RESUMO
Staphylococcal enterotoxin B (SEB), one of the exotoxins produced by Staphylococcus aureus, is the key toxin that causes poisoning reactions and toxic shock syndrome. In the current research work, a novel human antibody named LXY8 was screened from a human phage display antibody library, and LXY8 blocked the interaction between SEB and the T cell receptor (TCR). The binding activity between LXY8 and SEB was 0.525 nM. Furthermore, LXY8 could effectively inhibit the SEB-induced activation of peripheral blood mononuclear cells and release of cytokines. In the BALB/c mouse model, LXY8 effectively neutralized SEB toxicity in vivo. Finally, based on computer-guided molecular modeling, we designed a series of SEB mutation sites; these sites facilitated the determination of the key residues (i.e.176EFNN179) of SEB recognized by LXY8. The research revealed that the 176EFNN179 residues of SEB are important for specific antibody-antigen recognition. The results may be helpful for the development of antibody-based therapy for SEB-induced toxic shock syndrome.
Assuntos
Anticorpos Antibacterianos/análise , Anticorpos Monoclonais/análise , Anticorpos Neutralizantes/análise , Enterotoxinas/imunologia , Epitopos/imunologia , Animais , Células CHO , Proliferação de Células , Técnicas de Visualização da Superfície Celular , Cricetulus , Citocinas/metabolismo , Enterotoxinas/antagonistas & inibidores , Mapeamento de Epitopos , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Guanylate cyclase 2C (GC-C), encoded by the GUCY2C gene, is implicated in hereditary early onset chronic diarrhea. Several families with chronic diarrhea symptoms have been identified with autosomal dominant, gain-of-function mutations in GUCY2C. We have identified a Mennonite patient with a novel GUCY2C variant (c.2381A > T; p.Asp794Val) with chronic diarrhea and an extensive maternal family history of chronic diarrhea and bowel dilatation. Functional studies including co-segregation analysis showed that all family members who were heterozygous for this variant had GI-related symptoms. HEK-293 T cells expressing the Asp794Val GC-C variant showed increased cGMP production when stimulated with Escherichia coli heat-stable enterotoxin STp (HST), which was reversed when 5-(3-Bromophenyl)-5,11-dihydro-1,3-dimethyl-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (BPIPP; a GC-C inhibitor) was used. In addition, cystic fibrosis transmembrane conductance regulator (CFTR) activity measured with SPQ fluorescence assay was increased in these cells after treatment with HST, indicating a crucial role for CFTR activity in the pathogenesis of this disorder. These results support pathogenicity of the GC-C Asp794Val variant as a cause of chronic diarrhea in this family. Furthermore, this work identifies potential candidate drug, GC-C inhibitor BPIPP, to treat diarrhea caused by this syndrome.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diarreia/genética , Predisposição Genética para Doença , Receptores de Enterotoxina/genética , Adolescente , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/genética , Criança , Diarreia/tratamento farmacológico , Diarreia/patologia , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Feminino , Mutação com Ganho de Função/genética , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Linhagem , Adulto JovemRESUMO
Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/patogenicidade , Infecção Hospitalar/tratamento farmacológico , Enterocolite Pseudomembranosa/tratamento farmacológico , Enterotoxinas/antagonistas & inibidores , ADP-Ribosilação/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/deficiência , Actinas/genética , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Infecção Hospitalar/metabolismo , Infecção Hospitalar/microbiologia , Infecção Hospitalar/patologia , Endocitose/efeitos dos fármacos , Enterocolite Pseudomembranosa/metabolismo , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de ProteínaRESUMO
A polymer based dynamic combinatorial library (DCL) was generated through condensation between aldehyde functionalized linear poly(glycidol) (APG) and galactose containing acylhydrazide derivatives. Pentameric E. coli heat labile enterotoxin B subunit (LTB) was subsequently applied to the DCL as external stimulus, resulting in amplification of a specific acylhydrazone side chain that was further used for the synthesis of a multivalent LTB inhibitor. In the in vitro biological evaluation, this inhibitor exhibited strong inhibition properties as well as low cytotoxicity.
Assuntos
Aldeídos/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Técnicas de Química Combinatória , Enterotoxinas/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Galactose/farmacologia , Hidrazinas/farmacologia , Propilenoglicóis/farmacologia , Aldeídos/química , Toxinas Bacterianas/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Galactose/química , Humanos , Hidrazinas/química , Estrutura Molecular , Propilenoglicóis/química , Relação Estrutura-AtividadeRESUMO
Staphylococcal enterotoxin A (SEA) is an enterotoxin produced mainly by Staphylococcus aureus. In recent years, it has become the most prevalent compound for staphylococcal food poisoning (SFP) around the world. In this study, we isolate new dual-function single-stranded DNA (ssDNA) aptamers by using some new methods, such as the Taguchi method, by focusing on the detection and neutralization of SEA enterotoxin in food and clinical samples. For the asymmetric polymerase chain reaction (PCR) optimization of each round of systematic evolution of ligands by exponential enrichment (SELEX), we use Taguchi L9 orthogonal arrays, and the aptamer mobility shift assay (AMSA) is used for initial evaluation of the protein-DNA interactions on the last SELEX round. In our investigation the dissociation constant (KD) value and the limit of detection (LOD) of the candidate aptamer were found to be 8.5⯱â¯0.91 of nM and 5â¯ng/ml using surface plasmon resonance (SPR). In the current study, the Taguchi and mobility shift assay methods were innovatively harnessed to improve the selection process and evaluate the protein-aptamer interactions. To the best of our knowledge, this is the first report on employing these two methods in aptamer technology especially against bacterial toxin.
Assuntos
Aptâmeros de Nucleotídeos/química , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/análise , Análise de Alimentos/métodos , Ressonância de Plasmônio de Superfície , Humanos , Técnica de Seleção de AptâmerosRESUMO
The discovery, synthesis and preliminary structure-activity relationship (SAR) of a novel class of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB) is described. A high throughput screening (HTS) campaign resulted in the identification of moderately active screening hits 1-5 the most potent of which was compound 1 (IC50â¯=â¯0.77⯵M). In silico docking of an early analog offered suggestions for structural modification which resulted in the design and synthesis of highly potent analogs 13j(IC50â¯=â¯1â¯nM) and 13â¯l(IC50â¯=â¯7â¯nM) which were chosen as leads for further optimization.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/efeitos dos fármacos , Nucleotidases/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacocinética , Apoptose/efeitos dos fármacos , Células CHO , Cricetulus , Estabilidade de Medicamentos , Enterotoxinas/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Many reports have shown that crude extracts of the American cockroach have therapeutic effects on inflammation. In a previous study, our research group showed that an antimicrobial peptide (Periplanetasin-2) derived from the American cockroach via de novo transcriptome analysis inhibited apoptosis of human colonocytes and inflammatory responses of the mouse gut caused by Clostridium difficile toxin A. Here, we examined whether Periplanetasin-4 (Peri-4), another antimicrobial peptide identified via de novo transcriptome analysis of the American cockroach, could also inhibit the various toxicities induced by C. difficile toxin A. We found that Peri-4 significantly reduced the cell viability loss and cell apoptosis caused by toxin A in vitro. Peri-4 also ameliorated the severe inflammatory responses seen in the toxin A-induced mouse enteritis model, rescuing the villus disruption and interleukin-6 production induced by luminal injection of toxin A into the mouse gut. Mechanistically, we found that Peri-4 could reduce toxin A-induced reactive oxygen species production to inhibit the activations of p38MAPK and p21Cip1/Waf1 , which are critical for the cell damages induced by toxin A. These results collectively suggest that the Peri-4 may be a potential therapeutic agent for treating toxin A-induced pseudomembranous colitis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Anti-Inflamatórios/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Enterite/tratamento farmacológico , Enterotoxinas/antagonistas & inibidores , Proteínas de Insetos/farmacologia , Animais , Toxinas Bacterianas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Enterite/imunologia , Enterite/metabolismo , Enterotoxinas/farmacologia , Células HT29 , Humanos , Íleo/efeitos dos fármacos , Íleo/imunologia , Íleo/patologia , Camundongos , Periplaneta/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-ß chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.
Assuntos
Anticorpos Monoclonais/imunologia , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/uso terapêutico , Cristalografia por Raios X , Enterotoxinas/química , Mapeamento de Epitopos , Epitopos/imunologia , Imunoterapia , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Infecções Estafilocócicas/terapia , Staphylococcus aureus/química , Superantígenos/químicaRESUMO
Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.
Assuntos
Antibacterianos/efeitos adversos , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Antitoxinas/farmacologia , Infecções por Clostridium/tratamento farmacológico , Vancomicina/efeitos adversos , Animais , Antibacterianos/administração & dosagem , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/biossíntese , Anticorpos Amplamente Neutralizantes , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/patogenicidade , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/mortalidade , Convalescença , Cricetulus , Modelos Animais de Doenças , Progressão da Doença , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/biossíntese , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Vancomicina/administração & dosagemRESUMO
3,39-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-g secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Apoptose/efeitos dos fármacos , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/toxicidade , Indóis/farmacologia , MicroRNAs/biossíntese , Linfócitos T/efeitos dos fármacos , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Síndrome de Vazamento Capilar/patologia , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Citocinas/sangue , Citocinas/metabolismo , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infiltração de Neutrófilos/efeitos dos fármacosRESUMO
Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.
Assuntos
Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Infecções por Clostridium/microbiologia , Enterotoxinas/metabolismo , Animais , Anticorpos Neutralizantes , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/genética , Chlorocebus aethiops , Clostridioides difficile/classificação , Clostridioides difficile/genética , Cricetinae , Modelos Animais de Doenças , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/genética , Deleção de Genes , Células HT29 , Humanos , Testes de Neutralização , Células Vero , Virulência/genéticaRESUMO
BACKGROUND: Recent studies have revealed that Staphylococcus aureus and its components participate in the pathogenesis of eosinophilic airway diseases, such as chronic rhinosinusitis with nasal polyps. OBJECTIVE: We sought to determine whether staphylococcal protein A (SpA) from S aureus regulated cellular responses in nasal polyps, especially when coupled to immunoglobulins in immune complexes (ICs). METHODS: Dispersed nasal polyp cells (DNPCs) or peripheral blood monocytes were cultured in vitro with SpA in the presence or absence of IgG, and IL-5, IL-13, IFN-γ, IL-17A, and IL-10 levels were measured in the supernatants. The effect of SpA exposure on staphylococcal enterotoxin B-induced cytokine production by DNPCs in the presence and absence of IgG, IgA, and autologous serum was also examined. RESULTS: Exposure to SpA induced DNPCs to produce significantly higher IL-10, IL-13, and IL-17A levels than DNPCs without SpA, although the magnitude of the IL-17A increase was less than that of IL-10 and IL-13. SpA induced IL-10 production mainly from adherent DNPCs, and this was significantly enhanced in the presence of IgG; similar results were observed in peripheral blood monocytes. IC formation between SpA and IgG (SpA-IgG ICs) was confirmed by using native polyacrylamide gel electrophoresis. SpA-IgG ICs, but not SpA alone, almost completely suppressed staphylococcal enterotoxin B-induced IL-5, IL-13, IFN-γ, and IL-17A production by DNPCs; similar inhibition was observed in DNPCs treated with SpA in the presence of either IgA or autologous serum. CONCLUSIONS: Our results suggest that SpA can regulate the pathogenesis of enterotoxin-induced inflammation in patients with chronic rhinosinusitis with nasal polyps through coupling to immunoglobulins.
Assuntos
Complexo Antígeno-Anticorpo/biossíntese , Enterotoxinas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Proteína Estafilocócica A/farmacologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Enterotoxinas/antagonistas & inibidores , Feminino , Humanos , Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Cavidade Nasal/imunologia , Cavidade Nasal/patologia , Cavidade Nasal/cirurgia , Pólipos Nasais/complicações , Pólipos Nasais/patologia , Pólipos Nasais/cirurgia , Cultura Primária de Células , Rinite/complicações , Rinite/patologia , Rinite/cirurgia , Sinusite/complicações , Sinusite/patologia , Sinusite/cirurgiaRESUMO
BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Jejuno/efeitos dos fármacos , Piperidinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/antagonistas & inibidores , Receptores de Peptídeos/antagonistas & inibidores , Adenilil Ciclases/metabolismo , Adulto , Animais , Toxinas Bacterianas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia , Escherichia coli Enterotoxigênica , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Jejuno/citologia , Jejuno/metabolismo , Modelos Biológicos , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Adulto JovemRESUMO
Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.
Assuntos
Anticorpos Antibacterianos/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/química , Enterotoxinas/química , Epitopos/química , Anticorpos de Cadeia Única/química , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Clostridioides difficile/genética , Clostridioides difficile/imunologia , Cristalografia por Raios X , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/genética , Enterotoxinas/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Estrutura Terciária de Proteína , Anticorpos de Cadeia Única/imunologia , Relação Estrutura-AtividadeRESUMO
Both the endogenous antisecretory factor (AF) protein and peptide AF-16, which has a sequence that matches that of the active N-terminal region of AF, inhibit the increase in the epithelial transport of fluid and electrolytes induced by bacterial toxins in animal and ex vivo models. We conducted a study to investigate the inhibitory effect of peptide AF-16 against the increase of transcellular passage and paracellular permeability promoted by the secreted autotransporter toxin (Sat) in a cultured cellular model of the human intestinal epithelial barrier. Peptide AF-16 produced a concentration-dependent inhibition of the Sat-induced increase in the formation of fluid domes, in the mucosal-to-serosal passage of D-[1-(14)C]mannitol, and in the rearrangements in the distribution and protein expression of the tight junction (TJ)-associated proteins ZO-1 and occludin in cultured human enterocyte-like Caco-2/TC7 cell monolayers. In addition, we show that peptide AF-16 also inhibits the cholera toxin-induced increase of transcellular passage and the Clostridium difficile toxin-induced effects on paracellular permeability and TJ protein organization in Caco-2/TC7 cell monolayers. Treatment of cell monolayers by the lipid raft disorganizer methyl-ß-cyclodextrin abolished the inhibitory activity of peptide AF-16 at the transcellular passage level and did not modify the effect of the peptide at the paracellular level.
Assuntos
Toxina da Cólera/antagonistas & inibidores , Enterócitos/efeitos dos fármacos , Enterotoxinas/antagonistas & inibidores , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Radioisótopos de Carbono , Toxina da Cólera/toxicidade , Enterócitos/citologia , Enterócitos/metabolismo , Enterotoxinas/toxicidade , Humanos , Cinética , Manitol/metabolismo , Dados de Sequência Molecular , Neuropeptídeos/química , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a "double-hit" mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS.
Assuntos
Enterotoxinas/antagonistas & inibidores , Técnica de Seleção de Aptâmeros , Animais , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Biologia Computacional , Citocinas/biossíntese , Avaliação Pré-Clínica de Medicamentos , Feminino , Galactosamina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Choque Séptico/tratamento farmacológico , Choque Séptico/microbiologiaRESUMO
BACKGROUND: Clostridium difficile infection (CDI) is mediated by potent extracellular toxins and is spread largely via bacterial spores. We and others have shown that some antibiotics stimulate C. difficile toxin production in a strain-specific manner; however, the effects of newer anti-C. difficile antibiotics on this process remain to be investigated. METHODS: The effects of the protein synthesis inhibitor tigecycline on sporulation and toxin A and toxin B production were compared in historical (strain 9689) and hypervirulent BI/NAP1/027 (strain 5325) isolates of C. difficile in vitro. RESULTS: Tigecycline at 1/4× MIC stimulated an increased and earlier toxin A and/or B gene expression in both the historical and the hypervirulent strains, although a commensurate increase in toxin protein production was observed only in the 9689 strain. In fact, in the hypervirulent 5325 strain, toxin production was dramatically suppressed. By comparison, subinhibitory concentrations of vancomycin and metronidazole also stimulated increased protein toxin production by the historical, but not the hypervirulent, strain. In addition, tigecycline dose-dependently reduced viable spore production by both the 9689 and 5325 strains. Vancomycin treatment also suppressed spore formation in both C. difficile strains; however, metronidazole, while reducing spore formation in the 9689 strain, stimulated a near 2 log increase in spore production by the 5325 isolate. CONCLUSIONS: In summary, these findings suggest that the treatment of CDI patients with tigecycline could effectively both control disease progression and limit its spread by disrupting sporulation.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/efeitos dos fármacos , Enterotoxinas/antagonistas & inibidores , Minociclina/análogos & derivados , Esporos Bacterianos/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Minociclina/farmacologia , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , TigeciclinaRESUMO
Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Diterpenos/metabolismo , Enterotoxinas/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Exotoxinas/antagonistas & inibidores , Proteínas Hemolisinas/antagonistas & inibidores , Staphylococcus aureus/metabolismo , Abietanos , Animais , Western Blotting , Linhagem Celular , Hemólise , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.
Assuntos
Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Enterocolite Pseudomembranosa/tratamento farmacológico , Oxazolidinonas/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Acetamidas/farmacologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/biossíntese , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/metabolismo , Infecções por Clostridium/microbiologia , Infecções por Clostridium/mortalidade , Cricetinae , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/biossíntese , Feminino , Fluoroquinolonas/farmacologia , Humanos , Linezolida , Masculino , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Esporos Bacterianos/crescimento & desenvolvimento , Análise de Sobrevida , Vancomicina/farmacologiaRESUMO
Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 µM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1ß (IL-1ß) protein and mRNA expression. Treatment with fidaxomicin (20 µM) or its primary metabolite, OP-1118 (120 µM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1ß protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 µM) and metronidazole (20 µM) did not alter toxin A-induced histologic damage and IL-1ß protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.