key: cord-0752696-0aavshar
authors: Ji, Jeong Yeon; Jo, Ara; Won, Jina; Gil, Chan Hee; Shin, Haeun; Kim, Sujin; Jeon, Yung Jin; Kim, Hyun Jik
title: The nasal symbiont Staphylococcus species restricts the transcription of SARS-CoV-2 entry factors in human nasal epithelium
date: 2021-09-25
journal: iScience
DOI: 10.1016/j.isci.2021.103172
sha: 0cd69c4711d7ae4867695720ea3ae5aff243cc85
doc_id: 752696
cord_uid: 0aavshar

Emerging evidence indicates that severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is transmitted through the human nasal mucosa via the principal entry factors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which are highly expressed in the nasal epithelium. Therefore, the biologics targeting host entry factors on human nasal mucosa will be necessary for complete control of SARS-CoV-2. Our data reveal that ACE2 was more abundant in human nasal mucosa than lung tissue. Both ACE2 and TMPRSS2 transcriptions were significantly decreased in nasal epithelium in response to S. epidermidis and were relatively lower in human nasal mucus with large number of S. epidermidis. ACE2 transcription was also reduced in nasal epithelium in response to nasal symbiont S. aureus. This study propose that Staphylococcus species nasal commensals might potentially restrict SARS-CoV-2 entry to the nasal epithelium via down regulation of cellular receptors coupled with reduction of principal host protease.

respiratory syndrome-related coronavirus-2 (SARS-CoV-2), which causes 48 SARS-CoV-2 in the human nasal epithelium, we investigated gene expression of 125 ACE2 depending on nasal epithelial cell subset through single-cell RNA sequencing 126 (scRNA-seq) ( Figure S2 ). We confirmed increased normalized ACE2 expression in 127 both suprabasal cells and secretory-like NHNE cells ( Figure 1D ). As a next step, we 128 determined the gene expression level of TMPRSS2 in human nasal mucosa, lung 129 tissue, NHNE and NHBE cells. Real-time PCR results revealed that the gene 130 expression of TMPRSS2 was rather lower in human nasal mucosa and NHNE cells 131 than lung and NHBE cells (Figure 1E ) and scRNA-seq data showed that TMPRSS2 132 gene expression across cell types of NHNE cells was much stronger with a certain 133 specificity for suprabasal cells ( Figure 1F ). While principal serine protease 134 expression for SARS-CoV-2 invasion was relatively lower, the cellular receptor 135 required for SARS-CoV-2 to enter the human respiratory tract was more distributed 136 in human nasal mucosa. These data suggest that the nasal epithelium is the primary 137 target of SARS-CoV-2 invasion, and SARS-CoV-2 infection is spread to the 138 respiratory tract after intracellular entry via nasal epithelial cells. 139 epidermidis in restricting host entry factors of SARS-CoV-2. The terms associated 149 with serine-type peptidase activity were also examined, and the results revealed top 150 significant terms of "virus receptor activity," "serine-type endopeptidase inhibitor 151 activity," "serine-type peptidase activity," "peptidase activity," and "receptor binding" 152 (Figure 2A) . 153

Significant gene populations (fold change > or < 1.5 and normalized data (log2) > or 154 < 2.0) in S. epidermidis-inoculated NHNE cells were compared to those from NHNE 155 cells without S. epidermidis inoculation. The scatter plot data of genes associated 156 with virus receptor activity revealed lower ACE2 gene expression in S. epidermidis-157 inoculated NHNE cells relative to the control ( Figure 2B ). We also analyzed 158 significant gene expression associated with serine-type peptidase activity and scatter 159 plot data showed that PLAU, TMPRSS11E, KLK7, KLK8, and HTRA2 mRNA levels 160 were higher in S. epidermidis-inoculated NHNE cells. However, the principal host 161 protease for SARS-CoV-2 invasion, TMPRSS2 transcription was reduced in NHNE 162 cells after S. epidermidis inoculation (0.59-fold decrease) relative to the control 163 (Figure 2C at 1 dpi (4.210 9 ; Figure 3A ). The mean colony forming unit (CFU) of S. epidermidis 187 was significantly increased in the supernatant of NHNE cells until 1 day (2.4x10 4 188 CFU/ml) after S. epidermidis inoculation ( Figure 3B ). Subsequently, we tested 189 whether S. epidermidis-inoculated NHNE cells exhibited the decrease of ACE2 and 190 TMPRSS2 as shown in scRNA-seq data. Real-time PCR results showed that ACE2 191 mRNA was reduced significantly at 1 day after S. epidermidis inoculation ( Figure 3C ) 192 and ACE2 protein expression was also significantly decreased in the cell lysate of S. 193 epidermidis-inoculated NHNE cells ( Figure 3D ). In addition, a gradual decrease of 194 The present study is somewhat limited. We did not perform any experiments with 306 SARS-CoV-2 and did not prove whether S. epidermidis and S. aureus could reduce 307 the host entry of SARS-CoV-2 to nasal epithelium or not. Furthermore, we did not 308 directly demonstrate the key signaling between reduction of ACE2 and TMPRSS2 Library construction was performed using 10X Chromium Single Cell 3' reagent kits 500 v3.1. Samples were sequenced using the Illumina NovaSeq 6000 platform, and 501 preliminary sequencing results were converted to FASTQ files using the Cell Ranger 502 pipeline. We followed the 10x Genomics standard sequence protocol by trimming the 503 barcode and unique molecular identifier (UMI) end to 26 bp and the mRNA end to 98 504 bp. Then, the FASTQ files were aligned to the human reference genome (GRCh38). 505

Subsequently, we applied Cell Ranger for preliminary data analysis and generated a 506 file that contained a barcode table, a gene table, 2% SDS, 4% β-mercaptoethanol, 0.02% bromophenol blue, and 10% glycerol). Cell 541 lysate (30 μg of protein) was electrophoresed in 10% SDS gels and transferred to 542 7.5), 150 mM NaCl) for 1 h at room temperature. The membrane was incubated 544 overnight with primary antibody (1:500) in Tween-Tris buffered saline (TTBS; 0.5% 545 Tween-20 in TBS). After washing with TTBS, the blot was incubated for 1 h at room 546 temperature with secondary anti-rabbit antibody (1:1000, Cell Signaling, Beverly, MA, 547 USA) in TTBS and was visualized using an ECL system (Amersham, Little Chalfont, 548 UK). 549 550 Immunohistochemistry 551

Human nasal mucosa was obtained from the middle turbinate of healthy subjects 552 who had nasal surgery due to nasal obstruction, and paraffin block slides were 553 prepared for immunostaining. Immunohistochemical analysis was performed using 554 ACE2 antibody (1:200, Cell Signaling Technology, MA, USA) to determine protein 555 level in the nasal mucosa. Briefly, 5-μm sections were fixed in acetone for 10 min at 556 room temperature (RT). Non-specific protein staining was blocked with goat serum. 557

Slides were treated with 0.5% hydrogen peroxidase to eliminate endogenous 558 peroxidase for 10 min at RT and incubated with primary antibody overnight at RT. 559

After washing with Tris-buffered saline (TBS, pH 7.5), slides were incubated with 560 horseradish peroxidase-conjugated secondary antibody (Thermo, Asheville, NC, 561 USA) for 30 min at RT. Chromogen (3-amino-9-ethylcarbazole) was applied for 562 visualization. Glass cover slides were mounted and examined with optical 563 microscopy and ACE2 protein was detected with DAB (3,3'-diaminobenzidine) 564 chromogen staining. The same procedures were performed using non-immunized 565 mouse IgG (purified IgG, Sigma) instead of primary antibody as a negative control. 

Initial Influenza Virus Replication Can Be Limited in Allergic Asthma Through References 580

Integrating 581 single-cell transcriptomic data across different conditions, technologies, and species

serine-type 620 peptidase activity" in S. epidermidis-inoculated NHNE cells. Scatter plots indicating 621 enriched genes related with (B) virus receptor activity, and (C) serine-type peptidase 622 in S. epidermidis-inoculated NHNE cells. Heatmap depicting the expression levels of 623 genes related with (D) virus receptor activity and (E) serine-type peptidase activity 624 differentially expressed in S

Figure 3. S. epidermidis promotes reduction of ACE2 and TMPRSS2 628 expression in the nasal epithelium

NHNE cells from five healthy volunteers were inoculated with human nasal S

epidermidis at an MOI of 0.25. (A) femA mRNA level was monitored by real-time

(B) S. epidermidis CFUs were determined over the course 632 of 24 h in the supernatant of S. epidermidis-inoculated NHNE cells. (C) ACE2 mRNA 633 level was determined by real-time PCR until 24 h. (D) TMPRSS2 mRNA level was 634 determined by real-time

Schematic illustration of step-by-step experiments from S

epidermidis isolation to inoculation of NHNE cells

For bacterial colony isolation, nasal mucus of healthy subjects was obtained using a 710 cotton swab and was used to inoculate Lysogeny Broth (LB) plates. After 2 days of 711 incubation, bacterial colonies were obtained from the LB plates, S. epidermidis 712 colonies were identified using GS-FLX 454 pyrosequencing by 16S rRNA gene 713 amplification, and S