ABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) targets mainly the respiratory tract. In addition to respiratory symptoms, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. 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. The spike protein of SARS-CoV-2 is a heavily N- and O-glycosylated trimer. 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. This study 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. For this purpose, the S1 protein was expressed in COS-1 cells and its glycosylation modified using N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located alpha-glucosidases I and II, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located alpha-mannosidase I. The intracellular and secreted S1 proteins were analyzed by endoglycosidase H treatment. Similarly, ACE2 trafficking to the brush border membrane of intestinal Caco-2 cells was also assessed in the presence or absence of the inhibitors. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitation. Our data show that NB-DNJ significantly reduced the secretion of S1 proteins in COS-1 cells, while dMM affected S1 secretion to a lesser extent. Moreover, NB-DNJ and dMM differentially affected ACE2 trafficking and sorting to the brush border membrane of intestinal Caco-2 cells. 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 candidates for SARS-CoV-2 treatment. This work has been supported by a grant from the German Research Foundation (DFG) grant NA331/15-1 to HYN. M.K. was supported by a scholarship from the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI) and by the DFG grant NA331/15-1.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
RATIONALE: Treatments for the coronavirus disease of 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through the interactions of its spike (S) protein with ACE2 and TMPRSS2 on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. Our finding that the airway administration of OM-85 inhibits Ace2 and Tmprss2 transcription in mouse lungs prompted us to investigate whether and how OM-85 may protect non-human primate and human epithelial cells against SARS-CoV-2 infection. METHODS: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein-pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2- transfected HEK293T cells treated with OM-85 in vitro. RESULTS: OM-85 significantly downregulated ACE2 and TMPRSS2 mRNA in epithelial cell lines and primary bronchial epithelial cells, and strongly inhibited SARS-CoV-2 S protein binding to, SARS-CoV-2 S proteinpseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on the downregulation of SARS-CoV-2 receptor expression. CONCLUSIONS: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of COVID-19.
ABSTRACT
The ongoing COVID-19 pandemic is caused by the severe acute respiratory corona virus-2 (SARS-CoV-2) which as of right now has infected 10% of world’s population and has caused >1.5 million deaths worldwide. In addition to respiratory symptoms, COVID-19 causes nausea, vomiting and diarrhea in more than half of infected subjects. This indicates that SARS-CoV-2 not only infects the respiratory tract, but also the gastrointestinal. Bats are thought to be the original reservoir for SARS-CoV-2, since SARS-CoV-2 is 96% identical to the bat coronavirus RatG13, which was identified in horseshoe bats. However, coronaviruses fail to cause overt disease in the bats, whereas strong cytopathic effects were observed in human respiratory and gastrointestinal epithelial cells upon SARS-CoV-2 infection. The goal of our research is to compare the response of primary intestinal epithelial cells of bats and humans to SARS-CoV-2 infection in order to better understand the cellular mechanism that allow bats to harbor coronaviruses without developing disease symptoms. To study the SARS-Co-V-2 infection in bats, we have, for the first time, established organoids lines from the stomach, proximal and distal small intestine of three adult Jamaican Fruit Bats (Artibeus jamaicensis). Organoids were successfully generated from both fresh and frozen tissue and could be passaged at least 25 times and frozen and thawed with no apparent changes in growth and morphology. Microscopic analysis showed that bat gastric and intestinal organoids were composed of a simple columnar epithelium and secreted variable amounts of mucus. We also observed spontaneous development of gland and crypt structures, indicating appropriate differentiation (Fig. 1). When seeded on transwell inserts, both gastric and intestinal organoid cells consistently developed a transepithelial resistance, demonstrating intact barrier function. Using confocal microscopy, we showed that both gastric and intestinal organoids from bats expressed angiotensin I converting enzyme 2 (ACE2), a key receptor for SARS-CoV-2 entry. Our innovative experimental platform will enable us to study multiple aspects of coronavirus infection including viral evolution and determinants of spillover events in a relevant primary cell model system. Importantly, we will utilize the bat organoid model to identify nonpathogenic cellular pathways that enable tolerance to SARS-CoV-2 in the reservoir hosts for this virus, potentially informing novel treatment strategies in human COVID-19 patients.