Biology subjects

Seibel, J.

Publications and source records attributed to Seibel, J..

13 recordsLinked to original sources

bioRxiv2026

Monitoring Sphingomyelin Biosynthesis at Nanoscale Resolution by Expansion Microscopy

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is synthesized by two isoenzymes, sphingomyelin synthase 1 and 2, located in the Golgi and in the plasma membrane. Abnormal sphingomyelin synthesis is associated with infections and diseases such as diabetes and cancer. Measuring cellular sphingomyelin synthase activity fosters our understanding of how these enzymes are involved in pathological processes and can be crucial for the identification of therapeutic compounds modifying sphingomyelin biosynthesis. We have developed a novel fluorometric assay that enables microscopic detection of cellular sphingomyelin synthase activity. We show that sphingomyelin synthases use propargyl choline and -NH2-{omega}-N3-C6-ceramide to generate trifunctional sphingomyelin, a lipid derivative detectable with spatial resolution via Forster resonance energy transfer. By combining this assay with expansion microscopy, a super-resolution imaging technique, we measured the distribution of de novo synthesized trifunctional sphingomyelin in cells at nanoscale resolution, thereby directly demonstrating sphingomyelin biosynthesis at the Golgi and the plasma membrane. By monitoring sphingomyelin biosynthesis and degradation in cells infected with the obligate intracellular pathogen Chlamydia trachomatis, we dissected the complex sphingolipid metabolization of these bacteria with unprecedented resolution. By correlating spatial metabolic information with lipidomics, we provide a powerful tool for investigating cellular sphingomyelin metabolism. TeaserCombining functional lipids, expansion microscopy and FRET helps visualise sphingolipid metabolism at unprecedented resolution.

biochemistry↗

bioRxiv2026

Chlamydia trachomatis deploys sphingolipids for genome organisation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and a leading cause of sexually transmitted infections worldwide. During its biphasic developmental cycle, infectious, non-replicative elementary bodies alternate with replicative reticulate bodies within a membrane-bound intracellular niche known as the inclusion. C. trachomatis relies heavily on host-derived metabolites, including sphingolipids, which are essential for inclusion integrity, bacterial growth and production of infectious progeny. Here, using expansion microscopy, we uncover an unexpected localization of sphingolipid derivatives within the highly condensed DNA nucleoids of elementary bodies. These sphingolipids are released from nucleoids prior to DNA decondensation during the elementary-to-reticulate body transition, the earliest phenotypic event in the complex developmental cycle of these bacteria. Thereafter, nucleoids undergo a characteristic DNA decondensation process that we visualized by expansion microscopy. By combining super-resolution imaging with a FRET-based metabolic tracking approach and lipidomics, we identified sphingomyelin derived from the sphingolipid analogues as the sphingolipid species predominantly associated with the condensing nucleoids of elementary bodies. Notably, reticulate bodies arrested in their developmental stage fail to accumulate sphingomyelin, suggesting a role for this lipid in stage-specific DNA condensation. Together, our findings suggest an unanticipated role for sphingolipids in bacterial DNA organization and developmental regulation in C. trachomatis.

microbiology↗

bioRxiv2024

Inter-kingdom signaling by the Legionella autoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP

The causative agent of Legionnaires disease, Legionella pneumophila, is an amoebae-resistant environmental bacterium, which replicates intracellularly in a distinct compartment, the "Legionella-containing vacuole" (LCV). L. pneumophila employs the -hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 promotes intracellular replication and inhibits the migration of mammalian cells and Dictyostelium discoideum. In this study, we revealed that LAI-1 and "clickable" azido-LAI-1 derivatives inhibit the migration of D. discoideum and localize to LCVs. Azido-LAI-1 colocalizes with the LCV markers calnexin, P4C, and AmtA, but not with mitochondrial or lipid droplet markers. Intriguingly, LAI-1 dependent inhibition of D. discoideum migration involves the single guanylate-binding protein (GBP), a member of the GBP family of large GTPases, which in metazoan organisms promote cell autonomous immunity. D. discoideum lacking GBP ({Delta}gnbp) allows more efficient intracellular replication of L. pneumophila, without apparently compromising LCV remodeling or integrity, and GBP-GFP localizes to the ER at LCV-ER membrane contact sites (MCS). However, the peri-LCV localization of LAI-1 and GBP is not mutually dependent. Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (but not vacuoles harboring avirulent L. pneumophila) in a GBP-dependent manner. Taken together, the work shows that LAI-1 localizes to LCVs, and LAI-1-dependent inter-kingdom signaling involves D. discoideum GBP, which localizes to LCV-ER MCS and acts as an antimicrobial factor by restricting the intracellular growth of L. pneumophila. Author SummarySmall molecule inter-kingdom signaling between pathogens and host cells represents a crucial but only partly understood aspect of microbial virulence. The amoeba-resistant opportunistic pathogen Legionella pneumophila employs the compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. In metazoan cells, the conserved and wide-spread family of guanylate-binding protein (GBP) large GTPases usually comprises several distinct paralogues, which are implicated in pathogen detection, inflammation, cell death pathways, and cell autonomous immunity. In the social amoeba Dictyostelium discoideum, only a single GBP gene of unknown function is present. Using approaches from organic chemistry, genetics, cell biology and infection biology, we reveal that GBP is involved in the inhibition of D. discoideum migration and pathogen vacuole expansion/remodeling by LAI-1 as well as in intracellular growth of L. pneumophila. This study provides a novel link between small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity.

microbiology↗

bioRxiv2024

AMPK/ULK1 activation downregulates TXNIP, Rab5, and Rab7 and inhibits endocytosis-mediated entry of human pathogenic viruses

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran-uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus and SARS-CoV-2 not only in mammalian and insect cells but in precision-cut lung slices and neuronal organoids. However, receptor presentation at the cytoplasmic membrane was unaffected, indicating that receptor-binding remained unaltered and later steps of endocytosis were targeted. Indeed, AMPK pathway activation reduced early endocytic factors TXNIP, Rab5 and the late endosomal marker Rab7 amounts. Furthermore, AMPK activation impaired SARS-CoV-2 late-replication steps by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction of early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acids and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.

microbiology↗

bioRxiv2023

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote inter-bacterial and inter-kingdom signaling

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires disease. The opportunistic pathogen employs the -hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 is produced by the autoinducer synthase LqsA, but it is not known, how LAI-1 is released by the pathogen. Here, we use a V. cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in E. coli generated LAI-1, which partitions to outer membrane vesicles (OMVs), and slightly reduces OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L. pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favour of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes inter-bacterial communication as well as interactions with eukaryotic host cells. Originality - Significance StatementInter-kingdom signaling involving low molecular weight bacterial compounds that are detected by eukaryotic cells represents an important, yet incompletely understood aspect of pathogen-host interactions. In many cases, the small signaling molecules are produced in only little amounts, their secretion mechanism is not known, and their effects on eukaryotic host cells are barely studied. Here, we reveal that the -hydroxyketone compound LAI-1 of L. pneumophila is released from the bacteria by outer membrane vesicles, which promote inter-bacterial communication as well as inter-kingdom signaling.

microbiology↗

bioRxiv2023

Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model

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 (Gb3). 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 CRISPR/Cas9 gene editing. We subjected iPSC to targeted peripheral neuronal differentiation and observed intra-lysosomal Gb3 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 Fabry cell lines as compared to the healthy control. Moreover, we demonstrate a drastic increase in Fabry sensory neuron Ca2+ levels at 39{degrees}C mimicking clinical fever (p < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons obtained from Fabry patients compared to 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 jam of mitochondrial trafficking at sphingolipid accumulations within Fabry sensory neurites utilizing a click-chemistry approach together with mitochondrial dysmorphism compared to healthy control cells. We pioneer 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.

neuroscience↗

bioRxiv2021

Food interactions observed in a pharmacokinetic investigation comparing two marketed cold preparations (BNO1016 and ELOM-080) after administration to male beagle dogs

The cold remedies Sinupret extract (BNO 1016) and Gelomyrtol forte (ELOM-080) represent the two top-selling cold remedies in Germany nowadays. Whereas BNO 1016 is a typical immediate release coated tablet, ELOM-080 is an enteric-coated soft gelatin capsule. The latter formulation, however, is at risk of pharmacokinetic interactions affecting absorption especially in the case of concomitant food intake. In the present pilot study, we investigated the risk of a possible food effect on BNO 1016 in comparison to ELOM-080 in three male beagle dogs. Single doses of BNO 1016 and ELOM-80 at 80 mg/kg and 160 mg/kg, respectively, were administered under fasting and fed conditions according to a 4-period, 4-treatment within-design. Blood sampling took place until up to 30 h p.a. and plasma concentrations of gentiopicroside, verbenalin and from a further marker analyte (BNO 1016 analytes) as well as 1,8-cineole, limonene and perillic acid (ELOM-080 analytes) were determined. Pharmacokinetic parameters focusing on rate and extent of absorption were derived. BNO 1016 analytes demonstrated a homogenous course in all animals in both, the fasted and fed state. Pharmacokinetic characteristics of a typical immediate release drug formulation were observed for all analytes and a food effect could be ruled out. ELOM-080 analytes also showed a homogeneous picture in the fasted state. However, lag-times (tlag) of up to 2 h p.a. with corresponding tmax values of 3 to 4 h were observed, reflecting a longer gastric residence time of the formulation. In the fed state, ELOM-080 showed significant pharmacokinetic characteristics suggesting a clear food effect. A major observation was a double peak phenomenon that could be observed in two out of three dogs. Furthermore, lag-times of some analytes up to 3-4 h and corresponding tmax values of up to 6-8 h occurred. In contrast to BNO 1016, these findings suggest that, as with other enteric-coated formulations, there may be a significant risk for food effects with ELOM-080 also in humans.

pharmacology and toxicology↗

bioRxiv2021

In vitro screening of herbal medicinal products for their supportive curing potential in the context of SARS-CoV-2

BackgroundHerbal medicinal products have a long-standing history of use in the therapy of common respiratory infections. In the COVID-19 pandemic, they may have the potential for symptom relief in non-severe or moderate disease cases. Here we describe the results derived by in vitro screening of five herbal medicinal products with regard to their potential to i) interfere with the binding of the human Angiotensin-converting enzyme 2 (ACE2) receptor with the SARS-CoV-2 Spike S1 protein, ii) modulate the release of the human defensin HBD1 and cathelicidin LL-37 from human A549 lung cells upon Spike S1 protein stimulation and iii) modulate the release of IFN-{gamma} from activated human peripheral blood mononuclear cells (PBMC). The investigated extracts were: Sinupret extract (SINx), Bronchipret thyme-ivy (BRO TE), Bronchipret thyme-primrose (BRO TP), Imupret (IMU), and Tonsipret (TOP). MethodsThe inhibitory effect of the herbal medicinal products on the binding interaction of Spike S1 protein and the human ACE2 receptor was measured by ELISA. The effects on intracellular IFN-{gamma} expression in stimulated human PBMCs were measured by flow cytometry. Regulation on HBD1 and LL-37 expression and secretion was assessed in 25d long-term cultured human lung A549 epithelial cells by RT-PCR and ELISA. ResultsIMU and BRO TE concentration-dependently inhibited the interaction between spike protein and the ACE2 Receptor. However, this effect was only observed in the cell-free assay at a concentration range which was later on determined as cytotoxic to human PBMC. SINx, TOP and BRO TP significantly upregulated the intracellular expression of antiviral IFN{gamma} from stimulated PBMC. Co-treatment of A549 cells with IMU or BRO TP together with SARS-CoV-2 spike protein significantly upregulated mRNA expression (IMU) and release (IMU and BRO TP) of HBD1 and LL-37 (BRO TP). ConclusionsThe in vitro screening results provide first evidence for an immune activating potential of some of the tested herbal medicinal extracts in the context of SARS-CoV-2. Whether these could be helpful in prevention of SARS-CoV-2 invasion or supportive in recovery from SARS-CoV-2 infection needs deeper understanding of the observations.

pharmacology and toxicology↗

bioRxiv2020

Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation, a nuclear export defect and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

microbiology↗

bioRxiv2020

The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. In these screenings, Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8{micro}g/ml significantly, and the EC50 was determined with 387ng/ml. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus. Fluoxetine treatment resulted in a decrease in viral protein expression. Furthermore, Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. We see the role of Fluoxetine in the early treatment of SARS-CoV-2 infected patients of risk groups.

molecular biology↗

bioRxiv2020

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 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) enabled imaging of sphingolipids and their interactions with proteins in the membrane of intracellular organelles with a spatial resolution of 10-20 nm. Because sphingolipids accumulated efficiently in pathogens we used 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 allowed us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 {+/-} 7.7 nm.

microbiology↗

bioRxiv2019

A central role of glutamine in Chlamydia infection

Obligate intracellular bacteria like Chlamydia trachomatis undergo a complex developmental cycle between infectious non-replicative (EBs) and non-infectious replicative (RBs) forms. EBs shortly after entering a host cell transform to RBs, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell it is currently unknown how the transition from EBs to RBs is initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine by the bacteria is critical to initiate EB-RB transition. These bacteria utilize glutamine to synthesize cell wall peptidoglycan which has recently been detected in the septa of replicating intracellular Chlamydia. The increased requirement for glutamine in infected cells is achieved by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine was effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limited growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 knockout mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infective strategies.

microbiology↗

bioRxiv2018

Bioorthogonal labeling with tetrazine-dyes forsuper-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

biochemistry↗