Discovery of a novel inhibitor of macropinocytosis with antiviral activity.

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using high-throughput microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as mpox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.

A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens.

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.

Azido-Ceramides, a Tool to Analyse SARS-CoV-2 Replication and Inhibition-SARS-CoV-2 Is Inhibited by Ceramides.

Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.

Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo.

Like human Th1 cells, mouse Th1 cells also secrete IFN-γ upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans.

Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis.

Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels.

Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy.

The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50-60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements.

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.

High-throughput measurement of the content and properties of nano-sized bioparticles with single-particle profiler.

We introduce a method, single-particle profiler, that provides single-particle information on the content and biophysical properties of thousands of particles in the size range 5-200 nm. We use our single-particle profiler to measure the messenger RNA encapsulation efficiency of lipid nanoparticles, the viral binding efficiencies of different nanobodies, and the biophysical heterogeneity of liposomes, lipoproteins, exosomes and viruses.

Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data.

Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear-nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

The Acid Ceramidase Is a SARS-CoV-2 Host Factor.

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

Superresolution Microscopy of Sphingolipids.

This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.

Sphingolipids: Effectors and Achilles Heals in Viral Infections?

As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention.

Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria.

Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.

Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM.

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell-cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus.

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1ß, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56brightCD16dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1ß, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites.

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the ß2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.

Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria.

Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C6 and long-chain C16-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C6-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω-azido-C6-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω-azido-C6-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.