Biology subjects

Kleuser, B.

Publications and source records attributed to Kleuser, B..

9 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↗

bioRxiv2026

Kidney disease reprograms microbiome-host signaling to promote heart failure

BackgroundHeart failure is prevalent in chronic kidney disease (CKD) and linked to chronic inflammation. CKD-typical gut microbiome dysbiosis may stimulate inflammation, as bacterial aromatic metabolites are highly abundant and engage transcriptional programs through the aryl hydrocarbon receptor (AhR). Whether this axis drives cardiac remodeling and is therapeutically targetable remains unknown. MethodsWe used the subtotal nephrectomy model (STNx) and microbiome depletion by oral antibiotics. We investigated cardiac and renal function, AhR activity, metabolite profiles, and immunophenotypes by flow cytometry and transcriptomics. Candidate metabolite indoxyl sulfate (IxS) was tested in experimental HFpEF. In vivo and in vitro AhR inhibition (AhRi) was performed using a clinically tested compound. Mechanistic studies were performed in primary human and murine cardiac fibroblasts and T cells, as well as translational validation using UK Biobank data. ResultsMicrobiome depletion lowered bacterial metabolites and attenuated cardiac fibrosis and diastolic dysfunction in STNx, identifying AhR-driven expansion of interleukin-17A (IL-17A)-producing T helper cells (TH17) as key effector. Plasma IL-17A was stage-dependently elevated in CKD patients, particularly in HFpEF, and associated with all-cause mortality. Bacterial metabolite IxS promoted TH17 polarization and exacerbated cardiac dysfunction in HFpEF. AhRi using a small molecule inhibitor reduced TH17 abundance and attenuated cardiac fibrosis in STNx. Mechanistically, AhR and IL-17A signaling synergistically induced a conserved pro-fibrotic phenotype in human and murine cardiac fibroblasts, and AhR inhibition blocked ECM production in response to CKD patient serum. ConclusionA microbiome-AhR-IL-17A axis drives CKD-associated cardiac fibrosis. AhRi prevents remodeling, highlighting a potential therapeutic avenue to prevent cardiorenal multimorbidity.

immunology↗

bioRxiv2024

A genome-wide genetic screen identified targets for destabilizing the parasitophorous vacuole of Chlamydia trachomatis

The bacterial pathogen Chlamydia trachomatis employs the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. While conducting a CRISPR knock-out screen for genes contributing to this response, we uncovered a mutant deficient for CpoS to display a markedly increased reliance on host cellular ceramide synthesis, compensating for its diminished ability to acquire sphingolipids via modulating membrane trafficking. Employing the power of the just recently established molecular genetic toolbox for Chlamydia, we developed an innovative microscopic reporter system that revealed the mutant to thrive in unstable parasitophorous vacuoles (inclusions), characterized initially by the release of individual bacteria from otherwise intact-appearing vacuoles. CpoS-deficient inclusions were further destabilized by disruptions in ceramide synthesis, while supplementation of sphingoid bases stabilized them, also preventing the defensive host cell death response. Notably, early inclusion destabilization, achieved by simultaneous disruption of two transport routes, caused infection clearance without damaging the host cells. Overall, this study highlights the inclusions role as a refuge, demonstrates CpoS to maintain inclusion integrity by ensuring sphingolipid supply, and provides directions for a future therapeutic exploitation. SIGNIFICANCEA wide range of clinically significant microbes evolved to hide from the intrinsic defenses of their host cells by thriving within membrane-enclosed pathogen-containing vacuoles. This raises the intriguing possibility that such vacuoles could be targeted therapeutically. The bacterial pathogen Chlamydia trachomatis could be an exceptionally well-suited target for such innovative medicines given its medical importance and strict dependence on host cells. However, progress has been stalled by the lack of sensitive tools for detecting inclusion damage. Here, we resolved this major technical roadblock and uncovered the pathogen to employ the secreted effector CpoS, a modulator of membrane trafficking, to stabilize its vacuole by ensuring adequate sphingolipid supply. These methodological advances and mechanistic insights should promote the development of vacuole-destabilizing therapeutics.

microbiology↗

bioRxiv2024

Fertilization by short-term stored sperm alters DNA methylation patterns in common carp (Cyprinus carpio) embryos at single-base resolution

Short-term storage of sperm in vitro is widely used for artificial fertilization in aquaculture. It has been shown that short-term storage affects sperm motility characteristics, resulting in diminished fertility. However, the detrimental effects of short-term sperm storage on embryos development have remained largely unexplored in single-base methylome resolution. The main aim of the present study was to investigate DNA methylation in the offspring of common carp (Cyprinus carpio) derived from short-term stored sperm. Sperm were stored in artificial seminal plasma on ice (0-2{degrees}C) for 0, 3 and 6 days in vitro, fertilization was performed using oocytes from a single female, and embryos were collected at the mid-blastula stage. Sperm and embryo DNA was extracted, analysed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and whole genome bisulfite sequencing (WGBS). DNA methylation was then assessed. Sperm storage showed negative effects on motility, viability and DNA integrity, but had no effect on global DNA methylation of spermatozoa and resulting embryos. Results from the WGBS showed that methylation of 3313 genes was affected in the embryos fertilized with the 6-day-stored sperm, and the differentially methylated regions (DMRs) identified were mainly involved in cell adhesion, calcium, mitogen-activated protein kinase and adrenergic signalling, melanogenesis, metabolism and RNA transport. Such results suggest that prolongation of storage time may have certain impacts on embryonic development. These initial results provide valuable information for future consideration of the DNA methylome in embryos generated from short-term stored sperm, which is widely used for genetic management of broodstock in aquaculture.

molecular biology↗

bioRxiv2023

Secondary bile acid production by gut bacteria promotes Western diet-associated colorectal cancer

Western diet is an important risk factor for the development of sporadic colorectal cancer (CRC). Dietary fat stimulates bile acid (BA) production by the host and their conversion to secondary BAs by 7-dehydroxylating (7DH+) bacteria, but causal proof of their tumor-promoting effects in vivo is lacking. To address this, we performed feeding studies in a genetically engineered pig model of CRC combined with multi-omics analyses and gnotobiotic mouse studies. Western diet worsened the disease phenotype in APC1311/+ pigs. This was accompanied by microbiota changes, increased levels of the secondary bile acid deoxycholic acid (DCA), and higher colonic epithelial cell proliferation. The latter was counteracted by using the BA-scavenging drug colestyramine. Metagenomic analysis across multiple cohorts revealed higher occurrence of bai (BA inducible) operons from Clostridium scindens and close relatives in stool of CRC subjects (n = 1,034). Using two gnotobiotic mouse models of CRC, we demonstrate that colonization with 7DH+ bacteria (C. scindens or Extibacter muris) increased colonic tumor loads. This work provides clear evidence for the causal role of microbiome-derived DCA production in CRC under detrimental dietary conditions, opening avenues for future preventive strategies.

microbiology↗

bioRxiv2022

Cell-intrinsic ceramides determine T cell function during melanoma progression

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Asmflox/flox/CD4cre mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Acflox/flox/CD4cre mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Acflox/flox/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

immunology↗

bioRxiv2022

The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis

Acid ceramidase (Ac) is part of the sphingolipid metabolism and responsible for the degradation of ceramide. As bioactive molecule, ceramide is involved in the regulation of many cellular processes. However, the impact of cell-intrinsic Ac activity and ceramide on the course of Plasmodium infection remains elusive. Here, we use Ac-deficient mice with ubiquitously increased ceramide levels to elucidate the role of endogenous Ac activity in a murine malaria model. Interestingly, ablation of Ac leads to alleviated parasitemia associated with decreased T cell responses in the early phase of Plasmodium yoelii (P. yoelii) infection. Mechanistically, we identified dysregulated erythropoiesis with reduced numbers of reticulocytes, the preferred host cells of P. yoelii, in Ac-deficient mice. Furthermore, we demonstrate that administration of the Ac inhibitor carmofur to wild type mice has similar effects on P. yoelii infection and erythropoiesis. Notably, therapeutic carmofur treatment after manifestation of P. yoelii infection is efficient in reducing parasitemia. Hence, our results provide evidence for the involvement of Ac and ceramide in controlling P. yoelii infection by regulating red blood cell development.

microbiology↗

bioRxiv2021

Acid sphingomyelinase deactivation post-ischemia/ reperfusion promotes cerebral angiogenesis and brain remodeling via small extracellular vesicles

Functional inhibitors of acid sphingomyelinase are clinically used as anti-depressants since [~]60 years. Here, we show that acid sphingomyelinase inhibition by the antidepressants amitriptyline, fluoxetine and desipramine protects from ischemia/reperfusion and elicits a profound brain remodeling response with increased angiogenesis, improved blood-brain barrier integrity, reduced brain leukocyte infiltration and increased neuronal survival. Angiogenesis is promoted by small extracellular vesicles with bona fide characteristics of exosomes, which are released from endothelial cells and which constitute an elegant target for the amplification of stroke recovery.

neuroscience↗