Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Correction: State-of-the-art analytical methods of viral infections in human lung organoids.

[This corrects the article DOI: 10.1371/journal.pone.0276115.].

Ex vivo infection model for Francisella using human lung tissue.

Introduction: Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. Method: In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Results: Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1ß, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. Conclusion: We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less- or non-virulent strains and can be used to investigate the role of specific virulence factors.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

CLEC12A Binds to Legionella pneumophila but Has No Impact on the Host's Antibacterial Response.

Legionella pneumophila is an intracellular pathogen that can cause severe pneumonia after the inhalation of contaminated aerosols and replication in alveolar macrophages. Several pattern recognition receptors (PRRs) have been identified that contribute to the recognition of L. pneumophila by the innate immune system. However, the function of the C-type lectin receptors (CLRs), which are mainly expressed by macrophages and other myeloid cells, remains largely unexplored. Here, we used a library of CLR-Fc fusion proteins to search for CLRs that can bind the bacterium and identified the specific binding of CLEC12A to L. pneumophila. Subsequent infection experiments in human and murine macrophages, however, did not provide evidence for a substantial role of CLEC12A in controlling innate immune responses to the bacterium. Consistently, antibacterial and inflammatory responses to Legionella lung infection were not significantly influenced by CLEC12A deficiency. Collectively, CLEC12A is able to bind to L. pneumophila-derived ligands but does not appear to play a major role in the innate defense against L. pneumophila.

A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine.

SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8 µM at a molecular weight of 211 g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease.

State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

Transcriptomic comparison of primary human lung cells with lung tissue samples and the human A549 lung cell line highlights cell type specific responses during infections with influenza A virus.

Influenza A virus (IAV) causes pandemics and annual epidemics of severe respiratory infections. A better understanding of the molecular regulation in tissue and cells upon IAV infection is needed to thoroughly understand pathogenesis. We analyzed IAV replication and gene expression induced by IAV strain H3N2 Panama in isolated primary human alveolar epithelial type II cells (AECIIs), the permanent A549 adenocarcinoma cell line, alveolar macrophages (AMs) and explanted human lung tissue by bulk RNA sequencing. Primary AECII exhibit in comparison to AM a broad set of strongly induced genes related to RIG-I and interferon (IFN) signaling. The response of AECII was partly mirrored in A549 cells. In human lung tissue, we observed induction of genes unlike in isolated cells. Viral RNA was used to correlate host cell gene expression changes with viral burden. While relative induction of key genes was similar, gene abundance was highest in AECII cells and AM, while weaker in the human lung (due to less IAV replication) and A549 cells (pointing to their limited suitability as a model). Correlation of host gene induction with viral burden allows a better understanding of the cell-type specific induction of pathways and a possible role of cellular crosstalk requiring intact tissue.

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Single-cell transcriptomics reveals common epithelial response patterns in human acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. METHODS: We performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 h after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. RESULTS: High-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs, and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. CONCLUSIONS: The study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Introducing quality measures in an academic research consortium: Lessons and recommendation from implementing an ad hoc quality management system for organ model research: Lessons and recommendation from implementing an ad hoc quality management system for organ model research.

Lessons from implementing quality control systems in an academic research consortium to improve Good Scientific Practice and reproducibility.

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics.

Single-cell ribonucleic acid sequencing is becoming widely employed to study biological processes at a novel resolution depth. The ability to analyse transcriptomes of multiple heterogeneous cell types in parallel is especially valuable for cell-focused lung research where a variety of resident and recruited cells are essential for maintaining organ functionality. We compared the single-cell transcriptomes from publicly available and unpublished datasets of the lungs in six different species: human (Homo sapiens), African green monkey (Chlorocebus sabaeus), pig (Sus domesticus), hamster (Mesocricetus auratus), rat (Rattus norvegicus) and mouse (Mus musculus) by employing RNA velocity and intercellular communication based on ligand-receptor co-expression, among other techniques. Specifically, we demonstrated a workflow for interspecies data integration, applied a single unified gene nomenclature, performed cell-specific clustering and identified marker genes for each species. Overall, integrative approaches combining newly sequenced as well as publicly available datasets could help identify species-specific transcriptomic signatures in both healthy and diseased lung tissue and select appropriate models for future respiratory research.

Analysis of Severe Acute Respiratory Syndrome 2 Replication in Explant Cultures of the Human Upper Respiratory Tract Reveals Broad Tissue Tropism of Wild-Type and B.1.1.7 Variant Viruses.

BACKGROUND: The upper respiratory tract (URT) is the primary entry site for severe acute respiratory syndrome 2 (SARS-CoV-2) and other respiratory viruses, but its involvement in viral amplification and pathogenesis remains incompletely understood. METHODS: In this study, we investigated primary nasal epithelial cultures, as well as vital explanted tissues, to scrutinize the tropism of wild-type SARS-CoV-2 and the recently emerged B.1.1.7 variant. RESULTS: Our analyses revealed a widespread replication competence of SARS-CoV-2 in polarized nasal epithelium as well as in the examined URT and salivary gland tissues, which was also shared by the B.1.1.7 virus. CONCLUSIONS: In our analyses, we highlighted the active role of these anatomic sites in coronavirus disease 2019.

Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors.

Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1ß and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses.

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals.

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.