Enterohemorrhagic E. coli (EHEC) Secreted Serine Protease EspP‐Induced Injury and Regeneration is Guided by Desert Hedgehog and WNT2B in Human Colonoids

Abstract

IntroductionEnterohemorrhagic E. coli (EHEC) is a major foodborne pathogen in developed countries. In humans, EHEC colonizes the colon and can cause life threatening hemorrhagic colitis and hemolytic uremic syndrome. The colon pathology typically shows hemorrhagic mucosa with apoptotic crypts; however, the epithelium regenerates after bacterial clearance. We have demonstrated that human colonoids are a relevant pathophysiological model to study early molecular events of EHEC infection. The goal of this study was to elucidate the mechanism of epithelial regeneration after EHEC‐induced injury.MethodsHuman colonoids derived from colonic biopsies obtained from healthy donors were treated overnight with a high dose of recombinant serine protease EspP (the major EHEC‐secreted cytotoxin) to induce epithelial apoptosis. Non‐treated cultures served as controls. Following incubation, EspP was washed from the cultures and both EspP‐treated and control populations were lysed. The protein signatures of the colonoids were identified and quantified with tandem mass spectrometry and iTRAQ. The proteome was validated by immunoblot or qRT‐PCR of selected genes in the absence of suitable antibodies.ResultsOvernight incubation of colonoids with EspP induced significant cell death, seen by propidium iodide staining. Based on the downstream targets that were upregulated, the proteome suggests that the WNT and Hedgehog pathways are essential for regeneration following EspP‐induced injury. Sonic and indian hedgehog are highly expressed under basal conditions in colonoids, but are downregulated during regeneration. Instead, desert hedgehog (DHH), which is below detection under basal conditions, was upregulated 3x fold in regenerating colonoids. Colonoids treated with the smoothened agonist (stimulator of hedgehog signaling) or recombinant DHH had increased survivability. WNT2B was also upregulated (2x fold) during regeneration. Inhibition of WNT secretion with IWP‐2 (porcupine inhibitor) had no effect on basal colonoid growth, but prevented regeneration of EspP‐injured colonoids. Knockout of WNT2B by CRISPR gene editing also halted regeneration.ConclusionsThe results of the current study demonstrate that 1) WNT and hedgehog pathways are important modulators of regeneration following EspP‐induced injury but 2) through Desert Hedgehog and WNT2B stimulation specifically, suggesting the various hedgehog ligands (sonic, indian, desert) are not redundant. 3) WNT2B is produced by the epithelia and is not essential for homeostasis, but is indispensable during regeneration. These signaling pathways are related, yet distinct from unchecked proliferation seen in colon cancers, suggesting that colonic stem cells specifically respond to bacterial‐mediated injury. This provides a new understanding of the mechanisms related to stem cell response in diarrheal diseases.Support or Funding InformationNIH grants: P01 AI125181, K01 DK106323, P30 DK089502, and U01 DK103168