RESUMO
A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and ß-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity.
Assuntos
Clostridioides difficile/química , Clostridioides difficile/genética , Engenharia de Proteínas/métodos , Toxoides/química , Toxoides/genética , Animais , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Vacinas Bacterianas , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Enterotoxinas/química , Epitopos , Etildimetilaminopropil Carbodi-Imida/química , Imunização , Mesocricetus , Proteínas Recombinantes , Succinimidas/química , Ressonância de Plasmônio de SuperfícieRESUMO
Staphylococcus aureus and other staphylococci cause severe human disease, and there are currently no vaccines available. We evaluated whether manganese transport protein C (MntC), which is conserved across the staphylococcal species group, could confer protection against S. aureus and Staphylococcus epidermidis. In vivo analysis of S. aureus MntC expression revealed that expression occurs very early during the infectious cycle. Active immunization with MntC was effective at reducing the bacterial load associated with S. aureus and S. epidermidis infection in an acute murine bacteremia model. Anti-MntC monoclonal antibodies have been identified that can bind S. aureus and S. epidermidis cells and are protective in an infant rat passive protection model and induce neutrophil respiratory burst activity. This is the first description of a protein that has the potential to provide protection across the staphylococcal species group.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Proteínas de Membrana/imunologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Bacteriemia/imunologia , Bacteriemia/prevenção & controle , Bacteriemia/terapia , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Modelos Animais de Doenças , Feminino , Imunização Passiva , Proteínas de Membrana/genética , Camundongos , Coelhos , Ratos , Ratos Sprague-Dawley , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/terapia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Resultado do TratamentoRESUMO
An efficient three-step construction of the highly oxygenated D-ring of the kinamycin antibiotics is reported for a simple model system. A comparison of the spectroscopic characteristics of the synthetic models with those of natural kinamycin F, which is suspected to be the bioactive form of the kinamycins, leads to the conclusion that the favored D-ring conformation of kinamycin F differs from that of the other partially or fully acylated variants.
Assuntos
Antibacterianos/química , Antibacterianos/síntese química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Quinonas/síntese química , Quinonas/química , Streptomyces/químicaRESUMO
Inhibition of translation by small molecule ligands has proven to be a useful tool for understanding this complex cellular mechanism, as well as providing drugs of significant medical importance. Many small molecule ligands inhibit translation by binding to RNA or RNA/protein components of the ribosomal subunits and usurping their function. A class of peptidomimetics [aminoglycoside-arginine conjugates (AAC)] has recently been designed to inhibit HIV TAR/tat interaction and in experiments aimed at assessing the inhibitory effects of AACs on TAR-containing transcripts, we found that AACs are general inhibitors of translation. Experiments reported herein aim at characterizing these novel properties of AACs. We find that AACs are inhibitors of eukaryotic and prokaryotic translation and exert their effects by blocking peptide chain elongation. Structure/activity relationship studies suggest that inhibition of translation by AACs is directly related to the number of arginine groups present on the aminoglycoside backbone and to the nature of the core aminoglycoside. AACs are therefore attractive tools for understanding and probing ribosome function.