Kellnerová Sára, Chatterjee Sneha, Bayarri-Olmos Rafael, Justesen Louise, Talasz Heribert, Posch Wilfried, Kenno Samyr, Garred Peter, Orth-Höller Dorothea, Grasse Marco, Würzner Reinhard
Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstraβe 41, A-6020 Innsbruck, Austria.
Laboratory of Molecular Medicine, Department of Clinical Immunology Section 7631, Rigshospitalet, University of Copenhagen, Ole Maaloesvej 26, 2200 Copenhagen, Denmark.
Toxins (Basel). 2020 Dec 24;13(1):8. doi: 10.3390/toxins13010008.
Enterohemorrhagic (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.
肠出血性大肠杆菌（EHEC）感染可通过其主要毒力因子志贺毒素（Stxs）引发与EHEC相关的溶血尿毒综合征（eHUS）。据报道，补体参与了eHUS的进展过程。本研究的目的是调查毒素最有效的亚型Stx2a与关键补体蛋白C3b和C5之间的相互作用。该研究进一步检测了Stx2a刺激对这些补体蛋白在人靶细胞系中转录和合成的影响。通过酶联免疫吸附测定（ELISA）评估Stx2a与C3b和C5的结合情况。用不同浓度的Stx2a刺激肾和肠道细胞系（HK-2和HCT-8）。随后通过实时定量聚合酶链反应（qPCR）研究补体基因转录情况，并进行ELISA和蛋白质免疫印迹法检测受刺激的HK-2细胞上清液和裂解物中C3和C5的蛋白质合成。Stx2a显示出与C3b和C5的特异性结合。在这两种细胞系中，随着Stx2a浓度的增加，C3和C5的基因转录上调，但蛋白质合成未上调。本研究证明了Stx2a与补体蛋白C3b和C5的结合，这可能在eHUS感染期间参与调节补体，支持进一步研究以阐明补体在eHUS发病机制中的作用。
