RESUMEN
Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Modelos Animales de Enfermedad , Hepatocitos/efectos de los fármacos , Hepatocitos/parasitología , Inmunización , Inmunización Pasiva , Estadios del Ciclo de Vida , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Ratones , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas RecombinantesRESUMEN
The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Endotoxinas/biosíntesis , Endotoxinas/genética , Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/genética , Pseudomonas fluorescens/genética , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Endotoxinas/aislamiento & purificación , Fermentación , Expresión Génica , Genes Bacterianos , Proteínas Hemolisinas/aislamiento & purificación , Cuerpos de Inclusión/química , Insectos/patogenicidad , Plantas Modificadas Genéticamente , Plásmidos/genética , Pseudomonas fluorescens/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Zea mays/genética , Zea mays/parasitologíaRESUMEN
BACKGROUND: In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). RESULTS: The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). CONCLUSION: We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.