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


Read preprint (2 votes)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage

Also in the molecular biology category: