TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

Ruochen Zang, Maria F.G. Castro, Broc T. McCune, Qiru Zeng, Paul W. Rothlauf, Naomi M. Sonnek, Zhuoming Liu, Kevin F. Brulois, Xin Wang, Harry B. Greenberg, Michael S. Diamond, Matthew A. Ciorba, Sean P.J. Whelan, Siyuan Ding

Preprint posted on 23 April 2020

Article now published in Science Immunology at

A gut feeling: intestinal organoids model COVID-19 pathogenesis illuminating a novel mode of infection where SARS-CoV-2 invades human enterocytes via TMPRSS2 and TMPRSS4 action

Selected by Brooke Chambers


Recently, a global pandemic erupted due to a novel severe acute respiratory syndrome coronavirus, SARS-CoV-2, which initiated a cluster of lethal pneumonia cases in Wuhan, China (Rabi et al., 2020). This viral infection is commonly known as coronavirus disease 2019 (COVID-19) and has claimed over 217,000 lives to date. Symptoms of infection can range from mild to severe, and patients can present with fever, cough, difficulty breathing, headache, and progressive pneumonia. SARS-CoV-2 belongs to the Coronaviridae family, which project crown-like spikes from their surface and are enveloped, positive-sense RNA viruses. Upon sequencing the SARS-CoV-2 genome, researchers found it exhibited 79.6% homology with SARS-CoV-1 that had previously caused an outbreak in 2002-2003. Further, at the whole-genome level, SARS-CoV-2 is 96% identical to a bat coronavirus, identifying the likely host species of zoonotic origin. This specific virus particle is coated in spike glycoproteins (S) that contain a variable receptor-binding domain that enables entry into human target cells. Specifically, this domain has a high affinity for the angiotensin-converting enzyme-2 receptor (ACE-2), which is expressed in heart, lung, kidney, and intestinal tissue (Zhou et al., 2020). After receptor attachment, SARS-CoV-2 S protein is processed by TMPRSS2, a transmembrane serine protease, to infiltrate the host cytoplasm (Hoffmann et al., 2020). Interestingly, TMPRSS2 is highly enriched in intestinal epithelial cells (IECs) and 20-50% of COVID-19 patients report gastrointestinal symptoms. To this point, Zang and colleagues (2020) investigate if SARS-CoV-2 displays features of enteric viruses to shed light on potential mechanisms of infection, transmission, and pathogenesis.

Key Findings:

SARS-CoV-2 infects and replicates within human intestinal epithelial cells (IECs)

To address whether SARS-CoV-2 is capable of infecting human IECs, the investigators cleverly employed an intestinal organoid culture system. Organoid technology has exploded onto the research scene as a 3D culture method to grow miniaturized organs in a dish that accurately recapitulate aspects of human tissue development and disease. Their system consisted of isolating tissue from the small intestinal crypts of healthy colonoscopy patients. Patient cells were seeded in a 3D Matrigel matrix and subjected to conditioned media containing growth factors to promote differentiation and organoid assembly. In the field, these specific organoids are considered ‘intestinal enteroids’ because they were derived from an adult stem cell source (Yoo & Donowotiz, 2019). Intestinal enteroids were subjected to VSV-SARS-CoV-2-S-GFP, which is a newly developed fluorescent strategy to track viral entry and cell tropism. SARS-CoV-2 infection and replication was evident, as GFP+ IECs were scattered throughout intestinal enteroids and a ~10,000-fold increase of viral RNA occurred within 24 hours.

Upon scrutinizing the mode of viral entry, the researchers discovered SARS-CoV-2 exclusively targets the apical surface of mature villous enterocytes that highly express the ACE2 chaperone protein. Goblet, enteroendocrine, and Paneth cells exhibited no evidence of viral penetration. Notably, infected enterocytes formed syncytia and exhibited luminal shedding of viral progeny, providing important insights to potential mechanisms of gastrointestinal pathogenesis in COVID-19 patients. Collectively, these analyses pinpointed the specific intestinal cell type that is susceptible to SARS-CoV-2 infection, which is indispensable information moving forward.

ACE2, TMPRSS2, and TMPRSS4 function in concert to regulate SARS-CoV-2 viral entry

Previous studies indicate that TMPRSS2 cleaves the SARS-CoV-2 S protein and mediates entry into the host cell. However, two closely related serine proteases, TMPRSS4 and ST14, are also present in intestinal tissue. To interrogate whether these other proteases enhance viral infection, the investigators devised a HEK293 ectopic expression strategy. In an ACE2-dependent manner, the induction of TMPRSS4 expression initiated a significant surge in viral RNA levels and increased cleavage and fusogenic activity of the S protein. Coexpression of TMPRSS4 and TMPRSS2 produced a synergistic elevation of viral titers, and ST14 had no effect on infectivity. In agreement with this data, CRISPR-mediated deletion of TMPRSS4 in intestinal enteroids led to a marked decrease in SARS-CoV-2 replication, even more severe than TMPRSS2 knockdown. Pharmacological pretreatment of enteroids with camostat mesylate, a TMPRSS inhibitor, attenuated viral infection whereas blocking cathepsin pathways were ineffective. Here, the authors divulged a unique element of SARS-CoV-2 as compared to other coronaviruses, in that it does not rely on cathepsin-mediated endocytosis as an alternative route of entry in human IECs. Although the virus can robustly infect enteroids, it was quickly degraded when exposed to colonic fluids; therefore, fecal-oral transmission is highly unlikely. Taken together, this study provides groundbreaking evidence that SARS-CoV-2 can infect intestinal epithelium. The authors reveal for the first time that TMPRSS4 action may amplify COVID-19 pathogenesis.

What I like about this paper:

What drew me to this study was the authors’ goal of addressing overlooked questions central to understanding COVID-19 pathogenesis: 1) Can SARS-CoV-2 can actively infect gastrointestinal cells? 2) If so, what mechanisms does it use to hijack host cells? 3) Is there evidence pointing to fecal-oral transmission? A large proportion of emerging studies have focused on how the virus behaves in lung tissue and the resulting immune response. I appreciate how this body of work pioneers a new arena of COVID-19 research and believe that the data identifies the intestine as likely site of viral replication, which could accelerate systemic disease. Additionally, this study further establishes organoids as a valuable system to model SARS-CoV-2 infection and evaluate cellular tropism within an organ of interest.

Open Questions:

  1. Because organoids are amenable to high throughput drug screening, have you considered applying this strategy to your SARS-CoV-2 intestinal enteroid model? It would be exciting to test an FDA-approved chemical library on infected enteroids to facilitate the rapid discovery of compounds that could be repurposed to combat viral infection.
  2. There are reports from the University of Tokyo that nafamostat mesylate can inhibit membrane fusion at a much smaller concentration than camostat mesylate. Is there a reason why you preferentially tested camostat in your system?
  3. Based on your findings that expose a novel role for TMPRSS4 in intestinal infection, do you believe that this protease also regulates SARS-CoV-2 entry into other tissue types like the nasopharyngeal mucosa and lungs?


  • Rabi, F. A., Al Zoubi, M. S., Kasasbeh, G. A., Salameh, D. M., & Al-Nasser, A. D. (2020). SARS-CoV-2 and Coronavirus Disease 2019: What We Know So Far. Pathogens, 9(3), 231.
  • Zhou, P., Yang, X. L., Wang, X. G., Hu, B., Zhang, L., Zhang, W., … & Chen, H. D. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579(7798), 270-273.
  • Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., … & Müller, M. A. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell.
  • Yoo, J. H., & Donowitz, M. (2019). Intestinal enteroids/organoids: A novel platform for drug discovery in inflammatory bowel diseases. World Journal of Gastroenterology, 25(30), 4125.



Tags: covid19, immunology, intestine, organoids, virology, virus

Posted on: 29 April 2020


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