Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine

Abulmagd, Shaimaa; Emara, Mohamed; Aziz, Shuokr Qarani; El-Domany, Ramadan A.

doi:10.4103/0255-0857.108702

Cited by 7 publications

(6 citation statements)

References 18 publications

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“…Diphtheria toxin, made naturally by Corynebacterium diphtheria , is a secreted polypetide that is cleaved into A and B fragments upon entry into eukaryotic cells. The B chain is responsible for binding of the toxin to markers present on the cell surface, while the A chain is responsible for the catalytic activity of the toxin and inhibition of protein synthesis in the target cell 4,7 . Notably, DTA released from dead cells is not able to enter bystander cells, due to the lack of the cell-targeting B chain.…”

Section: Discussionmentioning

confidence: 99%

“…One commonly used suicide gene is the catalytic diphtheria toxin fragment A gene (DTA). Diphtheria toxin (DT) is a 62 kDa protein secreted by the gram positive bacillus, Corynebacterium diphtheria 4,5 . The A fragment inhibits eukaryotic translation elongation factor 2 (eEF2) through adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death 4–7 .…”

Section: Introductionmentioning

confidence: 99%

“…Diphtheria toxin (DT) is a 62 kDa protein secreted by the gram positive bacillus, Corynebacterium diphtheria 4,5 . The A fragment inhibits eukaryotic translation elongation factor 2 (eEF2) through adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death 4–7 . Biosynthesis of diphthamide consists of stepwise modifications to His715 by a series of proteins, Dph1-7 7 .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of DTA-Encoding Lentiviruses

Lange

Lyddon

Johnson

2019

Sci Rep

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Suicide genes have been widely investigated for their utility as therapeutic agents and as tools for in vitro negative selection strategies. Several methods for delivery of suicide genes have been explored. Two important considerations for delivery are the quantity of delivered cargo and the ability to target the cargo to specific cells. Delivery using a lentiviral vector is particularly attractive due to the ability to encode the gene within the viral genome, as well as the ability to limit off-target effects by using cell type-specific glycoproteins. Here, we present the design and validation of a diphtheria toxin A (DTA)-encoding lentiviral vector expressing DTA under the control of a constituitive promoter to allow for expression of DTA in a variety of cell types, with specificity provided via selection of glycoproteins for pseudotyping of the lentiviral particles. DTA exerts its toxic activity through inhibition of eukaryotic translation elongation factor 2 (eEF2) via adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death. Thus, we also detail development of DTA-resistant cell lines, engineered through CRISPR/Cas9-mediated knockout of the diphthamide 1 (DPH1) gene, which enable both robust virus production by transfection and evaluation of DTA-expressing virus infectivity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of DTA-Encoding Lentiviruses

Lange

Lyddon

Johnson

2019

Sci Rep

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“…C. diphtheriae Park William strain (ATCC [American Type Culture Collection] 13812) was cultured at 35℃ for 48 hours with shaking at 140 rpm in Linggood medium for vaccine production and source of the dt and dtb genes [ 19 ]. E. coli DH5α and E. coli BL21were used for transformation and protein expression after cloning of the dt and dtb genes using a bacterial expression system pET29a (+) vector (Novagen, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Abulmagd

Khattab

Zedan

2022

Clin Exp Vaccine Res

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Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli , the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

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“…The immunization can trigger an immune response and the formation of human antibodies as protection against diphtheria which is mainly mediated by binding to the B subunit. Mutations and changes in the genetic structure of tox B allow the failure to recognize antibodies that cause diphtheria infection to persist even after vaccination (13)(14)(15). Mutations in the tox B gene can prevent the antitoxin from attaching and lead to failure of the neutralisation process, with eventual cell damage (16).…”

Section: Introductionmentioning

confidence: 99%

Genetic characterization of diphtheria tox B to evaluate vaccine efficacy in Indonesia

Rosana

Lusiana

Yasmon

2022

IJM

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Background and Objectives: Blocking the attachment of diphtheria toxins to host cells through the intact receptor binding site (tox B) was the initial mechanism of action of the diphtheria vaccine. Diphtheria outbreaks in populations with good vaccination coverage can be caused by mutations or changes in the genetic structure of the tox B protein. The aim of this study was to characterize the Tox B protein produced by Corynebacterium diphtheriae isolated from 2018 to 2019 in patients in Jakarta who had already received the diphtheria vaccine. Materials and Methods: Of the 89 throat swab specimens of patients with a clinical diagnosis of diphtheria, 10 were posi- tive for diphtheria and toxin. PCR was used to amplify the tox B DNA fragment in the 10 positive isolates. DNA sequencing was conducted with overlapping primers and the DNA sequences were analysed by using SeqScape V2.7. Results: Of the 10 isolates, nine isolate showed a DNA mutation (G30A), but the mutation did not change the amino acid encoding arginin (silent mutation). Our findings indicate that the efficacy of the diphtheria vaccine used in Indonesia has not decreased because of mutations in the tox B genes not change the amino acid. Conclusion: Overall, there are no amino acid changes in the tox B protein, indicating that the outbreaks are not affected by mutation in tox B. Another possible mechanism – overexpression of the toxin – is likely responsible for causing diphtheria in patients who have a complete history of immunization in Indonesia.

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Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine

Cited by 7 publications

References 18 publications

Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of DTA-Encoding Lentiviruses

Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of DTA-Encoding Lentiviruses

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Genetic characterization of diphtheria tox B to evaluate vaccine efficacy in Indonesia

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