David Holthaus1, Martin R Kraft1, Susanne M Krug2, Silver Wolf3, Antonia Müller1, Estefania Delgado Betancourt1, Madeleine Schorr4, Gudrun Holland5, Felix Knauf4, Joerg-Dieter Schulzke2, Toni Aebischer1, Christian Klotz6. 1. Department of Infectious Diseases, Unit 16 Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany. 2. Department of Gastroenterology, Rheumatology and Infectious Diseases, Clinical Physiology Nutritional Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany. 3. MF 1 Bioinformatics, Robert Koch-Institute, Berlin, Germany. 4. Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Berlin, Germany. 5. Advanced Light and Electron Microscopy, Centre for Biological Threats and Special Pathogens, Robert Koch-Institute, Berlin, Germany. 6. Department of Infectious Diseases, Unit 16 Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany. Electronic address: klotzc@rki.de.
Abstract
BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.
BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.
Authors: David Warschkau; Estefanía Delgado-Betancourt; David Holthaus; Antonia Müller; Gudrun Kliem; Susanne M Krug; Joerg-Dieter Schulzke; Toni Aebischer; Christian Klotz; Frank Seeber Journal: Bio Protoc Date: 2022-01-20