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

Abstract

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2⁺ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

Fig. 1. VSV-SARS-CoV-2 infects human small intestinal enteroids.

Mouse small intestinal cells were analyzed by single-cell RNA-sequencing and resolved into 20 clusters based on gene expression profiles (left panel). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Clusters 10 and 18: intraepithelial lymphocytes; clusters 1, 2, 5, 8, 9, 17, 19, 20: enterocytes; cluster 3: goblet cells; cluster 4: entero-endocrine cells; cluster 7: Tuft cells; cluster 11: crypt stem cells; cluster 12: Paneth cells. Each dot represents a single cell. Note that Ace2^high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (A) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bar: 32 μm. (B) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5X10⁵ plaque forming units (PFU) of VSV-SARS-CoV-2 (MOI=0.3) for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) media or differentiation (DIFF) media for 3 days and infected with 2.2X10⁵ PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bar: 50 μm. (D) Same as (C) except that virus titers were measured using an TCID50 assay instead of viral RNA levels by QPCR. (E) Same as (D) except that human ileum enteroids were used instead. Scale bar: 80 μm. (F) Same as (D) except that human colon enteroids were used instead. Scale bar: 80 μm. For all figures except A, experiments were repeated at least three times with similar results. Figure 1A was performed once with small intestinal tissues pooled from three mice. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

Fig. 2. VSV-SARS-CoV-2 and wild-type SARS-CoV-2 replicate in ACE2⁺ human mature enterocytes.

(A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to a TCID50 assay to measure the amount of infectious virus. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5X10⁵ PFU of infectious SARS-CoV-2 virus (MOI=0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a Taqman assay and normalized to that of GAPDH. (D) Differentiated ileum enteroids in monolayer were apically or basolaterally infected with 2.5X10⁵ PFU of infectious SARS-CoV-2 virus (MOI=0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a Taqman assay and normalized to that of GAPDH. (E) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bar: 32 μm. SARS-CoV-2 infected ACE2 positive cells are enlarged in the inset (yellow box). (F) SARS-CoV-2 infected duodenum monolayers were imaged along the z-stacks and sectioned for YZ planes (top panel) and reconstructed for 3D images (bottom panel). For all figures except C, D, and E, experiments were repeated at least three times with similar results. Figure 2C, 2D, and 2E were performed twice with technical duplicates in each experiment. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

Fig. 3. TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 S mediated entry.

(A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. Cells were treated with trypsin at 0.5 μg/ml for 10 min. The levels of S and GAPDH were measured by Western blot. The intensity of bands was quantified by ImageJ and shown as percentage of the bottom band versus the top band in each lane. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8X10⁵ PFU of VSV-SARS-CoV-2 on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37°C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia. Scale bar: 100 μm. For all figures except A, experiments were repeated at least three times with similar results. RNA-seq in Fig. 3A was performed once with duplicate samples. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

Fig. 4. TMPRSS2 and TMPRSS4 promote VSV-SARS-CoV-2 infection in enteroids.

(A) Schematic diagram of SARS-CoV-2 infection of human mature enterocytes. SARS-CoV-2 particles, host proteins, and host cells are not proportional to the true sizes. Based on the scRNA-seq data (Fig. S3A), TMPRSS4 is more highly expressed than TMPRSS2 on mature absorptive enterocytes and TMPRSS2 is more highly expressed on secretory cells than TMPRSS4. (B) GFP-expressing HEK293 cells were mixed at 1:1 ratio and co-cultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hours (right panel). Note the formation of cell-cell fusion (yellow), highlighted by black arrows. (C) Images in (B) were quantified based on the intensity of yellow signals. T2: TMPRSS2, T4: TMPRSS4. (D) Human duodenum enteroids in 3D Matrigel were transduced with lentiviruses encoding Cas9 and sgRNA against TMPRSS2 or TMPRSS4 (oligonucleotide information in Table S1). Gene knockout enteroids were seeded into monolayers and infected with 1.5X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Human duodenum, ileum, and colon enteroids were infected with 2.9X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The levels of indicated viral and host transcripts were measured by RT-qPCR and normalized to that of GAPDH. (F) Human duodenum enteroids seeded into collagen-coated 96-well plates were differentiated for 3 days, pre-treated with 50 μg/ml of soybean trypsin inhibitor (SBTI), 10 μM of camostat mesylate, or 10 μM of E-64d for 30 min, and infected with 1.5X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

Fig. 5. SARS-CoV-2 rapidly lose infectivity in the human GI tract.

(A) 2.5X10⁵ PFU of SARS-CoV-2-mNeonGreen virus was incubated with M199 media, simulated human small intestinal (SI) fluid, or simulated human large intestinal (LI) fluid for indicated time points at 37°C. The virus was subsequently serially diluted and added to MA104 cells for 24 hours and GFP signals were scanned by Typhoon 5. (B) Stool specimens from 10 COVID-19 patients were collected and subjected to QPCR experiments to quantify the absolute levels of SARS-CoV-2 N gene. Figure 5A was performed once in quadruplicate. Figure 5B was performed once. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).