scholarly journals Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 638 ◽  
Author(s):  
Mary K. Estes ◽  
Khalil Ettayebi ◽  
Victoria R. Tenge ◽  
Kosuke Murakami ◽  
Umesh Karandikar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Host Factors ◽  
Viral Gastroenteritis ◽  
Intestinal Epithelial ◽  
Human Stem Cell ◽  
Human Intestinal Epithelial Cell ◽  
Major Barrier ◽  
Cultivation System ◽  
Human Intestinal Epithelial Cells ◽  
Human Intestinal Epithelium

Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.

scholarly journals The Antibiotic Bacitracin Protects Human Intestinal Epithelial Cells and Stem Cell-Derived Intestinal Organoids from Clostridium difficile Toxin TcdB

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Ziyu Zhu ◽  
Leonie Schnell ◽  
Bastian Müller ◽  
Martin Müller ◽  
Panagiotis Papatheodorou ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Inhibitory Effect ◽  
Target Cells ◽  
Intestinal Epithelial ◽  
Intestinal Organoids ◽  
Human Intestinal Epithelial Cells ◽  
Protein Toxins ◽  
Dependent Transport

Bacitracin is an established antibiotic for local application and inhibits the cell wall synthesis of Gram-positive bacteria. Recently, we discovered a completely different mode of action of bacitracin and reported that this drug protects human cells from intoxication by a variety of medically relevant bacterial protein toxins including CDT, the binary actin ADP-ribosylating toxin of Clostridium (C.) difficile. Bacitracin prevents the transport of CDT into the cytosol of target cells, most likely by inhibiting the transport function of the binding subunit of this toxin. Here, we tested the effect of bacitracin towards TcdB, a major virulence factor of C. difficile contributing to severe C. difficile-associated diseases (CDAD) including pseudomembranous colitis. Bacitracin protected stem cell-derived human intestinal organoids as well as human gut epithelial cells from intoxication with TcdB. Moreover, it prevented the TcdB-induced disruption of epithelia formed by gut epithelium cells in vitro and maintained the barrier function as detected by measuring transepithelial electrical resistance (TEER). In the presence of bacitracin, TcdB was not able reach its substrate Rac1 in the cytosol of human epithelial cells, most likely because its pH-dependent transport across cell membranes into the cytosol is decreased by bacitracin. In conclusion, in addition to its direct antibiotic activity against C. difficile and its inhibitory effect towards the toxin CDT, bacitracin neutralizes the exotoxin TcdB of this important pathogenic bacterium.

scholarly journals Rotavirus Infection Induces an Increase in Intracellular Calcium Concentration in Human Intestinal Epithelial Cells: Role in Microvillar Actin Alteration

2000 ◽  
Vol 74 (5) ◽  
pp. 2323-2332 ◽  
Author(s):  
Jean-Philippe Brunet ◽  
Jacqueline Cotte-Laffitte ◽  
Catherine Linxe ◽  
Anne-Marie Quero ◽  
Monique Géniteau-Legendre ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Progressive Increase ◽  
Intracellular Calcium Concentration ◽  
Viral Gastroenteritis ◽  
Intestinal Epithelial ◽  
Rotavirus Infection ◽  
Human Intestinal Epithelial Cells ◽  
Intracellular Organelles ◽  
Necessary And Sufficient

ABSTRACT Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228–7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca2+]i) in Caco-2 cells, since Ca2+ is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca2+]i from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca2+]i was related only to an increase in Ca2+ permeability of plasmalemma. At a late stage of infection, [Ca2+]i elevation was due to both extracellular Ca2+ influx and Ca2+ release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca2+]i increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca2+ release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca2+]i of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca2+from the ER and by extracellular Ca2+ influx. These supernatants induced a Ca2+-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.

scholarly journals Norovirus replication in human intestinal epithelial cells is restricted by the interferon-induced JAK/STAT signalling pathway and RNA Polymerase II mediated transcriptional responses

2019 ◽  
Author(s):  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Komal Nayak ◽  
Frederic Sorgeloos ◽  
Bon-Kyoung Koo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intestinal Epithelial Cells ◽  
Interferon Response ◽  
Viral Gastroenteritis ◽  
Intestinal Epithelial ◽  
Transcriptional Responses ◽  
Human Noroviruses ◽  
Human Intestinal Epithelial Cells

AbstractHuman noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa derived intestinal epithelial organoids (IEOs), has transformed our capability to dissect the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggest that the innate immune response may play no role in the restriction of HuNoV replication in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication, but enhanced replication to a greater degree compared to blocking of JAK signalling directly. Furthermore, we demonstrate for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, are more permissive to HuNoV infection. Together our work identifies the IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrates that the inhibition of these responses by modifications to the culture conditions can greatly enhance the robustness of the norovirus culture system.ImportanceNoroviruses are a major cause of gastroenteritis worldwide yet the challenges associated with their growth culture has greatly hampered the development of therapeutic approaches and has limited our understanding of cellular pathways that control infection. Here we show that human intestinal epithelial cells, the first point of entry of human noroviruses into the host, limit virus replication by the induction of the innate responses. Furthermore we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms to enhance the robustness of norovirus culture.

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