RESUMO
OBJECTIVES: To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. RESULTS: In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. CONCLUSIONS: Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.
Assuntos
Proteínas de Bactérias/imunologia , Clostridium botulinum tipo B/imunologia , Animais , Proteínas de Bactérias/metabolismo , Botulismo/diagnóstico , Botulismo/imunologia , Botulismo/prevenção & controle , Clostridium botulinum/classificação , Clostridium botulinum/imunologia , Clostridium botulinum tipo B/isolamento & purificação , Clostridium botulinum tipo B/metabolismo , Reações Cruzadas , Meios de Cultura/metabolismo , Soros Imunes/imunologia , Camundongos , ProteômicaRESUMO
Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.
Assuntos
Astrócitos/fisiologia , Hipocampo/citologia , Neurogênese/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Astrócitos/ultraestrutura , Clostridium botulinum tipo B/genética , Clostridium botulinum tipo B/metabolismo , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Transportador 1 de Aminoácido Excitatório/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Neurogênese/genética , Neurônios/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Serina/farmacologia , Cloreto de Sódio/farmacologia , Sinapses/genética , Sinapses/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Tamoxifeno/farmacologiaRESUMO
Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.
Assuntos
Toxinas Botulínicas/genética , Botulismo/microbiologia , Clostridium botulinum tipo B/genética , Clostridium botulinum tipo B/patogenicidade , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidade , Clostridium botulinum tipo B/química , Clostridium botulinum tipo B/metabolismo , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Inoculações Seriadas , VirulênciaRESUMO
We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.
