level of the antibacterial activity by B. subtilis B38. A first screening step showed that lactose, ammonium succinate, and manganese most influenced both cell growth and antibacterial activity production. These three factors varied at two levels in eight experiments using full factorial design. Results indicated that maximum cell growth (OD = 10.2) and maximum production of antibacterial activity (360 AU/mL) were obtained in a modified medium containing 1.5% (w/v) lactose, 0.15% (w/v) ammonium succinate, and 0.3 mg/L manganese. Depending on the indicator strain used, the antibacterial activity was 2- to 4-fold higher in the modified culture medium than in TSB medium under the same conditions. Thin layer chromatography-bioautography assay showed the presence of three active spots with R(f) values of 0.47, 0.7, and 0.82 in TSB medium. However, the inhibition zone of two spots (R(f) values of 0.7 and 0.82) was slightly larger in the modified medium. Moreover, a large zone of inhibition with an R(f) value of 0.3, was observed in this modified medium, instead of the spot having an R(f) value of 0.47. These results suggest that the nutrients act as environmental factors, quantitatively and qualitatively affecting the production of antibacterial compounds by B. subtilis B38.
Assuntos
Antibacterianos/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacteriocinas/metabolismo , Técnicas Microbiológicas/métodos , Análise de Variância , Autorradiografia , Bacillus subtilis/metabolismo , Carboidratos , Proliferação de Células , Cromatografia em Camada Fina , Meios de Cultura , Lactose , Manganês , Testes de Sensibilidade Microbiana , Minerais , Nitrogênio , Análise de Regressão , Ácido SuccínicoRESUMO
B38 bacterial strain, isolated from Tunisian soil showed a strong antimicrobial activity. Based on biochemical characterization and 16S rDNA sequence analysis, B38 strain was identified as Bacillus subtilis. Cell culture supernatant showed antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several Gram-positive and Gram-negative bacteria. Antifungal activity against phytopathogenic fungi was also observed. Antibacterial activity production started at early exponential growth phase, and maximum activity was reached at the stationary phase. This antibacterial activity was neither affected by proteases, lipase, and organic solvents, nor by surfactants. It was stable over a wide pH range and still active after autoclaving at 121 degrees C during 20 min. Thin layer chromatography followed by bioautography assay allowed the detection of four active spots with R(f) values of 0.30, 0.47, 0.70, and 0.82. The single spot with R (f) 0.30 showed antifungal activity, whereas the spots with R(f) values of 0.47, 0.70, and 0.82 exhibited antibacterial activity.