Structural Basis of Subtilase Cytotoxin SubAB Assembly

Nours, Jérôme Le; Paton, Adrienne W.; Byres, E.; Troy, S.; Herdman, Brock P; Johnson, Matthew D.; Paton, James C.; Rossjohn, Jamie; Beddoe, Travis

doi:10.1074/jbc.m113.462622

Cited by 21 publications

(26 citation statements)

References 30 publications

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“…1) However, in the case of autoimmune diseases or chronic inflammatory diseases, excessive and prolonged production of cytokines is a major causative agent, which leads to tissue damage, hemodynamic changes, and multiple organ failure with a wide range of clinical manifestations and complications. 2,3) Subtilase cytotoxin (SubAB), a member of the AB 5 toxin family, was identified by Paton et al in an enterohemorrhagic Escherichia coli (EHEC) O113 : H21 that was associated with an outbreak in Australia. [4][5][6] SubAB is formed by an enzymatically active A subunit with a molecular weight of 35 kDa; it cleaves host endoplasmic reticulum (ER) chaperon protein BiP and pentameric B subunit (each monomer 13 kDa) that binds to the cell surface receptor, and mediates uptake into a target cell.…”

Section: Introductionmentioning

confidence: 99%

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages

Harada

Tsutsuki

Zhang

et al. 2020

CHEMICAL & PHARMACEUTICAL BULLETIN

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Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/ nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

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Section: Introductionmentioning

confidence: 99%

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages

Harada

Tsutsuki

Zhang

et al. 2020

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…In contrast, the pentameric B-subunits typically contain a higher amount of β-sheets [31]. Crystal structure analyses of the functional SubAB holo-toxin complex by Le Nours et al [32] showed that this fundamental distribution of secondary structure throughout the protein complex is true for SubAB1-His. In the current study, secondary structure analyses of the separately expressed and purified SubAB subunits revealed that SubA1-His, SubA2-2-His, and SubA2-2 showed typical α-helical signals in the far-UV CD spectra ( Figure 3A).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, the alterations were rather evenly distributed throughout the whole protein. In total, 13 amino acids throughout SubA1 were identified to be part of the interaction regions of the toxin complex [32]. None of them were different in SubA2-2-His.…”

Section: Discussionmentioning

confidence: 99%

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

Krause

Sessler

Kaziales

et al. 2019

Toxins

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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.

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“…Among such factors are the AB 5 toxins, which are complexes between five copies of a B subunit employed for attachment to the host plasma membrane and a single catalytic A subunit, which subverts cellular functions once the toxin complex has been internalized (Merritt and Hol, 1995). Four AB 5 families have been reported based on the function of the catalytic subunit: the Shiga toxins, which interfere with protein biosynthesis; the pertussis and cholera toxins, which affect regulation of fluid secretion; and the subtilase cytotoxins, which have a subtilisintype serine proteinase as A subunit that cleaves an endoplasmic reticulum chaperone (Beddoe et al, 2010;Le Nours et al, 2013). More recently, a fifth family was identified in diarrhea-causing strains of Escherichia coli and Citrobacter freundii, represented by toxins EcxAB and CfxAB, respectively (Jansson et al, 2010;Karasawa et al, 2002).…”

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confidence: 99%