RESUMO
Protective antigen (PA) is the central receptor binding component of anthrax toxin, which translocates catalytic components of the toxin into the cytosol of mammalian cells. Ever since the crystal structure of PA was solved, there have been speculations regarding the possible role of calcium ions present in domain I of the protein. We have carried out a systematic study to elucidate the effect of calcium removal on the structural stability of PA using various optical spectroscopic techniques, limited proteolysis and mutational analysis. Urea denaturation studies clearly suggest that the unfolding pathway of the protein follows a non-two state transition with apo-PA being an intermediate species, whereas the folding pathway shows that calcium ions may be critical for the initial protein assembly.
Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Cálcio/química , Substituição de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Apoproteínas/química , Apoproteínas/genética , Bacillus anthracis/química , Bacillus anthracis/imunologia , Toxinas Bacterianas/genética , Cálcio/metabolismo , Linhagem Celular , Dicroísmo Circular , Immunoblotting , Dose Letal Mediana , Camundongos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Tripsina/química , Ureia/químicaRESUMO
Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.
Assuntos
Antígenos de Bactérias , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , DNA/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Células COS , Citosol/metabolismo , DNA/genética , Proteínas de Ligação a DNA , Escherichia coli/metabolismo , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Anthrax toxin consists of three proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to mammalian cells and is the major component of human anthrax vaccine. Immunization with PA elicits the production of neutralizing antibodies that form a major component of the protective immunity against anthrax. Recent reports have shown that neutralizing antibody titres can serve as a reliable surrogate marker for protection against anthrax. In the present study, the use of non-invasive routes such as bare skin and nose for immunization with PA on its protective immune response was investigated. Mice were inoculated intranasally (i.n.), subcutaneously (s.c.) or through the skin on days 0, 15 and 28 with purified PA. Intranasal and subcutaneous immunization with PA resulted in high IgG ELISA titers. The predominant subclass in each group was IgG1. High titres of IgA were observed only in i.n. immunized mice. In a cytotoxicity assay these sera protected J774A.1 cells from lethal toxin challenge. The results suggest that non-invasive nasal immunization may be useful in improving vaccination strategies against anthrax.
Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Administração Intranasal , Animais , Vacinas contra Antraz/imunologia , Bacillus anthracis/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Camundongos , Testes de NeutralizaçãoRESUMO
Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.