ABSTRACT
Subunit B of diphtheria toxin (DT), which consists of two domains: R (receptor-binding) and T (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit A to the cell cytosol. Recombinant analogues of the subunit B are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. In the development of these protein constructs understanding of the role of each of the DT fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required. We have studied in this work the T-domain effect on intracellular transport of recombinant fragments of DT. We have compared intracellular transport of the R-domain and the subunit B, the last one consisted of both R-domain and T-domain. Recombinant fragments of DT used in this work were labeled with fluorescent proteins, which allowed applying colocalization technique for our study. Application of confocal microscopy technique revealed differences in transportation of recombinant derivates of DT in Vero cells: R-domain moved faster than subunit B to tubular compartments. Analysis of R-domain and subunit B transportation confirmed almost linear increase of their colocalization with the time regarding to Pearsons correlation coefficient (PCC). However, amount of colocalized with R-domain subunit B were not linearly increased with time according to Manders coefficient (M1), this could indicate the ability of subunit B to transport to such compartments that R-domain do not reach. Possible role of the T-domain in intracellular transportation and compartmentalization of the toxin may be associated with the ability of the T-domain to form a proton channels and its ability to interact with COPI complex.
Subject(s)
Cell Nucleus/metabolism , Cytosol/metabolism , Diphtheria Toxin/metabolism , Mutant Chimeric Proteins/metabolism , Peptide Fragments/metabolism , Protein Subunits/metabolism , Animals , Binding Sites , Chlorocebus aethiops , Diphtheria Toxin/chemistry , Diphtheria Toxin/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Mutant Chimeric Proteins/chemistry , Mutant Chimeric Proteins/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Binding , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Transport , Regression Analysis , Vero Cells , Red Fluorescent ProteinABSTRACT
Diphtheria toxin is the main pathogenicity factor of causative agent of diphtheria Corynebacterium diphtheriae. Due to the small molecule size, it is of considerable interestfor the development of synthetic protein molecules with transporting function, e.g. immunotoxins. Expression and characterization of nontoxic recombinant fluorescent derivates of diphtheria toxin and its nontoxic mutant CRM 197 were described in this article. Obtained proteins may be applied in studies of receptor-binding and transporting functions of the toxin in cells, for determination of toxin receptor proHB-EGF expression level, immunization and antibody generation against the toxin and in development of diagnostic test-systems, detection of diphtheria toxin and antitoxic antibodies.
Subject(s)
Diphtheria Toxin/genetics , Luminescent Proteins/genetics , Protein Engineering , Recombinant Proteins/genetics , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/immunology , Cloning, Molecular , Corynebacterium diphtheriae/genetics , Corynebacterium diphtheriae/pathogenicity , Diphtheria Toxin/chemistry , Humans , Immunization , Luminescent Proteins/biosynthesis , Luminescent Proteins/chemistry , Models, Molecular , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistryABSTRACT
Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Cattle Diseases/diagnosis , Immunoenzyme Techniques/statistics & numerical data , Mycobacterium bovis/immunology , Statistical Distributions , Tuberculosis, Bovine/diagnosis , Animals , Cattle , Cattle Diseases/microbiology , Immunoenzyme Techniques/methods , Recombinant Proteins/immunology , Tuberculosis, Bovine/microbiologyABSTRACT
The recombinant fluorescent derivative of diphtheria toxin (EGFP-SbB) obtained by the replacement of toxin A subunit by enhanced green fluorescent protein (EGFP) has been used for visualization of the interaction of diphtheria toxin (DT) with sensitive and insensitive cells. It was shown that EGFP-SbB could interact with cell surface of both toxin-sensitive monkey cells (Vero cell line) and toxin-resistant mouse cells (3T3 cell line). The affinity of this protein for receptors of Vero cells was three times higher as compared with 3T3 cells. It was demonstrated that fluorescent derivate was able to interact with receptors of both cell lines and to internalize into these cells. Internalization of EGFP-SbB into the cells was inhibited by endocytosis inhibitor phenyl arsine oxide. We suppose that diverse sensitivity to DT of monkey and mouse cells can be explained not only by differences in their receptor affinity for DT but also by the processes that occur after internalization of the toxin into the cells.
Subject(s)
Cell Membrane/metabolism , Diphtheria Toxin/metabolism , Host Specificity , Intercellular Signaling Peptides and Proteins/metabolism , Recombinant Fusion Proteins/metabolism , 3T3 Cells , Animals , Arsenicals/pharmacology , Chlorocebus aethiops , Cloning, Molecular , Diphtheria Toxin/chemistry , Diphtheria Toxin/genetics , Endocytosis/drug effects , Enzyme Inhibitors/pharmacology , Escherichia coli , Flow Cytometry , Fluorescence , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heparin-binding EGF-like Growth Factor , Kinetics , Mammals , Mice , Microscopy, Confocal , Recombinant Fusion Proteins/genetics , Vero CellsABSTRACT
Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.
Subject(s)
Diphtheria Toxin/metabolism , Green Fluorescent Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Binding, Competitive , Cell Culture Techniques , Chlorocebus aethiops , Diphtheria Toxin/genetics , Flow Cytometry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Genetic Vectors , Green Fluorescent Proteins/genetics , Heparin-binding EGF-like Growth Factor , Microscopy, Confocal , Peptide Fragments/genetics , Peptide Fragments/metabolism , Plasmids , Protein Binding , Recombinant Fusion Proteins/genetics , Vero CellsABSTRACT
Development of complications during diphtheria depends to a large extent on toxin-neutralizing antibodies level in the patient's blood. Active immunization of people with diphtheria anatoxin is widely used for diphtheria prevention and passive immunization with hyperimmune antitoxic horse serum is used for diphtheria treatment. A traditional component of anti-diphtheria vaccines--diphtheria anatoxin has a number of serious disadvantages, which are mainly associated with complicated procedure of its production. Thus, the search for new antigen substances, which can effectively stimulate protective humoral response to diphtheria toxin, is an urgent task in anti-diphtheria vaccine development. Furthermore, one of the most important objects is the development of new in vitro methods for estimation of diphtheria toxin-neutralizing polyclonal and monoclonal antibodies, which allow to avoid using active diphtheria toxin and toxin-sensitive laboratory animals. Comparative studies of toxin-neutralizing antibodies induction after immunization of laboratory animals with recombinant subunits A and B of diphtheria toxin were carried out. The new method for detection of protective antibodies in serum was proposed. This method is based on the ToBI test (Toxin Binding Inhibition test); namely on the property of anti-diphtheria antibodies to inhibit the biding of toxin subunit B fused with enhanced green fluorescent protein (EGFP) to the sensitive to diphtheria toxin Vero cells. The ability of subunit B to induce toxin-neutralizing antibodies in laboratory animals (rabbits and guinea pigs) was confirmed by the intradermal test, which is traditionally used to detect protective antitoxic antibodies in the serum, and by flow cytometry method, developed for this purpose. The results suggest that diphtheria toxin recombinant subunit B may be used for the induction of the protective immune response. The new developed approach for estimation diphtheria toxin-neutralizing antibodies is more ethical and safe and can substitute successfully the traditional methods.
Subject(s)
Antibodies, Bacterial/immunology , Diphtheria Toxin/immunology , Peptide Fragments/immunology , Recombinant Proteins/immunology , Animals , Antibodies, Bacterial/blood , Cell Culture Techniques , Chlorocebus aethiops , Diphtheria Toxoid/immunology , Escherichia coli/genetics , Flow Cytometry , Green Fluorescent Proteins/genetics , Guinea Pigs , Immunization/methods , Immunoenzyme Techniques , Rabbits , Recombinant Proteins/biosynthesis , Skin Tests , Vero CellsABSTRACT
The aim of this work was to obtain the panel of recombinant single-chain Fv-antibodies against diphtheria toxin B subunit, the main diagnostic and pathogenic antigen of Corynebacterium diphtheriae. For this purpose we have constructed the immune library of murine immunoglobulin genes. A number of scFv specific to diphtheria toxin B subunit were acquired from the obtained library after one round of selection by phage-display. ScFv encoding DNA-fragments of eight clones were subcloned into plasmid pET-22b(+). It was shown that selected scFv were highly specific to diphtheria toxin B subunit, with affinity constant for different clones ranged from 10(7) to 10(9) M(-1).
Subject(s)
Diphtheria Toxin/immunology , Peptide Library , Single-Chain Antibodies/genetics , Animals , Antibody Affinity , Antibody Specificity , Cells, Cultured , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/immunology , Diphtheria/diagnosis , Diphtheria/immunology , Escherichia coli/genetics , Female , Mice , Mice, Inbred BALB C , Protein Subunits , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Single-Chain Antibodies/immunology , Single-Chain Antibodies/isolation & purification , Spleen/cytology , Spleen/immunologyABSTRACT
The B subunit of diphtheria toxin (DT) is responsible for interaction with receptor on the cell surface and translocation of the catalytically active A subunit across endosomal membrane into the cell cytosole. Receptor for DT and its B subunit is membrane-anchored precursor of heparin-binding epidermal growth factor-like growth factor (pro-HB-EGF), which under the action of metalloproteases turns into soluble form (sHB-EGF), which acts as a potent mitogen for different cell types. Since free B subunit of DT has no catalytic activity it is considered to be nontoxic. However its influence on the cells in vitro remains to be investigated. The aim of this study was to examine the influence of diphtheria toxin B subunit on viability of diphtheria-sensitive cells using B subunit recombinant analogues. It was shown that diphtheria toxin B subunit recombinant analogue at a concentration of 12.8 x 10(-7) M had a cytotoxic effect on the human histocytic lymphoma cell line U937, which expresses a large amount of sHB-EGF. Besides, the similar cytotoxic effect had a fusion protein which consisted of a B subunit and an enhanced green fluorescent protein (EGFP). However recombinant EGFP alone didnot influence the cell viability. Annexin-V-FITC/PI staining demonstrated that maximal cytotoxic effect had been elicited after 48 hours of cultivation. Cytotoxic test with trypan blue and propidium iodide staining excluded the direct influence of investigated proteins on the integrity of plasma membrane because of the ability of B subunit to pore formation. So, we offer a hypothesis that realization of cytotoxic effect of diphtheria toxin B subunit and its derivative on the U937 cell culture occurs via inhibition of mitogenic activity of sHB-EGF resulted in induction of apoptosis.
Subject(s)
Diphtheria Toxin/pharmacology , Cell Culture Techniques , Cell Survival/drug effects , Diphtheria Toxin/genetics , Diphtheria Toxin/metabolism , Dose-Response Relationship, Drug , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/pharmacology , Heparin-binding EGF-like Growth Factor , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , U937 CellsABSTRACT
Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.
