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Toxins 2017, 9(10), 299; https://doi.org/10.3390/toxins9100299

Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

1
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
2
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA
*
Author to whom correspondence should be addressed.
Academic Editors: Emmanuel Lemichez and Michel R. Popoff
Received: 31 August 2017 / Revised: 20 September 2017 / Accepted: 20 September 2017 / Published: 22 September 2017
(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)
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Abstract

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo. View Full-Text
Keywords: membrane translocation; pH-dependent refolding; bilayer insertion; tryptophan fluorescence; depth-dependent fluorescence quenching membrane translocation; pH-dependent refolding; bilayer insertion; tryptophan fluorescence; depth-dependent fluorescence quenching
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Ladokhin, A.S.; Vargas-Uribe, M.; Rodnin, M.V.; Ghatak, C.; Sharma, O. Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain. Toxins 2017, 9, 299.

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