RESUMO
Infections induced by fungi, especially the drug-resistant fungi, are difficult clinical problems. Conventional antifungal treatment is effective but due to resistance, treatment failure, and treatment-related toxicity, there is a need for new antifungal drugs. In this study, SA-2 (YYRRLLRVLRRRW) was derived from Cystatin-SA, a saliva protein with a molecular weight of 14 kDa. Meanwhile, the structure-activity of SA-2 and its mutants was also studied. We detected the antimicrobial activity and cytotoxicity of SA-2 and found that SA-2 had a low cytotoxicity toward mammalian cells but a good inhibitory effect on Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), with MIC values of 16-64 µg/mL and 8-32 µg/mL, respectively. Interestingly, SA-2 effectively killed fluconazole-resistant C. neoformans and C. albicans within 12 h. This antifungal activity against fluconazole-resistant fungi was comparable to that of amphotericin B. In addition, the C. neoformans-infected mice model was established to evaluate the anti-infective activity of SA-2 in vivo. Results showed that SA-2 significantly reduced the counts of fungi in lung and brain tissues to protect fluconazole-resistant C. neoformans-infected mice from death without changing mice body weights. Moreover, the dramatically increased pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß induced by intranasal infection of C. neoformans could be obviously declined due to the treatment of SA-2, which may be attributed to the elimination of C. neoformans in time in the infected tissue. For the mode of actions underlying SA-2 against C. neoformans, we found that the cationic peptide SA-2 could adhere to the negatively charged fungal cell membrane to increase the surface potential of C. neoformans in a dose-dependent manner, and finally disrupted the integrity of fungal cell membrane, reflecting as a 60% positive rate of propidium iodide uptake of C. neoformans cells after SA-2 (4 × MIC) treatment. Our study indicated that SA-2 has the potential to develop as a new therapeutic agent against infection induced by drug-resistant fungi.
Assuntos
Cryptococcus neoformans , Cistatinas , Animais , Camundongos , Antifúngicos/farmacologia , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Candida albicans , Cistatinas/farmacologia , MamíferosRESUMO
Cbf-14 (RLLRKFFRKLKKSV), a designed antimicrobial peptide derived from the cathelicidin family, is effective against drug-resistant bacteria. Structurally related peptide impurities in peptide medicines probably have side effects or even toxicity, thus impurity profiling research during the entire production process is indispensable. In this study, a simple liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method using a quadrupole time-of-flight (Q-TOF) mass spectrometer was developed for separation, identification, and characterization of structurally related peptide impurities in Cbf-14. A total of one process-related impurity and thirty-two degradation products were identified, and seven of them have been synthesized and confirmed. These impurities have not been declared in custom synthetic peptides. The degradation products were divided into five categories: fifteen Cbf-14 hydrolysates, five Cbf-14 isomers, four acetyl-Cbf-14 isomers, two aldimine derivatives, and six oxidized impurities. Combined with the peptide synthesis and the stress-testing studies, the origins and the formation mechanisms of these impurities were elucidated, which provides a unique insight for the follow-up quality study of Cbf-14 and other peptide products.
Assuntos
Peptídeos Antimicrobianos , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Medicamentos , Peptídeos , Espectrometria de Massas em Tandem/métodosRESUMO
Helicobacter pylori (H. pylori) infection is highly prevalent, and has developed antimicrobial resistance to virtually all existing antibiotics. Currently, treatment of H. pylori infection (involving proton pump inhibitors and broad-spectrum antibiotics) is suboptimal, with high failure rates. Thus, there is a pressing need to develop new anti-H. pylori therapies. Cbf-K16, a cathelicidin-like antimicrobial peptide, presented broad antimicrobial activity during our previous research. This study further evaluated the therapeutic potential and the mode of action underlying Cbf-K16 against clarithromycin- and amoxicillin-resistant H. pylori SS1. The MIC and MBC of Cbf-K16 against the tested H. pylori were 16 and 32⯵g/ml, respectively, and its killing kinetics was time-dependent, reflecting the thorough elimination of drug-resistant bacteria within 24â¯h. This peptide also protected H. pylori-infected gastric epithelial cells (GES-1) from death by reducing the cell supernatant and intracellular bacterial counts by 1.9 and 2.9-log10 units, respectively. These data indicated the powerful antimicrobial effects of Cbf-K16in vitro. Meanwhile, notable antimicrobial activity in the mouse gastritis model was observed, with decreasing bacterial counts by 3.9-log10 units in stomach tissues and Cbf-K16 could effectively suppress the secretion of inflammatory cytokine IL-8. For its mode of action, Cbf-K16 not only neutralized the negative potential and increased the membrane uptake of NPN and PI by 78.5% and 85.1%, respectively, but also bound to genomic DNA, which in turn downregulated the expression of adhesion genes (alpA and alpB) and virulence gene (cagA), indicating its effective activities on membrane disruption, DNA-binding and gene expression. The data above demonstrated that Cbf-K16 possessed effective antimicrobial and anti-inflammatory activities and downregulated the expression of adhesion- and cytotoxin-associated genes of drug-resistant H. pylori SS1, making it a potential candidate for anti-infective therapy.
Assuntos
Adesinas Bacterianas/efeitos dos fármacos , Catelicidinas/farmacologia , Infecções por Helicobacter , Helicobacter pylori/efeitos dos fármacos , Interleucina-8/efeitos dos fármacos , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Antígenos de Bactérias/efeitos dos fármacos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Farmacorresistência Bacteriana , Genes Bacterianos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
OBJECTIVES: To investigate the effect of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) inhibitor on bone microstructure and bone density in rats with femoral head necrosis. METHODS: Eighty Sprague-Dawley (SD) rats were selected and randomly divided into two groups. One group was selected for femoral head necrosis modeling. Then the modeled rats were randomly divided into two groups, one group was injected with 11ß-HSD1 inhibitor as the treatment group, and the other group was used as the model. The unmodeled rats were also randomly divided into two groups, one group was injected with 11ß-HSD1 inhibitor as the control group, and the other group was taken as the normal group. The bone microstructure and femoral bone density of 4 groups of rats were observed. RESULTS: There were no significant differences in bone microstructure and bone density between the treatment group and the model group before injection (P>0.050), but they were significantly improved after injection (P<0.001). There was no significant difference in superoxide dismutase (SOD) and malondialdehyde (MDA) between the control group and the normal group (P>0.050). SOD increased significantly, and MDA decreased significantly after injection in the treatment group (P<0.001). CONCLUSIONS: 11ß-HSD1 inhibitor can effectively improve the bone microstructure of femoral head necrosis rats and increase bone density, which can be used as a new scheme for the treatment of femoral head necrosis in the future.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Densidade Óssea/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Necrose da Cabeça do Fêmur/patologia , Animais , Feminino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 µM and 32.8 µM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 µM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.
Assuntos
Antivirais/farmacologia , Ácido Benzoico/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/química , Ácido Benzoico/síntese química , Ácido Benzoico/química , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Cbf-14 (RLLRKFFRKLKKSV), a designed peptide derived from cathelicidin family AMP, has proven to be potent against drug-resistant bacteria. In the present study, we investigated the anti-cryptococcal activity of Cbf-14 in vitro and in a pulmonary infection mouse model. Sensitivity test indicated that Cbf-14 possessed effective antifungal activity against Cryptococcus neoformans with an MIC of 4-16⯵g/ml, and killing experiments showed that fungicidal activity was achieved after only 4â¯h treatment with Cbf-14 at 4× MIC concentrations in vitro. Meanwhile, Cbf-14 was effective at prolonging the survival of infected mice when compared with controls, and significantly inhibited the secretion of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, suggesting its anti-inflammatory activity against fungal infections. As a positively charged peptide, Cbf-14 was proven to neutralize the negative zeta potential of the fungal cell surface, disrupt the capsule polysaccharide of fungi, and further damage cell membrane integrity. These results were confirmed by flow cytometry analysis of the fluorescence intensity after PI staining, while cell membrane damage could be clearly observed by transmission electron microscopy after Cbf-14 (4× MIC) treatment for 1â¯h. In addition, Cbf-14 increased the IL-10 levels in cultured RAW 264.7 cells, which were stimulated by C. neoformans infection. The obtained data demonstrated that Cbf-14 could rapidly kill C. neoformans cells in vitro, effectively inhibit C. neoformans induced-infection in mice, and inhibit inflammation in vitro / vivo. Therefore, Cbf-14 could potentially be used for the treatment of fungal infections clinically.
Assuntos
Criptococose/tratamento farmacológico , Cryptococcus neoformans/patogenicidade , Peptídeos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antifúngicos/uso terapêutico , Criptococose/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Fator de Necrose Tumoral alfa/metabolismoRESUMO
S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Trifosfato de Adenosina/metabolismo , Citratos/metabolismo , Metionina Adenosiltransferase/genética , Metionina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Reatores Biológicos , Meios de Cultura/química , Estudos de Viabilidade , Fermentação , Isocitrato Desidrogenase/metabolismo , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/biossíntese , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Citrato de SódioRESUMO
S-Adenosyl-L-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.
Assuntos
Bactérias/genética , Fungos/genética , Engenharia Metabólica/métodos , S-Adenosilmetionina/metabolismo , Bactérias/crescimento & desenvolvimento , Vias Biossintéticas , Fermentação , Fungos/crescimento & desenvolvimento , Genes Bacterianos , Genes Fúngicos , MutaçãoRESUMO
We isolated and characterized a novel virulent bacteriophage, IME-EFm1, specifically infecting multidrug-resistant Enterococcus faecium. IME-EFm1 is morphologically similar to members of the family Siphoviridae. It was found capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 p.f.u. per cell. These biological characteristics suggested that IME-EFm1 has the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 was 42â597 bp, and was linear, with terminally non-redundant dsDNA and a G+C content of 35.2âmol%. The termini of the phage genome were determined with next-generation sequencing and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited a low similarity to other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponded to 90.7â% of the genome; 70 putative ORFs were deduced and, of these, 29 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-ß-lactamase gene was detected in the genome sequence. The identification of an antibiotic resistance gene emphasizes the necessity for complete genome sequencing of a phage to ensure it is free of any undesirable genes before use as a therapeutic agent against bacterial pathogens.
Assuntos
Bacteriófagos/genética , Bacteriófagos/patogenicidade , Enterococcus faecium/virologia , Bacteriófagos/isolamento & purificação , Sequência de Bases , DNA Viral/genética , Farmacorresistência Bacteriana Múltipla , Enterococcus faecium/efeitos dos fármacos , Genoma Viral , Especificidade de Hospedeiro , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Siphoviridae/genética , Virulência/genéticaRESUMO
Scutellaria baicalensis Georgi, a Chinese herbal decoction, has been used for the treatment of the common cold, fever and influenza virus infections. In previous studies, we found that oral administration of baicalein resulted in the inhibition of influenza A virus replication in vivo, which was linked to baicalin in serum. However, the effective dose and underlying mechanisms of the efficacy of baicalin against influenza A virus have not been fully elucidated. In this study, the antiviral effects of baicalin in influenza-virus-infected MDCK cells and mice were examined. The neuraminidase inhibition assay was performed to investigate the mechanism of action of baicalin. In vitro results showed that baicalin exhibited a half-maximal effective concentration (EC50) of 43.3 µg/ml against the influenza A/FM1/1/47 (H1N1) virus and 104.9 µg/ml against the influenza A/Beijing/32/92 (H3N2) virus. When added to MDCK cell cultures after inoculation with influenza virus, baicalin demonstrated obvious antiviral activity that increased in a dose-dependent manner, indicating that baicalin affected virus budding. Baicalin had clear inhibitory effects against neuraminidases, with half-maximal inhibitory concentration (IC50) of 52.3 µg/ml against the influenza A/FM1/1/47 (H1N1) virus and 85.8 µg/ml against the influenza A/Beijing/32/92 (H3N2) virus. In vivo studies showed that an intravenous injection of baicalin effectively reduced the death rate, prolonged the mean day to death (MDD) and improved the lung parameters of mice infected with influenza A virus. These results demonstrate that baicalin acts as a neuraminidase inhibitor, with clear inhibitory activities that are effective against different strains of influenza A virus in both cell culture and a mouse model, and that baicalin has potential utility in the management of influenza virus infections.
Assuntos
Antivirais/farmacologia , Flavonoides/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Administração Intravenosa , Animais , Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Técnicas de Cultura de Células , Modelos Animais de Doenças , Cães , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Flavonoides/isolamento & purificação , Flavonoides/uso terapêutico , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Concentração Inibidora 50 , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Orthomyxoviridae/tratamento farmacológico , Scutellaria/química , Análise de Sobrevida , Resultado do Tratamento , Liberação de Vírus/efeitos dos fármacosRESUMO
Shikimic acid (SA) is an industrially important chiral compound used in diverse commercial applications, and the insufficient supply by isolation from plants and expensive chemical synthesis of SA has increased the importance of developing strategies for SA synthesis. In our previous studies, glycerol was observed to be an effective carbon source for SA accumulation in E. coli DHPYAAS-T7, where the PTS operon (ptsHIcrr) and aroL and aroK genes were inactivated, and the tktA, glk, aroE, aroF (fbr) , and aroB genes were overexpressed. For further investigation of the effects of glycerol aerobic fermentation on SA accumulation in E. coli BL21(DE3), the glpD, glpK genes and tktA, glk, aroE, aroF (fbr) , aroB genes were overexpressed simultaneously. The results indicated that SA production was increased 5.6-fold, while the yield was increased 5.3-fold over that of parental strain in shake flasks. It is demonstrated that the aerobic fermentation of glycerol associated with glpD and glpK gene overexpression increased glycerol flux, resulting in higher SA accumulation in E. coli BL21(DE3)-P-DK.
Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Glicerol Quinase/metabolismo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Engenharia Metabólica , Ácido Chiquímico/metabolismo , Aerobiose , Escherichia coli/genética , Fermentação , Glicerol Quinase/genética , Glicerolfosfato Desidrogenase/genéticaRESUMO
Polyphenolic compound-modified hydrogel wound dressings with excellent wet tissue adhesion, antimicrobial properties, stretchability, and full-thickness skin healing properties are still extremely rare so far. Polyphenolic compounds such as tannic acid or dopamine can improve the antibacterial and bioadhesive properties of hydrogels, and are also polymerization inhibitors for free radical polymerization. In this study, polyacrylic acid (PAA) aqueous solution was first synthesized, and then antibacterial PAA-TA hydrogel was prepared by mixing it with tannic acid (TA) and the crosslinker 1,6-hexanediol bis(2-methyl-1-propionic acid azide) (HBMAP). This method avoids the hindrance of the phenolic hydroxyl groups in TA on acrylic acid polymerization, and we were able to obtain a series of TA hydrogels (in the range of 0-15 wt.%. We applied these PAA-TA hydrogels to wound dressings and found that they had excellent adhesion to biological tissues, and the tensile strength and elongation at break of PAA-TA hydrogels with 15 wt.%TA content were as high as 1.72 MPa and 1446.3% in tensile strength evaluation. In addition, microbiological analysis showed that wound dressings had significant antimicrobial activity against Staphylococcus aureus and Escherichia coli. In vitro wound healing experiments confirmed that the wound dressing was biocompatible and could significantly promote the healing of full-thickness skin defects in the guinea pig model. Our work describes an injectable, self-healing, antimicrobial hydrogel that may have promising clinical applications as a wound dressing material.
Assuntos
Resinas Acrílicas , Anti-Infecciosos , Hidrogéis , Polifenóis , Animais , Cobaias , Antibacterianos/farmacologia , Bandagens , Escherichia coliRESUMO
IMPORTANCE: S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin ß1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.
Assuntos
Streptococcus pneumoniae , Estreptolisinas , Humanos , Streptococcus pneumoniae/genética , Estreptolisinas/genética , Proteínas de Bactérias/genética , Macrófagos/metabolismoRESUMO
Antimicrobial peptides are promising therapeutic agents for treating drug-resistant bacterial disease due to their broad-spectrum antimicrobial activity and decreased susceptibility to evolutionary resistance. In this study, three novel cathelicidin antimicrobial peptides were identified from Thamnophis sirtalis, Balaenoptera musculus, and Lipotes vexillifer by protein database mining and sequence alignment and were subsequently named TS-CATH, BM-CATH, and LV-CATH, respectively. All three peptides exhibited satisfactory antibacterial activity and broad antibacterial spectra against clinically isolated E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii in vitro. Among them, TS-CATH displayed the best antimicrobial/bactericidal activity, with a rapid elimination efficiency against the tested drug-resistant gram-negative bacteria within 20 min, and exhibited the lowest cytotoxicity toward mammalian cells. Furthermore, TS-CATH effectively enhanced the survival rate of mice with ceftazidime-resistant E. coli bacteremia and promoted wound healing in meropenem-resistant P. aeruginosa infection. These results were achieved through the eradication of bacterial growth in target organs and wounds, further inhibiting the systemic dissemination of bacteria and the inflammatory response. TS-CATH exhibited direct antimicrobial activity by damaging the inner and outer membranes, resulting in leakage of the bacterial contents at super-MICs. Moreover, TS-CATH disrupted the bacterial respiratory chain, which inhibited ATP synthesis and induced ROS formation, significantly contributing to its antibacterial efficacy at sub-MICs. Overall, TS-CATH has potential for use as an antibacterial agent.
RESUMO
Antimicrobial peptides (AMPs) have the potential to treat multidrug-resistant bacterial infections. Cathelicidins are a class of cationic antimicrobial peptides that are found in nearly all vertebrates. Herein, we determined the mature peptide region of Alligator sinensis cathelicidin by comparing its cathelicidin peptide sequence with those of other reptiles and designed nine peptide mutants based on the Alligator sinensis cathelicidin mature peptide. According to the antibacterial activity and cytotoxicity screening, the peptide AS-12W demonstrated broad-spectrum antibacterial activity and exhibited low erythrocyte hemolytic activity. In particular, AS-12W exhibited strong antibacterial activity and rapid bactericidal activity against carbapenem-resistant Pseudomonas aeruginosa in vitro. Additionally, AS-12W effectively removed carbapenem-resistant P. aeruginosa from blood and organs in vivo, leading to improved survival rates in septic mice. Furthermore, AS-12W exhibited good stability and tolerance to harsh conditions such as high heat, high salt, strong acid, and strong alkali, and it also displayed high stability toward trypsin and simulated gastric fluid (SGF). Moreover, AS-12W showed significant anti-inflammatory effects in vitro by inhibiting the production of proinflammatory factors induced by lipopolysaccharide (LPS). Due to its antibacterial mechanism against Escherichia coli, we found that this peptide could neutralize the negative charge on the surface of the bacteria and disrupt the integrity of the bacterial cell membrane. In addition, AS-12W has the ability to bind to the genomic DNA of bacteria and stimulate the production of reactive oxygen species (ROS) within bacteria, which is believed to be the reason for the good antibacterial activity of AS-12W. These results demonstrated that AS-12W exhibits remarkable antibacterial activity, particularly against carbapenem-resistant P. aeruginosa. Therefore, it is a potential candidate for antibacterial drug development.
RESUMO
New Delhi metallo-beta-lactamase-1(NDM-1)-carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin-BF (BF-30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf-K(16) and Cbf-A(7) A(13) were obtained by mutating Lys(16), Ala(7), and Ala(13) of BF-30, respectively. To investigate their antimicrobial activities against NDM-1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)-NDM-1 with high NDM-1 activity was constructed by inserting the Klebsiella pneumoniae NDM-1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM-1-carrying E. coli, and the minimum inhibitory concentrations of Cbf-K(16) and Cbf-A(7) A(13) were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf-K(16) and Cbf-A(7) A(13) treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled ß-galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf-K(16) treatment (8 µg/ml). These data demonstrated that Cbf-K(16) and Cbf-A(7) A(13) possess effective antimicrobial activity against drug-resistant strains, including NDM-1 carrying E. coli BL21 (DE3)-NDM-1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM-1-carrying bacterial infections.
Assuntos
Antibacterianos/farmacologia , Catelicidinas/farmacologia , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Catelicidinas/química , Catelicidinas/metabolismo , Permeabilidade da Membrana Celular , DNA Bacteriano/química , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Minociclina/análogos & derivados , Minociclina/farmacologia , Dados de Sequência Molecular , Ligação Proteica , Staphylococcus aureus , Tigeciclina , Resistência beta-Lactâmica , beta-Lactamas/farmacologiaRESUMO
Chronic hepatitis B virus (HBV) infection is a worldwide public health problem that causes significant liver-related morbidity and mortality. In our previous study, Strongylocentrotus nudus eggs polysaccharide (SEP), extracted from sea urchins, had immunomodulatory and antitumor effects. Whether SEP has anti-HBV activity is still obscure. This study demonstrated that SEP decreased the secretion of hepatitis B surface antigen (HBsAg) and e antigen (HBeAg), as well as the replication and transcription of HBV both in vitro and in vivo. Immunofluorescence and immunohistochemistry results showed that the level of HBV core antigen (HBcAg) was clearly reduced by SEP treatment. Mechanistically, RT-qPCR, western blot, and confocal microscopy analysis showed that SEP significantly increased the expression of toll-like receptor 4 (TLR4) and co-localization with TLR4. The downstream molecules of TLR4, including NF-κb and IRF3, were activated and the expression of IFN-ß, TNF-α, IL-6, OAS, and MxA were also increased, which could suppress HBV replication. Moreover, SEP inhibited other genotypes of HBV and hepatitis C virus (HCV) replication in vitro. In summary, SEP could be investigated as a potential anti-HBV drug capable of modulating the innate immune.
Assuntos
Hepatite B Crônica , Strongylocentrotus , Animais , Humanos , Vírus da Hepatite B , Receptor 4 Toll-Like/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Polissacarídeos/farmacologia , Polissacarídeos/metabolismo , Antígenos E da Hepatite B/metabolismo , Strongylocentrotus/metabolismo , Replicação ViralRESUMO
Polymyxin resistance is conferred by MCR-1 (mobile colistin resistance 1)-induced lipopolysaccharide (LPS) modification of G- bacteria. However, the peptide MSI-1 exerts potent antimicrobial activity against mcr-1-carrying bacteria. To further investigate the potential role of MCR-1 in improving bacterial virulence and facilitating immune evasion, and the immunomodulatory effect of peptide MSI-1, we first explored outer membrane vesicle (OMV) alterations of mcr-1-carrying bacteria in the presence and absence of sub-MIC MSI-1, and host immune activation during bacterial infection and OMV stimulation. Our results demonstrated that LPS remodelling induced by MCR-1 negatively affected OMV formation and protein cargo by E. coli. In addition, MCR-1 diminished LPS-stimulated pyroptosis but facilitated mitochondrial dysfunction, further aggravating apoptosis in macrophages induced by OMVs of E. coli. Similarly, TLR4-mediated NF-κB activation was markedly alleviated once LPS was modified by MCR-1. However, peptide MSI-1 at the sub-MIC level inhibited the expression of MCR-1, further partly rescuing OMV alteration and attenuation of immune responses in the presence of MCR-1 during both infection and OMV stimulation, which can be exploited for anti-infective therapy.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Lipopolissacarídeos , Evasão da Resposta Imune , Colistina/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptídeos/farmacologia , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The interactions between active pharmaceutical ingredients (APIs) and excipients may lead to API degradation, thereby affecting the safety and efficacy of drug products. Cbf-14 is a synthetic peptide derived from Cathelicidin-BF, showing potential for bacterial and fungal infections. In order to assess impurities in Cbf-14 gel, we developed a two-dimensional liquid chromatography coupled with quadrupole/time-of-flight mass spectrometric method. A total of eleven peptide degradation impurities were identified and characterized. Furthermore, the compatibility tests were conducted to evaluate the interactions of Cbf-14 with glycerol and methylcellulose, respectively. The results revealed that the impurities originated from condensation reactions between Cbf-14 and aldehydes caused by glycerol degradation. Several aldehydes were employed to validate this hypothesis. The formation mechanisms were elucidated as Maillard reactions between primary amino groups of Cbf-14 and aldehydes derived from glycerol degradation. Additionally, the compatibility of Cbf-14 with glycerol from different sources and with varying storage times was investigated. Notably, the interaction products in the gel increased with extended storage time, even when fresh glycerol for injection was added. This study offers unique insights into the compatibility study of peptides and glycerol, contributing to the ongoing quality study of Cbf-14 gel. It also serves as a reference for the design of other peptide preparations and excipients selections.
RESUMO
The heavy and rigid appearance of conventional burnt building tiles is not suitable for a global sustainable development strategy. Flexible facing tiles with lightweight and environmental materials are highly desirable for the construction industry today. In this work, water-based polymer emulsion-assisted flexible building tiles were prepared. Based on the method of achieving post crosslinking and improving adhesion with inorganic matrix-based materials, WPAs modified with GMA and KH570 display good chemical resistance and low solvent absorption (0.132 in water and 0.289 in ethanol respectively). The optimum mechanical performance of flexible building materials prepared with WPAs can strain 1.406% and stress 1.8658 MPa. The TGA, XRD, SEM and AFM results further indicate the excellent thermal stability and compatibility of flexible building tiles. Hence, flexible building tiles prepared with WPAs can be promising building materials for construction.