Abstract
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.
Highlights
The genes encoding the subtilase cytotoxin (SubAB) are present in various Shiga toxin-producingEscherichia coli (STEC) strains [1,2,3]
Seyahian et al [13] showed that SubAB1 injection in sublethal doses causes severe damages in kidneys, hearts, and livers in rats, which can contribute to hemolytic uremic syndrome (HUS) pathogenesis
SubAB may be considered as a pathogenicity factor, which contributes to the virulence of Shiga toxin-producing Escherichia coli (STEC)
Summary
The genes encoding the subtilase cytotoxin (SubAB) are present in various Shiga toxin-producingEscherichia coli (STEC) strains [1,2,3]. The genes encoding the subtilase cytotoxin (SubAB) are present in various Shiga toxin-producing. Several virulence profiling studies showed that subAB genes are often present in sheep and wild ruminant STEC isolates. SubAB genes are always detected in Shiga toxin (stx)-positive strains. It was hypothesized that the pathogenic function of SubAB is linked to that of Stx [5,11]. Seyahian et al [13] showed that SubAB1 injection in sublethal doses causes severe damages in kidneys, hearts, and livers in rats, which can contribute to HUS pathogenesis. SubAB may be considered as a pathogenicity factor, which contributes to the virulence of STEC
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