Enzymatic cleavage of Stx2a in the gut and identification of pancreatic elastase and trypsin as possible main cleavers

Authors: KELLNEROVA, SARA - Innsbruck Medical University; HUBER, SILKE - Innsbruck Medical University; MASSRI, MARIAM - Innsbruck Medical University; FLEISCHER, VERENA - Innsbruck Medical University; LOSSO, KLEMENS - Innsbruck Medical University; SARG, BETTINA - Innsbruck Medical University; KREMSER, LEOPOLD - Innsbruck Medical University; TALASZ, HERIBERT - Innsbruck Medical University; He, Xiaohua; VARRONE, ELISA - University Of Bologna, Italy; BRIGOTTI, MAURIZIO - University Of Bologna, Italy; ARDISSINO, GIANLUIGI - University Of Bologna, Italy; ORTHHOLLER, DOROTHEA - Innsbruck Medical University; WURZNER, REINHARD - Innsbruck Medical University

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/2/2023
Publication Date: 10/4/2023
Citation: Kellnerova, S., Huber, S., Massri, M., Fleischer, V., Losso, K., Sarg, B., Kremser, L., Talasz, H., He, X., Varrone, E., Brigotti, M., Ardissino, G., Orth-holler, D., Wurzner, R. 2023. Enzymatic cleavage of Stx2a in the gut and identification of pancreatic elastase and trypsin as possible main cleavers. Microorganisms. 11(10):2487. https://doi.org/10.3390/microorganisms11102487.
DOI: https://doi.org/10.3390/microorganisms11102487

Interpretive Summary: Shiga toxins (Stxs) are the major virulence factors in E. coli associated life-threatening hemolytic uremic syndrome (HUS). Understanding the function of Stx's structure in E. coli infection and HUS pathogenesis is critical for the development of efficient preventative or therapeutic strategies. In this study, evidence of the potential cleavage of Stx2a A subunit in intestine prior to reaching the target cells was raised. Two serine proteases, trypsin and chymotrypsin-like elastase 3B were found to play key roles in this cleavage. These results are valuble for us to further investigate the actual process of HUS pathogenesis during natural infections.

Technical Abstract: Shiga toxins (Stxs), and more specifically the Stx2a subtype, are the major virulence factors in-volved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS); a life-threatening disease causing acute kidney injury, especially in children. Cleavage of Stxs results in the liberation of the toxic A subunit causing further damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether cleavage of Stx2a A subunit could potentially occur in the intestine, even prior entering target cells, Stx2a structure was characterized after its exposure to specific host factors present in human stool, a noninvasive representative of the intestinal environment. The structure of Stx2a A subunit was determined by immunoblotting after electrophoretic separation of proteins under reducing conditions. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that may trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether this observation indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. Concomitantly, further research should also examine whether the host's protease enzymatic profile might determine biological characteristics of Stx2a, likely affecting disease development.