Infektion von Darmzellen durch SARS-CoV2 und Auswirkungen auf die Darmfunktion
Biochemie
Gastroenterologie
Zellbiologie
Zusammenfassung der Projektergebnisse
SARS-CoV-2 is the virus behind the COVID-19 pandemic that started in 2019. It targets mainly the respiratory tract and causes flu-like symptoms such as cough, runny nose, fever, and shortness of breath to name a few. It also targets other organ systems. In fact, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. The virus was also detected in rectal swabs in patients with confirmed negative pharyngeal swabs, and virus shedding from the intestine was found even when oral swabs had become negative. Therefore, investigating the fecal-oral route as a potential mode of transmission of SARS-CoV-2 would shed a light on the infection mechanisms in the intestine. SARS-CoV-2 binds initially to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its spike (S) protein before it undergoes endocytosis and fusion with the lysosomal membrane. This project aimed at investigating the interaction of the spike protein with intestinal cells, the identification of the intestinal receptor(s) as well as and the modulation of this interaction with the ultimate goal of studying the overall effects of SARS-CoV-2 infection on intestinal function. First, we established a reliable and straightforward biochemical assay by which the interaction between the S protein and the putative receptors can be assessed in an in vitro cellular model. These studies revealed ACE2 as the main receptor for the S1 protein in intestinal Caco-2 cells. One important objective in these studies aimed at elucidating the impact of glycosylation modulation on the trafficking of both S1 subunit and ACE2 as well as their interaction at the cell surface of intestinal epithelial cells. Glycosylation is an essential posttranslational modification in the life cycle of membrane and secretory proteins that affects their structural and functional characteristics as well as their trafficking and sorting patterns. Moreover, the spike protein of SARS-CoV-2 is a heavily N- and O-glycosylated trimer and it is very likely that modulation of the glycosylation pattern could negatively impact the binding capacities to the receptor. Two glycosylation modulators were utilized, N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located α-glucosidase I, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located α-mannosidase I to assess the trafficking and secretion of S1 as well as ACE2 trafficking to the apical membrane of intestinal Caco-2 cells. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitations. Our data show that NB-DNJ significantly reduced the secretion of the S1 proteins, while dMM affected S1 secretion to a lesser extent. Moreover, the trafficking of ACE2 to the apical membrane in Caco-2 cells was markedly delayed in the presence of NB-DNJ and to a lesser extent when dMM was utilized. Strikingly, the interaction between S1 and ACE2 was significantly reduced when both proteins were processed by the glycosylation inhibitors, rendering glycosylation and its inhibitors potential target candidates for SARS-CoV-2 treatment. We further investigated the effect of NB-DNJ, which is not only a glycosylation inhibitor but also an inhibitor of glucosylceramide synthase and thus sphingolipid synthesis, on lipid rafts in Caco-2 and ACE2 trafficking. Current results revealed a redistribution of Flotillin 2, a lipid rafts marker, in the lipid rafts fractions upon treatment of Caco-2 cells with NB-DNJ. We could also observe reduced levels of ACE2 in lipid rafts upon NB-DNJ treatment of Caco-2 cells (ongoing studies). The significant reduction in the secretion of the spike protein in the presence of the glycosylation inhibitor NB-DNJ and its reduced interaction with the ACE2 receptor in Caco-2 cells are strong assets for further investigations in virus-infected Caco-2 cells or intestinal organoids in the presence of NB-DNJ. The reproducibility of these data i.e. reduced virus exit or virus entry in Caco-2 cells or organoids in the presence of NB-DNJ may render this drug an interesting target for further studies with therapeutic potential that decreases the infectivity with the virus.
Projektbezogene Publikationen (Auswahl)
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Infection of intestinal cells by SARS-CoV2 and its effects on intestinal function. Poster presented at DFG Research Conference Pandemics; November 15, 2020, Germany
El Khoury M. and Naim, H.Y.
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Biochemical Characterization of SARS-CoV-2 Spike RBD Mutations and Their Impact on ACE2 Receptor Binding. Frontiers in Molecular Biosciences, 9 (2022, 5, 23).
Hoter, Abdullah & Naim, Hassan Y.
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The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2. The FASEB Journal, 36(S1).
El, Khoury Marianne; Wanes, Dalanda; Hoter, Abdullah & Naim, Hassan Y.
