RESUMO
Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin. To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine. Immunization by the i.p., i.n. and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed. These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.
Assuntos
Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Precursores de Proteínas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Toxinas de Bacillus thuringiensis , Feminino , Proteínas Hemolisinas , Imunidade nas Mucosas , Injeções Intraperitoneais , Intestino Grosso/imunologia , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Reto , Sistema Respiratório/imunologia , Vacinação/métodos , Vagina/imunologiaRESUMO
Bacillus thuringiensis (Bt), considered a safe insecticide, produces insecticidal proteins named Cry during sporulation, which possess exceptional immunological properties. In this work using an immunohistochemical test we demonstrated that Cry1Ac protoxin (pCry1Ac) binds to the mucosal surface of the mouse small intestine. Ligand blot assay allowed us to detect, under denaturing conditions, six pCry1Ac-binding polypeptides present in brush border membrane vesicles isolated from the small intestine. Moreover, this protein induced in situ temporal changes in the electrophysiological properties of the mouse jejunum. The data obtained indicate a possible interaction in vivo of Cry proteins with the animal bowel which could induce changes in the physiological status of the intestine.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Proteínas de Membrana/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Eletrofisiologia , Escherichia coli/metabolismo , Imunofluorescência , Proteínas Hemolisinas , Jejuno/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microvilosidades/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.
Assuntos
Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Imunoglobulina G/biossíntese , Mucosa Intestinal/imunologia , Animais , Anticorpos Antibacterianos/sangue , Toxinas de Bacillus thuringiensis , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Hemolisinas , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins
Assuntos
Animais , Feminino , Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Imunoglobulina G/biossíntese , Mucosa Intestinal/imunologia , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos BALB CRESUMO
The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.
Assuntos
Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Mucosa Intestinal/imunologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/administração & dosagem , Toxina da Cólera/imunologia , Relação Dose-Resposta Imunológica , Endotoxinas/administração & dosagem , Fezes/microbiologia , Feminino , Proteínas Hemolisinas , Imunização , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The Escherichia coli gene gusA was expressed in the methylotrophic yeast Pichia pastoris in a transcriptional fusion to the homologous methanol-inducible AOX1 promoter. Four recombinant clones were selected for expression studies in shake flask conditions and beta-D-glucuronidase (beta-GUS) activity was assayed each 24 h during the induction period. Regardless of the genomic integration patterns and the gene dosage, beta-GUS was functionally expressed and easily detected in all studied clones. The results obtained demonstrate the feasibility of using this bacterial enzyme as a reporter in Pichia pastoris.
Assuntos
Escherichia coli/genética , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Pichia/genética , Southern Blotting , Escherichia coli/enzimologia , Expressão Gênica , Proteínas Mitocondriais , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Pichia/enzimologia , Proteínas de Plantas , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Transformação GenéticaRESUMO
Cry proteins from Bacillus thuringiensis have insecticidal properties. The function of domains I and II has been described but domain III has so far eluded understanding. Domain III from Cry1Ab and Cry1Ac has been cloned, expressed in E. coli and injected to rabbits with the aid of characterizing them immunologically. Interestingly, polyclonal antibodies against Cry1Ab fragment did not recognize either the native Cry1Ab toxin or the Cry1Ac fragment while those against the latter did recognize either the native Cry1Ac toxin or the Cry1Ab protein fragment. A combination of information from sequence comparison and hydrophobicity profile indicates that these protein fragments possibly adopt different spatial dispositions within the respective toxins.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Endotoxinas/química , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Clonagem Molecular , Endotoxinas/genética , Endotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , SolubilidadeRESUMO
Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of delta-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.