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1.
Crit Care ; 24(1): 47, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041659

RESUMO

OBJECTIVES: The intestinal epithelium compartmentalizes the sterile bloodstream and the commensal bacteria in the gut. Accumulating evidence suggests that this barrier is impaired in sepsis, aggravating systemic inflammation. Previous studies reported that cathelicidin is differentially expressed in various tissues in sepsis. However, its role in sepsis-induced intestinal barrier dysfunction has not been investigated. DESIGN: To examine the role of cathelicidin in polymicrobial sepsis, cathelicidin wild-(Cnlp+/+) and knockout (Cnlp-/-) mice underwent cecal-ligation and puncture (CLP) followed by the assessment of septic mortality and morbidity as well as histological, biochemical, immunological, and transcriptomic analyses in the ileal tissues. We also evaluated the prophylactic and therapeutic efficacies of vitamin D3 (an inducer of endogenous cathelicidin) in the CLP-induced murine polymicrobial sepsis model. RESULTS: The ileal expression of cathelicidin was increased by three-fold after CLP, peaking at 4 h. Knockout of Cnlp significantly increased 7-day mortality and was associated with a higher murine sepsis score. Alcian-blue staining revealed a reduced number of mucin-positive goblet cells, accompanied by reduced mucin expression. Increased number of apoptotic cells and cleavage of caspase-3 were observed. Cnlp deletion increased intestinal permeability to 4kD fluorescein-labeled dextran and reduced the expression of tight junction proteins claudin-1 and occludin. Notably, circulating bacterial DNA load increased more than two-fold. Transcriptome analysis revealed upregulation of cytokine/inflammatory pathway. Depletion of Cnlp induced more M1 macrophages and neutrophils compared with the wild-type mice after CLP. Mice pre-treated with cholecalciferol (an inactive form of vitamin D3) or treated with 1alpha, 25-dihydroxyvitamin D3 (an active form of VD3) had decreased 7-day mortality and significantly less severe symptoms. Intriguingly, the administration of cholecalciferol after CLP led to worsened 7-day mortality and the associated symptoms. CONCLUSIONS: Endogenous cathelicidin promotes intestinal barrier integrity accompanied by modulating the infiltration of neutrophils and macrophages in polymicrobial sepsis. Our data suggested that 1alpha, 25-dihydroxyvitamin D3 but not cholecalciferol is a potential therapeutic agent for treating sepsis.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Mucosa Intestinal , Sepse , Animais , Peptídeos Catiônicos Antimicrobianos/fisiologia , Mucosa Intestinal/metabolismo , Macrófagos , Masculino , Camundongos , Camundongos Knockout , Neutrófilos , Sepse/fisiopatologia , Vitamina D/análogos & derivados , Vitamina D/farmacologia
2.
World J Microbiol Biotechnol ; 36(1): 18, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31912374

RESUMO

In this paper, we report the antimicrobial activity of AMEP412 (a protein elicitor from Bacillus subtilis) against Streptomyces scabiei, which is the potato common scab pathogen. The purified protein samples showed an obvious inhibition zone on an S. scabiei agar plate, and the minimum inhibition concentration detected was 50 µg mL-1. The fluorescence localization assay revealed that AMEP412 could bind to aerial mycelia and spores. The stability test showed that AMEP412 was stable at 60 °C for 30 min and in pH values from 5.0 to 10.0. Its antimicrobial activity was not sensitive to metal cations. However, its activity declined by 23% when treated with Proteinase K, and was completely abrogated with Tween 80 treatment. Three antimicrobial peptides (GS21, GY20 and GY23) were identified from AMEP412, which further verified its antimicrobial activity. This research reveals the antimicrobial function of AMEP412, which not only enriches the function of the protein elicitor, but also provides a candidate for the biocontrol of potato common scab.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/farmacologia , Streptomyces/efeitos dos fármacos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Micélio/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Esporos Bacterianos/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento
3.
Pestic Biochem Physiol ; 163: 102-107, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31973845

RESUMO

Anthracnose is a leaf spot, blossom blight, or fruit rot disease caused by Colletotrichum gloeosporioides (Penz.). It is the most prevalent disease in mango-growing countries worldwide. Lipopeptides, such as those in the iturin family, account for the majority of antifungal secondary metabolites in Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus velezensis, and includes bacillomycin D. Thus far, the mechanism of bacillomycin D's activity has not been clear. In this study, bacillomycin D was isolated from B. velezensis HN-2, which strongly inhibits C. gloeosporioides (Penz.). The median inhibitory concentration of bacillomycin D was 2.162 µg/mL, causing deformation and damage to C. gloeosporioides (Penz.). Bacillomycin D showed more potent activity against C. gloeosporioides (Penz.) than two common fungicides prochloraz and mancozeb. Scanning and transmission electron microscopy revealed that bacillomycin D could injure the cell wall and cell membrane of the hyphae and spores of C. gloeosporioides (Penz.), and the cytoplasm and organelles inside the cell were exuded and formed empty holes. This research clarifies the mechanism underlying bacillomycin D antifungal activity and reveals its high potential as a biopesticide to control phytopathogens.


Assuntos
Bacillus , Colletotrichum , Antifúngicos , Peptídeos Catiônicos Antimicrobianos , Doenças das Plantas
4.
Ann Otol Rhinol Laryngol ; 129(3): 245-255, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31646875

RESUMO

OBJECTIVES: Diesel exhaust particles (DEP)s are notorious ambient pollutants composed of a complex mixture of a carbon core and diverse chemical irritants. Several studies have demonstrated significant relationships between DEP exposure and serious nasal inflammatory response in vitro, but available information regarding underlying networks in terms of gene expression changes has not sufficiently explained potential mechanisms of DEP-induced nasal damage, especially in vivo. METHODS: In the present study, we identified DEP-induced gene expression profiles under short-term and long-term exposure, and identified signaling pathways based on microarray data for understanding effects of DEP exposure in the mouse nasal cavity. RESULTS: Alteration in gene expression due to DEP exposure provokes an imbalance of the immune system via dysregulated inflammatory markers, predicted to disrupt protective responses against harmful exogenous substances in the body. Several candidate markers were identified after validation using qRT-PCR, including S100A9, CAMP, IL20, and S100A8. CONCLUSIONS: Although further mechanistic studies are required for verifying the utility of the potential biomarkers suggested by the present study, our in vivo results may provide meaningful suggestions for understanding the complex cellular signaling pathways involved in DEP-induced nasal damages.


Assuntos
Expressão Gênica , Rinite/induzido quimicamente , Emissões de Veículos/toxicidade , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Biomarcadores/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Camundongos Endogâmicos BALB C , Modelos Animais , Testes de Provocação Nasal , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Rinite/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
5.
Environ Pollut ; 256: 113443, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733951

RESUMO

During their lifetime honey bees (Apis mellifera) rarely experience optimal conditions. Sometimes, a simultaneous action of multiple stressors, natural and chemical, results in even greater effect than of any stressor alone. Therefore, integrative investigations of different factors affecting honey bees have to be carried out. In this study, adult honey bees exposed to thiamethoxam in larval and/or adult stage and infected with Nosema ceranae were examined. Newly emerged bees from colonies, non-treated or treated with thiamethoxam, were organized in six groups and kept in cages. Thiamethoxam treated bees were further exposed to either thiamethoxam or Nosema (groups TT and TN), or simultaneously to both (group TTN). Newly emerged bees from non-treated colonies were exposed to Nosema (group CN). From both, treated and non-treated colonies two groups were organized and further fed only with sugar solution (groups C and TC). Here, we present the expression profile of 19 genes in adult worker honey bees comprising those involved in immune, detoxification, development and apoptosis response. Results showed that gene expression patterns changed with time and depended on the treatment. In group TC at the time of emergence the majority of tested genes were downregulated, among which nine were significantly altered. The same gene pattern was observed on day six, where the only significantly upregulated gene was defensin-1. On day nine most of analyzed genes in all experimental groups showed upregulation compared to control group, where upregulation of antimicrobial peptide genes abaecin, defensin-1 and defensin-2 was significant in groups TT and TTN. On day 15 we observed a similar pattern of expression in groups TC and TT exposed to thiamethoxam only, where most of the detoxification genes were downregulated. Additionally RNA loads of Nosema and honey bee viruses were recorded. We detected a synergistic interaction of thiamethoxam and Nosema, reflected in lowest honey bee survival.


Assuntos
Abelhas/fisiologia , Inseticidas/toxicidade , Nosema , Tiametoxam/toxicidade , Animais , Peptídeos Catiônicos Antimicrobianos , Abelhas/efeitos dos fármacos , Abelhas/microbiologia , Expressão Gênica , Proteínas de Insetos , Larva/efeitos dos fármacos , Larva/microbiologia , Larva/fisiologia , Microsporidiose/veterinária
6.
Chem Biol Interact ; 315: 108904, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31758921

RESUMO

Epinecidin-1 (epi) was identified from orange-spotted grouper (Epinephelus coioides) and exhibits diverse biological activities. The aims of this study were to investigate how the distribution of positively charged amino acid residues affects epi-mediated cytotoxicity and to examine the molecular mechanism underlying epi-induced cytotoxicity in U87MG human glioblastoma cells. MTS/PMS and trypan blue exclusion assay were used to measure cell viability. Necrotic cell death was confirmed by detecting cyclophilin A release and propidium iodide incorporation. DNA damage was evaluated by measuring phosphorylated H2AX. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry using dihydroergotamine. Mitochondrial membrane potential was detected by flow cytometry using tetramethylrhodamine, ethyl ester. Overall, we found that epi caused cytotoxicity in U87MG cells by inducing DNA damage and necrosis through mitochondrial hyperpolarization and subsequent ROS production. The proper folding of epi into an α-helical structure was essential for epi-mediated anti-glioblastoma effects. In addition, NFκB signaling was activated in U87MG cells after exposure to epi. Suppression of NFκB further enhanced epi-induced cytotoxicity, ROS generation and DNA damage, indicating that NFκB may play a protective role in epi-induced cytotoxicity. Our findings may be useful for the design and improvement of antimicrobial peptides with anti-cancer activity.


Assuntos
Aminoácidos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Proteínas de Peixes/farmacologia , Glioblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Protein Pept Lett ; 27(1): 60-66, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31362652

RESUMO

BACKGROUND: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. OBJECTIVE: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. METHODS: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. RESULTS: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, markers for antigen presentation, on primary chicken monocytes, whereas LL-37 did not. D-CATH- 2 also increased the MRC1 and MHC-II expression if a chicken macrophage cell line (HD11 cells) was used. In addition, LPS-induced NO production by HD11 cells was inhibited by CATH-2 and D-CATH-2. CONCLUSION: These results are a clear indication that CATH-2 (and D-CATH-2) affect the activation state of monocytes and macrophages, which leads to optimization of the innate immune response and enhancement of the adaptive immune response.


Assuntos
Biomarcadores/metabolismo , Catelicidinas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Galinhas , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
8.
Protein Pept Lett ; 27(1): 41-47, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31438823

RESUMO

BACKGROUND: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. OBJECTIVE: In this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated. METHODS: Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay. RESULTS: The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH. CONCLUSION: Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Bases , Fosfatos de Cálcio/química , Sobrevivência Celular , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção
9.
Protein Pept Lett ; 27(1): 4-16, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31438824

RESUMO

Antimicrobial peptides in recent years have gained increased interest among scientists, health professionals and the pharmaceutical companies owing to their therapeutic potential. These are low molecular weight proteins with broad range antimicrobial and immuno modulatory activities against infectious bacteria (Gram positive and Gram negative), viruses and fungi. Inability of micro-organisms to develop resistance against most of the antimicrobial peptide has made them as an efficient product which can greatly impact the new era of antimicrobials. In addition to this these peptides also demonstrates increased efficacy, high specificity, decreased drug interaction, low toxicity, biological diversity and direct attacking properties. Pharmaceutical industries are therefore conducting appropriate clinical trials to develop these peptides as potential therapeutic drugs. More than 60 peptide drugs have already reached the market and several hundreds of novel therapeutic peptides are in preclinical and clinical development. Rational designing can be used further to modify the chemical and physical properties of existing peptides. This mini review will discuss the sources, mechanism and recent therapeutic applications of antimicrobial peptides in treatment of infectious diseases.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Interações de Medicamentos , Resistência Microbiana a Medicamentos , Fungos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Peso Molecular , Resultado do Tratamento , Vírus/efeitos dos fármacos
10.
J Basic Microbiol ; 60(3): 268-280, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31851769

RESUMO

The sheath blight disease of rice caused by Rhizoctonia solani is widely prevalent and one of the most destructive diseases, affecting rice cultivation and loss worldwide. In the present study, a set of twenty Bacillus isolates from saline soil of Uttar Pradesh were tested for their biocontrol activity against R. solani with the aim to obtain a potential strain for the control of sheath blight disease toward ecofriendly and sustainable agriculture. The results of dual-culture assay and scanning electron microscopic studies showed that the strain RH5 exhibited significant antagonistic activity (84.41%) against the fungal pathogen R. solani. On the basis of 16S rDNA sequencing analysis, the potential biocontrol strain RH5 was identified as Bacillus subtilis. Furthermore, the strain RH5 was characterized by different plant growth-promoting (PGP) activities and induction of defense-related enzymes in rice plants against R. solani. The strain RH5 posses various PGP attributes (indole acetic acid, siderophore, hydrogen cyanide production and phosphate, Zn, K solubility), hydrolytic enzymatic (chitinase, protease, cellulase, xylanase) activity, and presence of antimicrobial peptide biosynthetic genes (bacylisin, surfactin, and fengycin), which support the strain for efficient colonization of hyphae and its inhibition. Finally, the results of the greenhouse study confirmed that strain RH5 significantly increased plant growth and triggered resistance in rice plants through the production of defense-related antioxidant enzymes.


Assuntos
Bacillus subtilis/fisiologia , Agentes de Controle Biológico , Oryza/microbiologia , Doenças das Plantas/microbiologia , Rhizoctonia/fisiologia , Antibiose , Peptídeos Catiônicos Antimicrobianos/genética , Bacillus subtilis/classificação , Bacillus subtilis/isolamento & purificação , Agentes de Controle Biológico/isolamento & purificação , Genes Bacterianos , Oryza/crescimento & desenvolvimento , Filogenia , Desenvolvimento Vegetal , RNA Ribossômico 16S/genética , Rhizoctonia/crescimento & desenvolvimento , Microbiologia do Solo
11.
Exp Parasitol ; 209: 107823, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31862270

RESUMO

Typically, antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. Discovering novel therapeutic agents is considered as an undeniable demand due to increasing microbial species with antibiotic resistance. In this direction, the unique ability of AMPs to modulate immune responses highlighted them as novel drug candidates in the field of microbiology. Patients affected by leishmaniasis; a neglected tropical disease, confront serious problems for their treatment including resistance to common drugs as well as toxicity and high cost of therapy. So, there is a need for development of new drug candidates to control the diseases. Jellein, a peptide derived from royal jelly of honeybee has been shown to have promising effect against several bacterial and fungal species. In current study, anti-leishmanial effect of Jellein and its lauric acid conjugated form was investigated against two forms of Leishmania major (L. major) parasite. Moreover, cytotoxic effect of these peptides was studied in THP1 cell line and human Red Blood Cells (RBCs). Furthermore, the mechanism of action of peptides on L. major promastigotes was assessed through different methods. The results demonstrated that, conjugation of lauric acid to Jellein not only had no effect on the elevation of antimicrobial activity but also halted it completely. Moreover, Jellein caused a limitation in the number of L. major promastigotes by pore formation as well as changing the membrane potential rather than induction of apoptosis or activation of caspases.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Oligopeptídeos/química , Antígenos de Diferenciação de Linfócitos B/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/toxicidade , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Ácidos Graxos/química , Citometria de Fluxo , Hemólise , Antígenos de Histocompatibilidade Classe II/farmacologia , Humanos , Ácidos Láuricos/farmacologia , Ácidos Láuricos/uso terapêutico , Ácidos Láuricos/toxicidade , Leishmania major/ultraestrutura , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/parasitologia , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Oligopeptídeos/toxicidade
12.
Eur J Med Chem ; 185: 111814, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678742

RESUMO

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa, coupled with shrinking antibiotic pipelines, has increased the demand for new antimicrobials with novel mechanisms of action. As the indiscriminate nature of broad-spectrum antimicrobial toxicity may have negative clinical consequences and increase the incidence of resistance, we have developed a P. aeruginosa-selective antimicrobial peptide capable of preferentially killing P. aeruginosa relative to benign microorganisms. A targeting peptide (PA2) that binds specifically to OprF porin on P. aeruginosa was identified by phage display peptide library screening, and a hybrid peptide was constructed by addition of the targeting peptide to GNU7, a potent antimicrobial peptide. The resulting hybrid peptide PA2-GNU7 exhibited potent antimicrobial activity against P. aeruginosa without causing host toxicity. Confocal laser scanning microscopy analysis and time-kill experiments demonstrated that PA2-GNU7 exhibited a high degree of specificity for P. aeruginosa, and rapidly and selectively killed P. aeruginosa cells in mixed cultures. In addition, in vivo treatment efficacy of PA2-GNU7 was significantly greater than that of conventional antibiotics in a mouse model of MDR P. aeruginosa infection. Taken together, the data suggest that PA2-GNU7 may be a promising template for further development as a novel anti-MDR P. aeruginosa therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/citologia , Relação Estrutura-Atividade
13.
Chemistry ; 26(7): 1511-1517, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31867761

RESUMO

Solid-state 19 F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19 F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI)3 are representatives of an α-helix and a ß-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis.


Assuntos
Alcenos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Espectroscopia de Ressonância Magnética , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Flúor/química , Conformação Proteica em alfa-Hélice , Coloração e Rotulagem/métodos
14.
Biosci Biotechnol Biochem ; 84(1): 143-153, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31549575

RESUMO

Brevinin-GR23 (B-GR23) was a brevinin-2 like antimicrobial peptide, which had antimicrobial activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 16 µM. B-GR23 increased the bacterial membrane permeation, leading to the damage of membrane integrity and the leakage of genomic DNA, then causing the cell death. The peptide nearly inhibited all plantonic bacteria to start the initial attachment of biofilm at the concentration of 1 × MIC. Whereas the disruption rates on immature and mature biofilm decreased from 60% to 20%. B-GR23 reduced the production of extracellular polysaccharides (EPS) in the planktonic growth of S. aureus, which is a crucial structure of biofilm formation. B-GR23 with the concentration of ½ × MIC inhibited 50% water-soluble EPS, and 48% water-insoluble EPS, which contributed to the antibiofilm activity. B-GR23 had no significant toxicity to human blood cells under-tested concentration (200 µM), making it a potential template for designing antimicrobial peptides.


Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Polissacarídeos Bacterianos/antagonistas & inibidores , Conformação Proteica em alfa-Hélice , Estabilidade Proteica/efeitos da radiação , Ranidae , Infecções Estafilocócicas/tratamento farmacológico
15.
Arch Insect Biochem Physiol ; 103(1): e21626, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31562754

RESUMO

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Insetos/farmacologia , Spodoptera/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Bacillus subtilis/efeitos dos fármacos , Beauveria/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
16.
Int J Nanomedicine ; 14: 9777-9792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849468

RESUMO

Purpose: Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. Methods: In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. Results: The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. Conclusion: This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Lisostafina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Lipossomos/química , Lipossomos/farmacologia , Lisostafina/genética , Lisostafina/farmacocinética , Camundongos , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Infecções Estafilocócicas/tratamento farmacológico
17.
J Insect Sci ; 19(6)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31865367

RESUMO

Helicobacter pylori (Marshall & Goodwin) is a widespread human pathogen that is acquiring resistance to the antibiotics used to treat it. This increasing resistance necessitates a continued search for new antibiotics. An antibiotic source that shows promise is animals whose immune systems must adapt to living in bacteria-laden conditions by producing antibacterial peptides or small molecules. Among these animals is the black soldier fly (BSF; Hermetia illucens Linnaeus), a Diptera that colonizes decomposing organic matter. In order to find anti-H. pylori peptides in BSF, larvae were challenged with Escherichia coli (Enterobacteriales: Enterobacteriaceae). Small peptides were extracted from hemolymph and purified using solid-phase extraction, molecular weight cutoff filtration and two rounds of preparative high performance liquid chromatography (HPLC). The anti-H. pylori fraction was followed through the purification process using the inhibition zone assay in brain-heart infusion agar, while peptides from uninoculated larvae had no activity. The inhibition halo of the active sample was comparable to the action of metronidazole in the inhibition zone assay. The purified sample contained four peptides with average masses of approximately 4.2 kDa that eluted together when analyzed by HPLC-mass spectrometry. The peptides likely have similar sequences, activity, and properties. Therefore, BSF produces inducible antibacterial peptides that have in vitro activity against H. pylori, which highlights BSF's position as an important target for further bioprospecting.


Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Dípteros/química , Helicobacter pylori , Animais , Bioprospecção , Escherichia coli , Larva/química , Testes de Sensibilidade Microbiana
18.
Nat Commun ; 10(1): 5731, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844052

RESUMO

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , /imunologia , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Genes Bacterianos/genética , Genes Bacterianos/imunologia , Testes de Sensibilidade Microbiana , Mutação
19.
PLoS One ; 14(12): e0227080, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877198

RESUMO

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFß) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Citocinas/imunologia , Orelha Média/imunologia , Otite Média com Derrame/imunologia , Otite Média com Derrame/microbiologia , Bactérias/isolamento & purificação , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Doença Crônica , Estudos de Coortes , Orelha Média/microbiologia , Feminino , Humanos , Lactente , Masculino , Otite Média com Derrame/complicações
20.
J Photochem Photobiol B ; 200: 111645, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31671371

RESUMO

Antimicrobial peptide W3R6 was derived from chensinin-1b and showed potential as a novel antibiotics. However, W3R6 was susceptible to protease cleavage, which limited its therapeutic application. To improve the proteolytic resistance of W3R6, D-amino acids were incorporated into its sequence by specific amino acid substitution or whole sequence substitution according to the specificity of the cleavage site. In this study, partially substituted analog D-Arg-W3R6 and completely substituted D-enantiomer D-W3R6 were synthesized. The resistance of D-Arg-W3R6 and D-W3R6 to cleavage by the tested protease increased, particularly of D-W3R6. The antimicrobial activity of D-Arg-W3R6 was almost the same as that of the parent peptide W3R6, but the antimicrobial activity of D-W3R6 was slightly decreased. The hemolytic activity of both D-Arg-W3R6 and D-W3R6 was negligible. The CD spectrum of D-W3R6 exhibited symmetry with that of W3R6 in a membrane-mimetic environment. The membrane interaction between the D-amino acid substituted analogs and a real/mimic bacterial cell membrane was examined. The outer membrane depolarization, inner membrane permeability and dye leakage in three types of liposomes treated with D-Arg-W3R6 and D-W3R6 were not obviously different from W3R6, which could be due to the similar physical and chemical properties. In addition, these three peptides showed the binding ability with LPS micelles detected by ITC, and their ability to disrupt the LPS micelles was examined by DLS experiment and even neutralize the surface negative charge of E. coli cells. These results suggest that D-Arg-W3R6 is a promising antibiotic molecule.


Assuntos
Aminoácidos/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/metabolismo , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Estabilidade Proteica
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