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Open AccessArticle

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro

1
Department of Food Microbiology and Hygiene, Institute of Food Science and Biotechnology, Garbenstraße 28, University of Hohenheim, 70599 Stuttgart, Germany
2
Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, Germany
3
Center for Integrated Protein Science Munich, Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85748 Garching, Germany
*
Author to whom correspondence should be addressed.
Toxins 2019, 11(12), 703; https://doi.org/10.3390/toxins11120703
Received: 12 August 2019 / Revised: 19 November 2019 / Accepted: 27 November 2019 / Published: 3 December 2019
(This article belongs to the Section Bacterial Toxins)
The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells. View Full-Text
Keywords: subtilase cytotoxin; AB5 toxin; complex formation; STEC; analytical ultracentrifugation; isothermal titration calorimetry; flow cytometry subtilase cytotoxin; AB5 toxin; complex formation; STEC; analytical ultracentrifugation; isothermal titration calorimetry; flow cytometry
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Krause, M.; Sessler, K.; Kaziales, A.; Grahl, R.; Noettger, S.; Barth, H.; Schmidt, H. Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro. Toxins 2019, 11, 703.

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