@article{BoertleinSchumacherKleuseretal.2019, author = {B{\"o}rtlein, Charlene and Schumacher, Fabian and Kleuser, Burkhard and D{\"o}lken, Lars and Avota, Elita}, title = {Role of neutral sphingomyelinase-2 (NSM 2) in the control of T cell plasma membrane lipid composition and cholesterol homeostasis}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, number = {226}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00226}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190596}, year = {2019}, abstract = {The activity of neutral sphingomyelinase-2 (NSM2) to catalyze the conversion of sphingomyelin (SM) to ceramide and phosphocholine at the cytosolic leaflet of plasma membrane (PM) is important in T cell receptor (TCR) signaling. We recently identified PKCζ as a major NSM2 downstream effector which regulates microtubular polarization. It remained, however, unclear to what extent NSM2 activity affected overall composition of PM lipids and downstream effector lipids in antigen stimulated T cells. Here, we provide a detailed lipidomics analyses on PM fractions isolated from TCR stimulated wild type and NSM2 deficient (ΔNSM) Jurkat T cells. This revealed that in addition to that of sphingolipids, NSM2 depletion also affected concentrations of many other lipids. In particular, NSM2 ablation resulted in increase of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) which both govern PM biophysical properties. Crucially, TCR dependent upregulation of the important T cell signaling lipid diacylglycerol (DAG), which is fundamental for activation of conventional and novel PKCs, was abolished in ΔNSM cells. Moreover, NSM2 activity was found to play an important role in PM cholesterol transport to the endoplasmic reticulum (ER) and production of cholesteryl esters (CE) there. Most importantly, CE accumulation was essential to sustain human T cell proliferation. Accordingly, inhibition of CE generating enzymes, the cholesterol acetyltransferases ACAT1/SOAT1 and ACAT2/SOAT2, impaired TCR driven expansion of both CD4\(^+\) and CD8\(^+\) T cells. In summary, our study reveals an important role of NSM2 in regulating T cell functions by its multiple effects on PM lipids and cholesterol homeostasis.}, language = {en} } @article{SolgerKunzFinketal.2020, author = {Solger, Franziska and Kunz, Tobias C. and Fink, Julian and Paprotka, Kerstin and Pfister, Pauline and Hagen, Franziska and Schumacher, Fabian and Kleuser, Burkhard and Seibel, J{\"u}rgen and Rudel, Thomas}, title = {A Role of Sphingosine in the Intracellular Survival of Neisseria gonorrhoeae}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {10}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2020.00215}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204111}, year = {2020}, abstract = {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.}, language = {en} } @article{GrafenSchumacherChithelenetal.2019, author = {Grafen, Anika and Schumacher, Fabian and Chithelen, Janice and Kleuser, Burkhard and Beyersdorf, Niklas and Schneider-Schaulies, J{\"u}rgen}, title = {Use of acid ceramidase and sphingosine kinase inhibitors as antiviral compounds against measles virus infection of lymphocytes in vitro}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, number = {218}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00218}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-196099}, year = {2019}, abstract = {As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90\%) in PBL and 70-80\% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5-6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.}, language = {en} } @article{PetersKaiserFinketal.2021, author = {Peters, Simon and Kaiser, Lena and Fink, Julian and Schumacher, Fabian and Perschin, Veronika and Schlegel, Jan and Sauer, Markus and Stigloher, Christian and Kleuser, Burkhard and Seibel, Juergen and Schubert-Unkmeir, Alexandra}, title = {Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria}, series = {Scientific Reports}, volume = {11}, journal = {Scientific Reports}, number = {1}, doi = {10.1038/s41598-021-83813-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259147}, pages = {4300}, year = {2021}, abstract = {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.}, language = {en} } @article{DerakhshaniKurzJaptoketal.2019, author = {Derakhshani, Shaghayegh and Kurz, Andreas and Japtok, Lukasz and Schumacher, Fabian and Pilgram, Lisa and Steinke, Maria and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, title = {Measles virus infection fosters dendritic cell motility in a 3D environment to enhance transmission to target cells in the respiratory epithelium}, series = {Frontiers in Immunology}, volume = {10}, journal = {Frontiers in Immunology}, number = {1294}, doi = {10.3389/fimmu.2019.01294}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201818}, year = {2019}, abstract = {Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit.}, language = {en} } @article{SchneiderSchauliesSchumacherWiggeretal.2021, author = {Schneider-Schaulies, Sibylle and Schumacher, Fabian and Wigger, Dominik and Sch{\"o}l, Marie and Waghmare, Trushnal and Schlegel, Jan and Seibel, J{\"u}rgen and Kleuser, Burkhard}, title = {Sphingolipids: effectors and Achilles heals in viral infections?}, series = {Cells}, volume = {10}, journal = {Cells}, number = {9}, issn = {2073-4409}, doi = {10.3390/cells10092175}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245151}, year = {2021}, abstract = {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.}, language = {en} } @article{EderHollmannMandasarietal.2022, author = {Eder, Sascha and Hollmann, Claudia and Mandasari, Putri and Wittmann, Pia and Schumacher, Fabian and Kleuser, Burkhard and Fink, Julian and Seibel, J{\"u}rgen and Schneider-Schaulies, J{\"u}rgen and Stigloher, Christian and Beyersdorf, Niklas and Dembski, Sofia}, title = {Synthesis and characterization of ceramide-containing liposomes as membrane models for different T cell subpopulations}, series = {Journal of Functional Biomaterials}, volume = {13}, journal = {Journal of Functional Biomaterials}, number = {3}, issn = {2079-4983}, doi = {10.3390/jfb13030111}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286130}, year = {2022}, abstract = {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.}, language = {en} }