RESUMO
Staphylococcal Enterotoxin B (SEB), produced by Staphylococcus aureus (S. aureus), is a powerful superantigen that induces severe immune disruption and toxic shock syndrome (TSS) upon binding to MHC-II and TCR. Despite its significant impact on the pathogenesis of S. aureus, there are currently no specific therapeutic interventions available to counteract the mechanism of action exerted by this toxin. In this study, we have identified a human monoclonal antibody, named Hm0487, that specifically targets SEB by single-cell sequencing using PBMCs isolated from volunteers enrolled in a phase I clinical trial of the five-antigen S. aureus vaccine. X-ray crystallography studies revealed that Hm0487 exhibits high affinity for a linear B cell epitope in SEB (SEB138-147), which is located distantly from the site involved in the formation of the MHC-SEB-TCR ternary complex. Furthermore, in vitro studies demonstrated that Hm0487 significantly impacts the interaction of SEB with both receptors and the binding to immune cells, probably due to an allosteric effect on SEB rather than competing with receptors for binding sites. Moreover, both in vitro and in vivo studies validated that Hm0487 displayed efficient neutralizing efficacy in models of lethal shock and sepsis induced by either SEB or bacterial challenge. Our findings unveil an alternative mechanism for neutralizing the pathogenesis of SEB by Hm0487, and this antibody provides a novel strategy for mitigating both SEB-induced toxicity and S. aureus infection.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Enterotoxinas , Enterotoxinas/imunologia , Enterotoxinas/antagonistas & inibidores , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Animais , Cristalografia por Raios X , Staphylococcus aureus/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Epitopos de Linfócito B/imunologia , Camundongos , Choque Séptico/imunologia , Choque Séptico/prevenção & controle , Feminino , Leucócitos Mononucleares/imunologia , Vacinas Antiestafilocócicas/imunologia , Anticorpos Antibacterianos/imunologia , Superantígenos/imunologiaRESUMO
Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Simulação por Computador , Enterotoxinas/antagonistas & inibidores , Peptídeos/farmacologia , Toxinas Bacterianas/química , Desenho de Fármacos , Enterotoxinas/química , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Reprodutibilidade dos TestesRESUMO
Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/enzimologia , Infecções por Clostridium/microbiologia , Enterotoxinas/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , Enterotoxinas/química , Enterotoxinas/metabolismo , Humanos , CinéticaRESUMO
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
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
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
Previous epidemiological studies have shown that enterotoxins from enterotoxigenic Escherichia coli (ETEC) appear to be the most important causes of neonatal piglet and porcine post-weaning diarrhoea (PWD). Thus, it is necessary to develop an effective vaccine against ETEC infection. In the present study, the Kil cassette was inserted into the pseudogene yaiT by homologous recombination to create an attenuated E. coli double selection platform O142(yaiT-Kil). After that, PRPL-Kil was replaced with a fusion gene (LTA1-STa13 -STb-LTA2-LTB-STa13 -STb) to establish oral vaccines O142(yaiT::LTA1-STa13 -STb-LTA2-LTB-STa13 -STb) (ER-T). Subsequently, BALB/c mice were orally immunized with ER-T. Results showed that serum IgG and faecal sIgA responded against all ETEC enterotoxins and induced F41 antibody in BALB/c mice by orogastrically inoculation with recombinant E. coli ER-T. Moreover, the determination of cellular immune response demonstrated that the stimulation index (SI) was significantly higher in immunized mice than in control mice, and a clear trend in the helper T-cell (Th) response was Th2-cell (IL-4) exceed Th1-cell (IFN-γ).Our results indicated that recombinant E. coli ER-T provides effective protection against ETEC infection.
Assuntos
Antígenos de Bactérias/imunologia , Diarreia/veterinária , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Doenças dos Suínos/prevenção & controle , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Diarreia/microbiologia , Diarreia/prevenção & controle , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/administração & dosagem , Fezes/química , Imunidade Celular , Imunoglobulina A Secretora/análise , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/microbiologia , Resultado do Tratamento , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
PURPOSE: Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus. METHODOLOGY: S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control. RESULTS: Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents. CONCLUSION: Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production.
Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/biossíntese , Meticilina/farmacologia , Oxazolidinonas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tetrazóis/farmacologia , Animais , Antibacterianos/administração & dosagem , Toxinas Bacterianas/análise , Toxinas Bacterianas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Enterotoxinas/análise , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/biossíntese , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Exotoxinas/análise , Exotoxinas/antagonistas & inibidores , Exotoxinas/biossíntese , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/biossíntese , Humanos , Leucocidinas/análise , Leucocidinas/antagonistas & inibidores , Leucocidinas/biossíntese , Linezolida/administração & dosagem , Linezolida/farmacologia , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Nafcilina/administração & dosagem , Nafcilina/farmacologia , Oxazolidinonas/administração & dosagem , Coelhos , Ovinos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Superantígenos/análise , Superantígenos/biossíntese , Tetrazóis/administração & dosagemRESUMO
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
Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VHH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (KD) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC50 of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.
Assuntos
Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Produtos Biológicos/metabolismo , Desenho de Fármacos , Enterotoxinas/antagonistas & inibidores , Fatores Imunológicos/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Anticorpos Neutralizantes/genética , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Fatores Imunológicos/genética , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/genética , Células VeroRESUMO
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
The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Epitopos/metabolismo , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Agregados Proteicos , Ligação ProteicaRESUMO
Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vß region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Choque Séptico/prevenção & controle , Anticorpos de Cadeia Única/farmacologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Técnicas de Visualização da Superfície Celular , Células Cultivadas , Citocinas/metabolismo , Enterotoxinas/genética , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Mutação , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Choque Séptico/imunologia , Choque Séptico/metabolismo , Choque Séptico/microbiologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Superantígenos/genética , Superantígenos/imunologia , Superantígenos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
BACKGROUND: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host. METHODS: A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed. RESULTS: MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis. CONCLUSIONS: MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Terapia Biológica/métodos , Clostridioides difficile/crescimento & desenvolvimento , Enterocolite Pseudomembranosa/prevenção & controle , Enterotoxinas/antagonistas & inibidores , Microbioma Gastrointestinal , Animais , Toxinas Bacterianas/metabolismo , Células CACO-2 , Clostridioides difficile/isolamento & purificação , Citoesqueleto/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/metabolismo , Fezes/química , Fezes/citologia , Fezes/microbiologia , Fibroblastos/patologia , Humanos , Camundongos Endogâmicos C57BLRESUMO
Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work.
Assuntos
Antidiarreicos/química , Enterotoxinas/metabolismo , Myrtaceae/química , Extratos Vegetais/química , Polifenóis/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Antidiarreicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Enterotoxinas/antagonistas & inibidores , Flavonoides/análise , Frutas/química , Frutas/metabolismo , Ácido Gálico/química , Ácido Gálico/metabolismo , Temperatura Alta , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Myrtaceae/metabolismo , Fenóis/análise , Polifenóis/química , Salmonella typhimurium/efeitos dos fármacosRESUMO
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
Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd.
Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Antitoxinas/uso terapêutico , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/microbiologia , Infecções por Clostridium/terapia , Adenoviridae/genética , Animais , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Modelos Animais de Doenças , Portadores de Fármacos , Avaliação Pré-Clínica de Medicamentos , Enterotoxinas/antagonistas & inibidores , Terapia Genética/métodos , Mesocricetus , Camundongos Endogâmicos C57BL , Suínos , Resultado do TratamentoRESUMO
Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases.