TY - JOUR A1 - Götz, Ralph A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Solger, Franziska A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Sauer, Markus T1 - Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy JF - Nature Communications N2 - 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 4x to 10x 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 10-20nm. 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 +/- 7.7nm. Imaging of lipid bilayers using light microscopy is challenging. Here the authors label cells using a short chain click-compatible ceramide to visualize mammalian and bacterial membranes with expansion microscopy. KW - nanoscale imaging KW - bacterial infection KW - sphingolipid expansion microscopy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231248 VL - 11 ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A Role of Sphingosine in the Intracellular Survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204111 SN - 2235-2988 VL - 10 ER - TY - JOUR A1 - Schneider-Schaulies, Sibylle A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schöl, Marie A1 - Waghmare, Trushnal A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kleuser, Burkhard T1 - Sphingolipids: effectors and Achilles heals in viral infections? JF - Cells N2 - 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. KW - glycosphingolipids KW - ceramides KW - sphingosine 1-phosphate KW - sphingomyelinase KW - HIV KW - SARS-CoV-2 KW - measles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245151 SN - 2073-4409 VL - 10 IS - 9 ER - TY - JOUR A1 - Becam, Jérôme A1 - Walter, Tim A1 - Burgert, Anne A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Schubert-Unkmeir, Alexandra T1 - Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria JF - Scientific Reports N2 - 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 C\(_{6}\) and long-chain C\(_{16}\)-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 C\(_{6}\)-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-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-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. KW - ceramide analogs KW - Neisseria KW - ceramide Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159367 VL - 7 ER - TY - JOUR A1 - Eder, Sascha A1 - Hollmann, Claudia A1 - Mandasari, Putri A1 - Wittmann, Pia A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Fink, Julian A1 - Seibel, Jürgen A1 - Schneider-Schaulies, Jürgen A1 - Stigloher, Christian A1 - Beyersdorf, Niklas A1 - Dembski, Sofia T1 - Synthesis and characterization of ceramide-containing liposomes as membrane models for different T cell subpopulations JF - Journal of Functional Biomaterials N2 - A fine balance of regulatory (T\(_{reg}\)) and conventional CD4\(^+\) T cells (T\(_{conv}\)) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the T\(_{reg}\)/T\(_{conv}\) balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse T\(_{reg}\) and T\(_{conv}\) with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C\(_{16}\)-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into T\(_{reg}\) and T\(_{conv}\) reflect differences in the ceramide content of cellular membranes. KW - liposome KW - ceramide KW - cell membrane model Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286130 SN - 2079-4983 VL - 13 IS - 3 ER - TY - JOUR A1 - Geiger, Nina A1 - Kersting, Louise A1 - Schlegel, Jan A1 - Stelz, Linda A1 - Fähr, Sofie A1 - Diesendorf, Viktoria A1 - Roll, Valeria A1 - Sostmann, Marie A1 - König, Eva-Maria A1 - Reinhard, Sebastian A1 - Brenner, Daniela A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Bodem, Jochen T1 - The acid ceramidase is a SARS-CoV-2 host factor JF - Cells N2 - 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. KW - SARS-CoV-2 KW - ceramides KW - ceramidase KW - fluoxetine KW - acid sphingomyelinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286105 SN - 2073-4409 VL - 11 IS - 16 ER - TY - JOUR A1 - Brenner, Daniela A1 - Geiger, Nina A1 - Schlegel, Jan A1 - Diesendorf, Viktoria A1 - Kersting, Louise A1 - Fink, Julian A1 - Stelz, Linda A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Bodem, Jochen A1 - Seibel, Jürgen T1 - Azido-ceramides, a tool to analyse SARS-CoV-2 replication and inhibition — SARS-CoV-2 is inhibited by ceramides JF - International Journal of Molecular Sciences N2 - 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. KW - ceramides KW - SARS-CoV-2 KW - azido-ceramides KW - sphingolipids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313581 SN - 1422-0067 VL - 24 IS - 8 ER -