Bacnet: a user-friendly platform for building multi-omics websites.

SUMMARY: To face up to the exponential growth of heterogeneous datasets of various organisms, we developed a user-friendly platform for building multi-omics websites, which is named Bacnet. This platform helps bioinformaticians to construct four key web interfaces: (i) an interactive genome viewer; (ii) an expression and protein atlas; (iii) an interface for analysis of co-expression network; (iv) an interface for exploring homolog presence. We believe our platform will help the bioinformaticians to construct personalized user interfaces dedicated to biologists studying non-reference organisms. AVAILABILITY AND IMPLEMENTATION: https://github.com/becavin-lab/bacnet; Java; Eclipse RAP; Eclipse RCP.

A Single-Cell Atlas of the Human Healthy Airways.

Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors.

High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.

Unraveling the evolution and coevolution of small regulatory RNAs and coding genes in Listeria.

BACKGROUND: Small regulatory RNAs (sRNAs) are widely found in bacteria and play key roles in many important physiological and adaptation processes. Studying their evolution and screening for events of coevolution with other genomic features is a powerful way to better understand their origin and assess a common functional or adaptive relationship between them. However, evolution and coevolution of sRNAs with coding genes have been sparsely investigated in bacterial pathogens. RESULTS: We designed a robust and generic phylogenomics approach that detects correlated evolution between sRNAs and protein-coding genes using their observed and inferred patterns of presence-absence in a set of annotated genomes. We applied this approach on 79 complete genomes of the Listeria genus and identified fifty-two accessory sRNAs, of which most were present in the Listeria common ancestor and lost during Listeria evolution. We detected significant coevolution between 23 sRNA and 52 coding genes and inferred the Listeria sRNA-coding genes coevolution network. We characterized a main hub of 12 sRNAs that coevolved with genes encoding cell wall proteins and virulence factors. Among them, an sRNA specific to L. monocytogenes species, rli133, coevolved with genes involved either in pathogenicity or in interaction with host cells, possibly acting as a direct negative post-transcriptional regulation. CONCLUSIONS: Our approach allowed the identification of candidate sRNAs potentially involved in pathogenicity and host interaction, consistent with recent findings on known pathogenicity actors. We highlight four sRNAs coevolving with seven internalin genes, some of which being important virulence factors in Listeria.

A riboswitch-regulated antisense RNA in Listeria monocytogenes.

Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs.

Evidence of IL-17, IP-10, and IL-10 involvement in multiple-organ dysfunction and IL-17 pathway in acute renal failure associated to Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria in India is characterized by high rates of severe disease, with multiple organ dysfunction (MOD)-mainly associated with acute renal failure (ARF)-and increased mortality. The objective of this study is to identify cytokine signatures differentiating severe malaria patients with MOD, cerebral malaria (CM), and cerebral malaria with MOD (CM-MOD) in India. We have previously shown that two cytokines clusters differentiated CM from mild malaria in Maharashtra. Hence, we also aimed to determine if these cytokines could discriminate malaria subphenotypes in Odisha. METHODS: P. falciparum malaria patients from the SCB Medical College Cuttack in the Odisha state in India were enrolled along with three sets of controls: healthy individuals, patients with sepsis and encephalitis (n = 222). We determined plasma concentrations of pro- and anti-inflammatory cytokines and chemokines for all individuals using a multiplex assay. We then used an ensemble of statistical analytical methods to ascertain whether particular sets of cytokines/chemokines were predictors of severity or signatures of a disease category. RESULTS: Of the 26 cytokines/chemokines tested, 19 increased significantly during malaria and clearly distinguished malaria patients from controls, as well as sepsis and encephalitis patients. High amounts of IL-17, IP-10, and IL-10 predicted MOD, decreased IL-17 and MIP-1α segregated CM-MOD from MOD, and increased IL-12p40 differentiated CM from CM-MOD. Most severe malaria patients with ARF exhibited high levels of IL-17. CONCLUSION: We report distinct differences in cytokine production correlating with malarial disease severity in Odisha and Maharashtra populations in India. We show that CM, CM-MOD and MOD are clearly distinct malaria-associated pathologies. High amounts of IL-17, IP-10, and IL-10 were predictors of MOD; decreased IL-17 and MIP-1α separated CM-MOD from MOD; and increased IL-12p40 differentiated CM from CM-MOD. Data also suggest that the IL-17 pathway may contribute to malaria pathogenesis via different regulatory mechanisms and may represent an interesting target to mitigate the pathological processes in malaria-associated ARF.

Multiparameter single-cell profiling of human CD4+FOXP3+ regulatory T-cell populations in homeostatic conditions and during graft-versus-host disease.

Understanding the heterogeneity of human CD4+FOXP3+ regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4+ effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-γ showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4+FOXP3+ T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4+FOXP3+ T cells, explains the remarkable complexity and functional diversity of human Tregs.

Asymptomatic Plasmodium falciparum infection in children is associated with increased auto-antibody production, high IL-10 plasma levels and antibodies to merozoite surface protein 3.

BACKGROUND: Mechanisms of acquired protection to malaria in asymptomatic Plasmodium falciparum carriers are only partially understood. Among them, the role plays by the self-reactive antibodies has not been clarified yet. In this study, the relationship between repertoires of circulating self-reactive and parasite-specific immunoglobulin G (IgG), their correlation with cytokine levels, and their association with protection against malaria was investigated in asymptomatic Plasmodium falciparum-infected Gabonese children. METHODS: The diversity of P. falciparum-specific antibody repertoire was analysed using a protein micro-array immunoassay, the total auto-antibody repertoire by quantitative immunoblotting and circulating cytokine levels were measured by ELISA in endemic controls (EC) and P. falciparum-infected children from Gabon with asymptomatic (AM) or mild malaria (MM). The association of self- and parasite-specific antibody repertoires with circulating cytokines was evaluated using single linkage hierarchical clustering, Kruskal-Wallis tests and Spearman's rank correlation. RESULTS: Children with AM exhibited an IgG response to merozoite surface protein 3 (MSP3) but not to MSP1-19, although their levels of total P. falciparum-specific IgG were similar to those in the MM group. Moreover, the asymptomatic children had increased levels of autoantibodies recognising brain antigens. In addition, a correlation between IL-10 levels and parasite load was found in AM and MM children. These two groups also exhibited significant correlations between plasma levels of IL-10 and IFN-γ with age and with total plasma IgG levels. IL-10 and IFN-γ levels were also associated with auto-antibody responses in AM. CONCLUSIONS: Altogether, these results indicate that a self-reactive polyclonal response associated with increased IgG to MSP3 and high plasma levels of IL-10 and IFN-γ may contribute to protective immune mechanisms triggered in asymptomatic P. falciparum infection in Gabonese children.

Impact of lactobacilli on orally acquired listeriosis.

Listeria monocytogenes is a foodborne pathogen that crosses the intestinal barrier and disseminates within the host. Here, we report a unique comprehensive analysis of the impact of two Lactobacillus species, Lactobacillus paracasei CNCM I-3689 and Lactobacillus casei BL23, on L. monocytogenes and orally acquired listeriosis in a gnotobiotic humanized mouse model. We first assessed the effect of treatment with each Lactobacillus on L. monocytogenes counts in host tissues and showed that each decreases L. monocytogenes systemic dissemination in orally inoculated mice. A whole genome intestinal transcriptomic analysis revealed that each Lactobacillus changes expression of a specific subset of genes during infection, with IFN-stimulated genes (ISGs) being the most affected by both lactobacilli. We also examined microRNA (miR) expression and showed that three miRs (miR-192, miR-200b, and miR-215) are repressed during L. monocytogenes infection. Treatment with each Lactobacillus increased miR-192 expression, whereas only L. casei association increased miR-200b and miR-215 expression. Finally, we showed that treatment with each Lactobacillus significantly reshaped the L. monocytogenes transcriptome and up-regulated transcription of L. monocytogenes genes encoding enzymes allowing utilization of intestinal carbon and nitrogen sources in particular genes involved in propanediol and ethanolamine catabolism and cobalamin biosynthesis. Altogether, these data reveal that the modulation of L. monocytogenes infection by treatment with lactobacilli correlates with a decrease in host gene expression, in particular ISGs, miR regulation, and a dramatic reshaping of L. monocytogenes transcriptome.

Comparative transcriptomics of pathogenic and non-pathogenic Listeria species.

Listeria monocytogenes is a human, food-borne pathogen. Genomic comparisons between L. monocytogenes and Listeria innocua, a closely related non-pathogenic species, were pivotal in the identification of protein-coding genes essential for virulence. However, no comprehensive comparison has focused on the non-coding genome. We used strand-specific cDNA sequencing to produce genome-wide transcription start site maps for both organisms, and developed a publicly available integrative browser to visualize and analyze both transcriptomes in different growth conditions and genetic backgrounds. Our data revealed conservation across most transcripts, but significant divergence between the species in a subset of non-coding RNAs. In L. monocytogenes, we identified 113 small RNAs (33 novel) and 70 antisense RNAs (53 novel), significantly increasing the repertoire of ncRNAs in this species. Remarkably, we identified a class of long antisense transcripts (lasRNAs) that overlap one gene while also serving as the 5' UTR of the adjacent divergent gene. Experimental evidence suggests that lasRNAs transcription inhibits expression of one operon while activating the expression of another. Such a lasRNA/operon structure, that we named 'excludon', might represent a novel form of regulation in bacteria.

Yersiniomics, a Multi-Omics Interactive Database for Yersinia Species.

The genus Yersinia includes a large variety of nonpathogenic and life-threatening pathogenic bacteria, which cause a broad spectrum of diseases in humans and animals, such as plague, enteritis, Far East scarlet-like fever (FESLF), and enteric redmouth disease. Like most clinically relevant microorganisms, Yersinia spp. are currently subjected to intense multi-omics investigations whose numbers have increased extensively in recent years, generating massive amounts of data useful for diagnostic and therapeutic developments. The lack of a simple and centralized way to exploit these data led us to design Yersiniomics, a web-based platform allowing straightforward analysis of Yersinia omics data. Yersiniomics contains a curated multi-omics database at its core, gathering 200 genomic, 317 transcriptomic, and 62 proteomic data sets for Yersinia species. It integrates genomic, transcriptomic, and proteomic browsers, a genome viewer, and a heatmap viewer to navigate within genomes and experimental conditions. For streamlined access to structural and functional properties, it directly links each gene to GenBank, the Kyoto Encyclopedia of Genes and Genomes (KEGG), UniProt, InterPro, IntAct, and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and each experiment to Gene Expression Omnibus (GEO), the European Nucleotide Archive (ENA), or the Proteomics Identifications Database (PRIDE). Yersiniomics provides a powerful tool for microbiologists to assist with investigations ranging from specific gene studies to systems biology studies. IMPORTANCE The expanding genus Yersinia is composed of multiple nonpathogenic species and a few pathogenic species, including the deadly etiologic agent of plague, Yersinia pestis. In 2 decades, the number of genomic, transcriptomic, and proteomic studies on Yersinia grew massively, delivering a wealth of data. We developed Yersiniomics, an interactive web-based platform, to centralize and analyze omics data sets on Yersinia species. The platform allows user-friendly navigation between genomic data, expression data, and experimental conditions. Yersiniomics will be a valuable tool to microbiologists.

An integrated cell atlas of the lung in health and disease.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Improving the efficiency of multidimensional scaling in the analysis of high-dimensional data using singular value decomposition.

MOTIVATION: Multidimensional scaling (MDS) is a well-known multivariate statistical analysis method used for dimensionality reduction and visualization of similarities and dissimilarities in multidimensional data. The advantage of MDS with respect to singular value decomposition (SVD) based methods such as principal component analysis is its superior fidelity in representing the distance between different instances specially for high-dimensional geometric objects. Here, we investigate the importance of the choice of initial conditions for MDS, and show that SVD is the best choice to initiate MDS. Furthermore, we demonstrate that the use of the first principal components of SVD to initiate the MDS algorithm is more efficient than an iteration through all the principal components. Adding stochasticity to the molecular dynamics simulations typically used for MDS of large datasets, contrary to previous suggestions, likewise does not increase accuracy. Finally, we introduce a k nearest neighbor method to analyze the local structure of the geometric objects and use it to control the quality of the dimensionality reduction. RESULTS: We demonstrate here the, to our knowledge, most efficient and accurate initialization strategy for MDS algorithms, reducing considerably computational load. SVD-based initialization renders MDS methodology much more useful in the analysis of high-dimensional data such as functional genomics datasets.

The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing.

Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

Immunopeptidomics-based design of mRNA vaccine formulations against Listeria monocytogenes.

Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen for Listeria peptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68 Listeria immunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

HMGA1-dependent and independent 7SK RNA gene regulatory activity.

The small nuclear 7SK RNA negatively controls transcription by inactivating positive transcription elongation factor b (P-TEFb) and is an integral component of Tat-dependent and independent HIV-1 transcription initiation complexes. 7SK RNA has recently been shown to also directly control HMGA1 transcription activity. HMGA1 is a master regulator of gene expression and its deregulation is associated with virtually any type of human cancer. The degree of HMGA1 over-expression thereby correlates with tumor malignancy and metastatic potential. 7SK snRNA directly interacts through its loop2 (7SK L2) with the first A/T-hook DNA binding motif of HMGA1. We have developed several 7SK L2 RNA chimera with the Epstein Barr Virus expressed RNA 2 (EBER2) to target HMGA1 function in transcription regulation. The efficiency of interfering with HMGA1 transcription activity by the chimeric 7SK L2-EBER2 fusions by large exceeds the efficiency of 7SK wild-type RNA due to the stronger EBER2 promoter activity. Furthermore, the 7SK L2-EBER2 chimera do not interfere with P-TEFb controlled transcription elongation or the formation of 7SK sn/hnRNPs. The comparison of the effects of wild-type 7SK RNA on cellular transcriptome dynamics with those induced by the two 7SK L2 mutants as well as the changes in gene expression following inhibition of HMGA1 allow the identification and characterization of HMGA1-dependent and independent effects of 7SK snRNA. We furthermore also present evidence for P-TEFb and HMGA1-independent 7SK RNA L2 regulatory activity.

Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Transcription within condensed chromatin: Steric hindrance facilitates elongation.

During eukaryotic transcription, RNA-polymerase activity generates torsional stress in DNA, having a negative impact on the elongation process. Using our previous studies of chromatin fiber structure and conformational transitions, we suggest that this torsional stress can be alleviated, thanks to a tradeoff between the fiber twist and nucleosome conformational transitions into an activated state named "reversome". Our model enlightens the origin of polymerase pauses, and leads to the counterintuitive conclusion that chromatin-organized compaction might facilitate polymerase progression. Indeed, in a compact and well-structured chromatin loop, steric hindrance between nucleosomes enforces sequential transitions, thus ensuring that the polymerase always meets a permissive nucleosomal state.

SARS-CoV-2 Entry Genes Are Most Highly Expressed in Nasal Goblet and Ciliated Cells within Human Airways.

The SARS-CoV-2 coronavirus, the etiologic agent responsible for COVID-19 coronavirus disease, is a global threat. To better understand viral tropism, we assessed the RNA expression of the coronavirus receptor, ACE2, as well as the viral S protein priming protease TMPRSS2 thought to govern viral entry in single-cell RNA-sequencing (scRNA-seq) datasets from healthy individuals generated by the Human Cell Atlas consortium. We found that ACE2, as well as the protease TMPRSS2, are differentially expressed in respiratory and gut epithelial cells. In-depth analysis of epithelial cells in the respiratory tree reveals that nasal epithelial cells, specifically goblet/secretory cells and ciliated cells, display the highest ACE2 expression of all the epithelial cells analyzed. The skewed expression of viral receptors/entry-associated proteins towards the upper airway may be correlated with enhanced transmissivity. Finally, we showed that many of the top genes associated with ACE2 airway epithelial expression are innate immune-associated, antiviral genes, highly enriched in the nasal epithelial cells. This association with immune pathways might have clinical implications for the course of infection and viral pathology, and highlights the specific significance of nasal epithelia in viral infection. Our findings underscore the importance of the availability of the Human Cell Atlas as a reference dataset. In this instance, analysis of the compendium of data points to a particularly relevant role for nasal goblet and ciliated cells as early viral targets and potential reservoirs of SARS-CoV-2 infection. This, in turn, serves as a biological framework for dissecting viral transmission and developing clinical strategies for prevention and therapy.

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.

We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.