Article ; Online: Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not.
PloS one
2019 Volume 14, Issue 3, Page(s) e0214313
Abstract: The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, ...
| Abstract | The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays-the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays. |
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| MeSH term(s) | Bacterial Toxins/genetics ; Bacterial Toxins/metabolism ; Biological Assay/methods ; DNA Damage/drug effects ; Escherichia coli/metabolism ; Haemophilus ducreyi/metabolism ; HeLa Cells ; Humans ; Mutagenesis ; Plasmids/metabolism ; Protein Subunits/genetics ; Protein Subunits/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/pharmacology ; Transfection |
| Chemical Substances | Bacterial Toxins ; Protein Subunits ; Recombinant Proteins ; cytolethal distending toxin |
| Language | English |
| Publishing date | 2019-03-28 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, Non-U.S. Gov't |
| ISSN | 1932-6203 |
| ISSN (online) | 1932-6203 |
| DOI | 10.1371/journal.pone.0214313 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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