A phospho-tyrosine-based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity.

Toll-like receptors (TLRs) recognize pathogen- and host-derived factors and control immune responses via the adaptor protein MyD88 and members of the interferon regulatory transcription factor (IRF) family. IRFs orchestrate key effector functions, including cytokine release, cell differentiation, and, under certain circumstances, inflammation pathology. Here, we show that IRF activity is generically controlled by the Src kinase family member LYN, which phosphorylates all TLR-induced IRFs at a conserved tyrosine residue, resulting in K48-linked polyubiquitination and proteasomal degradation of IRFs. We further show that LYN activity is controlled by the upstream kinase C-terminal Src kinase (CSK), whose activity, in turn, is controlled by the adaptor protein SPOP, which serves as molecular bridge to recruit CSK into the TLR signaling complex and to activate CSK catalytic activity. Consistently, deletion of SPOP or CSK results in increased LYN activity, LYN-directed IRF degradation, and inhibition of IRF transcriptional activity. Together, the data reveal a key regulatory mechanism for IRF family members controlling TLR biology.

A rapid and affordable point of care test for antibodies against SARS-CoV-2 based on hemagglutination and artificial intelligence interpretation.

Diagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as 'NanoSpot.ai', is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens.

A Rapid and Affordable Point-of-care Test for Detection of SARS-Cov-2-Specific Antibodies Based on Hemagglutination and Artificial Intelligence-Based Image Interpretation.

Diagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test (HAT) that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization (EUA) demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as 'NanoSpot.ai', is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens.

Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

Quantitative proteomic analysis of the influenza A virus nonstructural proteins NS1 and NS2 during natural cell infection identifies PACT as an NS1 target protein and antiviral host factor.

UNLABELLED: Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE: Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.

Induction of bone loss by pathobiont-mediated Nod1 signaling in the oral cavity.

Periodontitis is a common disease that is characterized by resorption of the alveolar bone and mediated by commensal bacteria that trigger host immune responses and bone destruction through unidentified mechanisms. We report that Nod1, an innate intracellular host receptor for bacterial peptidoglycan-related molecules, is critical for commensal-induced periodontitis in a mouse model. Mice lacking Nod1 exhibit reduced bone resorption as well as impaired recruitment of neutrophils to gingival tissues and osteoclasts to the alveolar bone, which mediate tissue and bone destruction. Further analysis showed that accumulation of a Nod1-stimulating commensal bacterium, NI1060, at gingival sites was sufficient to induce neutrophil recruitment and bone resorption. Genomic sequencing revealed that NI1060 is a mouse-specific bacterium that is related to bacteria associated with the development of aggressive periodontitis in humans. These findings provide insight into commensal-host interactions contributing to periodontitis and identify a potential target for preventing this common oral disease.

Identification of a TLR2-stimulating lipoprotein in Bacteroides fragilis JCM 11019 (NCTC 9343).

Bacteroides fragilis is found among the normal intestinal flora and is involved in host immunostimulation via TLR2. Its cell surface components, such as LPS and capsular polysaccharides, were reported to participate in host immunostimulation. In this study, we report on the existence of a lipoprotein that acts as a TLR2 stimulant in B. fragilis. The TLR2-stimulating lipoprotein was obtained using Triton X-114-water phase partitioning followed by preparative SDS-PAGE. Its N-terminal hydrophobic peptide, which was separated from a tryptic digest, was characterized as a triacylated lipopeptide, and the lipoprotein was identified as BF1333 by mass spectrometry of Asp-N-digested peptides. These results showed that the lipoprotein acts as a TLR2-stimulating component in B. fragilis.

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1ß in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.

Transitions in oral and intestinal microflora composition and innate immune receptor-dependent stimulation during mouse development.

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.

Characterization of N-terminal structure of TLR2-activating lipoprotein in Staphylococcus aureus.

Staphylococcus aureus is known to activate mammalian immune cells through Toll-like receptor 2 (TLR2). We recently demonstrated that a lipoprotein fraction obtained from S. aureus by Triton X-114 phase partitioning is a potent activator of TLR2. In this study, we separated TLR2-activating lipoproteins expressed in S. aureus and characterized an N-terminal structure. The lipoprotein fraction of S. aureus was prepared by glass bead disruption followed by Triton X-114 phase partitioning. The TLR2-activating molecules were mainly detected in the mass range of 30-35 kDa. Seven lipoproteins were identified by the mass spectra of their tryptic digests. Among them, three lipoproteins were separated by preparative SDS-PAGE and proved to activate TLR2. After digestion with trypsin in the presence of sodium deoxycholate, the N terminus of the lipopeptide was isolated from lipoprotein SAOUHSC_02699 by normal phase high pressure liquid chromatography and characterized as an S-(diacyloxypropyl)cystein-containing peptide using tandem mass spectra. The synthetic lipopeptide counterpart also stimulated the cells via TLR2. These results showed that the diacylated lipoprotein from S. aureus acts as a TLR2 ligand in mammalian cells.

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus.

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.

Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components.

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of Toll-like receptor 2 (TLR2). In this study, we demonstrated that lipoproteins obtained from S. aureus are potent activators of TLR2. A fraction obtained by Triton X-114 phase partitioning activated cells through TLR2. The fraction contained proteins and LTA. The activity was detected in compounds in a mass range of 12-40 kDa. Proteinase K digested the active compounds into lower molecular weight active materials <10 kDa. In contrast, hydrofluoric acid treatment, which decomposes LTA, did not alter the molecular mass of the active compounds. Further, most of the activity was abrogated by lipoprotein lipase digestion. These results suggested that lipoproteins are predominant TLR2 ligands in S. aureus cell wall components.