Neisseria meningitidis (Nm, meningococcus) is an obligate commensal in humans, colonizing the nasopharyngeal mucosa usually without affecting the host. After the onset of colonization, Nm strains occasionally penetrate the mucosal membrane and enter the bloodstream to cause severe septicemia. Following bacteremia, Nm may bind and subsequently cross the meningeal blood-cerebrospinal fluid barrier (BCSFB) to enter the subarachnoid space and cause meningitis.
BCSFB penetration and development of novel multicellular in vitro models
The group’s major research focus lies on molecular interactions of Nm with microvascular brain endothelial cells (BECs) and mechanisms of BCSFB penetration. For this purpose, we use a variety of tissue culture techniques and cellular models, as well as a wide spectrum of innovative molecular, biochemical methods and imaging techniques. One current research topic covers the development of human, multicellular models of the BCSFB, which includes stem cell-derived BECs and leptomeningeal cells.
Sphingolipids and meningococcal meningitis
Sphingolipid-enriched membrane microdomains contribute to a variety of cellular processes, including signal transduction and vesicle trafficking, and can be exploited by pathogens such as Nm for host cell entry. Current work in this area is focused on the role of the downstream metabolite sphingosine 1-phosphate (S1P) and S1P-producing Sphingosine kinases 1 and 2 (Sphk1/2).
Nasopharyngeal colonization and transmigration
Meningococcal colonization of the nasopharynx is a dynamic process. Nm must mediate complex interactions with host epithelium, and persist in the presence of normal microflora and host mucosal defenses. We are currently developing an air-liquid Interface (ALI) human epithelial model to study the impact of Nm on the architecture and physiology of polarized epithelium and aim to characterize the process of meningococcal penetration of this barrier.
State of the art:
N. meningitidis (the meningococcus) colonizes the nasopharynx mainly as a commensal, being carried asymptomatically by 5–10% of the healthy population in non-endemic times. In rare cases, N. meningitidis can cross the epithelium of the nasopharynx, gain access to the bloodstream and cause severe septicemia and/or meningitis. One of the main factors affecting the pathogenicity of N. meningitidis is the ability to penetrate the vascular endothelial cell layer of the blood-cerebrospinal fluid barrier (B-CSFB) and infect the meninges. Although the role of adhesins and invasins in the virulence of N. meningitidis has been demonstrated, the mechanisms that govern meningococcal penetration of brain endothelial cells (BECs) and subsequent interaction with leptomeningeal cells are still not fully understood.
In the first funding period of the GRK 2157 induced pluripotent stem cells (iPSC)-derived BEC like cells have been implemented as a novel cellular model for N. meningitidis infection in close collaboration with Dr. Antje Appelt-Menzel/PD Dr. M. Metzger (LS TERM Würzburg) and Dr. Brandon Kim (University of Alabama)/Prof. E. Shusta (University of Wisconsin) according to previously described methods [1-5]. By using gentamicin protection and immunofluorescence assays we confirmed that multiple N. meningitidis wildtype strains and mutants followed similar phenotypes to previously described models (Fig.1). Recruitment of the recently published pilus adhesion receptor CD147 underneath meningococcal microcolonies in iPSC-BECs was demonstrated. N. meningitidis was found to directly disrupt the three tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction was preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in TEER, and an increase in sodium fluorescein (NaF) permeability could be shown (Fig.1). Notably, bacterial transmigration correlated with junctional disruption, indicating that a paracellular route contributes for bacterial crossing of BECs. In addition, RNA-Seq data analyses of sorted, infected iPSC-BECs were established in collaboration with A. Westermann (HIRI Wuerzburg) providing expression data of N. meningitidis-responsive host genes not previously described thus far to play a role in N. meningitidis infection of BECs . However, while iPSC-BECs formed a reasonable barrier with high TEER values, these cells lack significant cytokine/chemokine secretion after challenge with N. meningitidis . Since the inflammatory response plays a primordial role in the pathogenesis of meningococcal meningitis, we will continue using both, iPSC-derived BEC-like cells as well as hCMEC/D3 cells, that have been established as state of the art in vitro model in recent years for N. meningitidis infection of the B-CSFB.
Working hypothesis and work plan:
The B-CSFB is composed of brain ECs surrounded by leptomeningeal cells. In the second application period we now use the current model and developed a human B-CSFB in vitro co-culture transfer model. The co-culture model is established consisting of a confluent layer of BECs (iPSC-derived BEC-like cells and hCMEC/D3) on the apical side and a monolayer of leptomeningeal cells on the basolateral side of a microporous membrane for use in mechanistic studies on bacterial transmigration, permeability changes (monitored by NaF transport) and TEER values. Leptomeningeal cells have already been received from our collaboration partner, Prof. M. Christodoulides (University of Southampton) and successfully implemented.
We currently characterize and monitor the quality of the in vitro human B-CSFB co-culture model using transmission electron microscopy (TEM) in collaboration with C. Stigloher (Imaging Core Facility Biozentrum, Würzburg). Sequential events of bacterial adhesion, cellular uptake, localisation and transcytosis are analysed in detail by TEM imaging as well as super-resolution microscopy (SIM and dSTORM/PALM) in collaboration with M. Sauer (Biozentrum, Würzburg).
RNA-Seq data analyses of sorted, infected BECs provided differential regulation of genes involved in hypoxia . Hypoxia is a common feature during inflammation associated with bacterial infection, however the role of hypoxia on N. meningitidis B-CSFB infection has not been elucidated so far. Since activation of hypoxia-inducible factor 1 (HIF-1) transcription factor is the most recognized pathway adopted by hypoxic cells we will determine activation of HIF-1 in N. meningitidis infected BECs and analyse the role of this pathway in maintaining/disturbing B-CSFB function. The contribution of bacterial components (LOS, Pili, NadA) directly involved in HIF-1 activation by the pathogen will be addressed. The different strategies (BECs/leptomeningeal co-cultures, dynamic exposure) are very promising to improve the current in vitro models and will help to reveal useful sub-cellular details to provide evidence for the mechanism of bacterial transmigration in the human B-CSFB model.
1. Appelt-Menzel A, Cubukova A, Gunther K, Edenhofer F, Piontek J, Krause G, et al. Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells. Stem cell reports. 2017;8(4):894-906. Epub 2017/03/28. doi: 10.1016/j.stemcr.2017.02.021. PubMed PMID: 28344002; PubMed Central PMCID: PMCPMC5390136.
2. Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A, et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nature biotechnology. 2012;30(8):783-91. Epub 2012/06/26. doi: 10.1038/nbt.2247. PubMed PMID: 22729031; PubMed Central PMCID: PMCPMC3467331.
3. Endres LM, Schubert-Unkmeir A, Kim BJ. Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells. Journal of visualized experiments : JoVE. 2020;(161). Epub 2020/08/04. doi: 10.3791/61400. PubMed PMID: 32744533.
4. Kim BJ, Schubert-Unkmeir A. In Vitro Models for Studying the Interaction of Neisseria meningitidis with Human Brain Endothelial Cells. Methods in molecular biology (Clifton, NJ). 2019;1969:135-48. Epub 2019/03/17. doi: 10.1007/978-1-4939-9202-7_10. PubMed PMID: 30877675.
5. Doran KS, Fulde M, Gratz N, Kim BJ, Nau R, Prasadarao N, et al. Host-pathogen interactions in bacterial meningitis. Acta neuropathologica. 2016;131(2):185-209. Epub 2016/01/09. doi: 10.1007/s00401-015-1531-z. PubMed PMID: 26744349; PubMed Central PMCID: PMCPMC4713723.
6. Martins Gomes SF, Westermann AJ, Sauerwein T, Hertlein T, Forstner KU, Ohlsen K, et al. Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection. Frontiers in microbiology. 2019;10:1181. Epub 2019/06/14. doi: 10.3389/fmicb.2019.01181. PubMed PMID: 31191497; PubMed Central PMCID: PMCPMC6548865.
Scientific exchange with Tiantong Zhao from the research group of Prof. Jerry Wells, Chair Host Microbe Interactomics, Wageningen University and Research –
Both groups have a major research interests finding out how bacteria cross the blood-brain/blood-cerebrospinal fluid barrier.
Picture: Left Fatemeh Nosratabadi (PhD student research group Prof. A. Schubert-Unkmeir) right Tiantong Zhao (PhD student research group Prof. Jerry Wells).
Leo Endres hat seine neuesten Ergebnisse über die Entwicklung neuartiger mutlizellulärer in vitro Modelle der Blut-Liquor Schranke zur Untersuchung von Neisseria meningitidis Infektionsprozessen publiziert. Hierfür wurden spezielle Hirnendothelzellen aus induziert pluripotenten Stammzellen differenziert und auf einer porösen Membran mit Leptomeningealzellen kultiviert. Diese neuen Modelle zeichnen sich durch ihre erhöhte Vergleichbarkeit mit der menschlichen Physiologie aus und eignen sich im Infektionskontext besonders, um pathogene Veränderung und Überwindung der Blut-Liquor-Barriere zu untersuchen. In dieser Studie wurde festgestellt, dass Meningokokken Infektion zu verringerter Expression von Zellverbindungsproteinen im Hirnendothel führt und die allgemeine Barrierefunktion schädigt. Außerdem zeigten sich Indizien, die für eine Kombination aus trans- und parazellulärer Transmigration der Bakterien durch die Barriere sprechen.
Simon Peters presented his current work on the “Impact of glycosphingolipids on meningococcal pathogenicity in an Air-Liquid-Interface model of the nasopharyngeal epithelial barrier” during this year’s DGHM (German Society for Hygiene and Microbiology) Congress, which has taken place in Berlin from 05.09. – 07.09.2022.
Congratulations to Leo Endres, who received an award for his poster at the prestigious Gordon Research Conference ‘Barriers of the CNS’, which took place in New Hampshire, USA, from June 12-17. Poster prizes of up to $1000 were awarded to 6 out of the 110 posters presented at the conference. On his poster, Leo presented data from his project “Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection”, recently submitted for publication.
In a collaboration project between researchers from IHM (AG Schubert-Unkmeir) together with the groups of Prof. J. Seibel, Prof. M. Sauer, Prof. C. Stigloher and Prof. B. Kleuser, 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 in Neisseria meningitidis at subcellular level. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of the Gram-negative bacterium as an underlying cause of their antimicrobial activity.
Reference: Peters, S., Kaiser, L., Fink, J. Schumacher, F., Perschin, V., Schlegel, J. , Sauer, M., Stigloher, C., Kleuser, B., Seibel, J. and Schubert-Unkmeir, A. Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria. Sci Rep 11, 4300 (2021). https://doi.org/10.1038/s41598-021-83813-w
Funding FOR2123/DFG and GRK2581/DFG
Tel.: +49 931 31-46944
Tel.: +49 931 31-46944
Tel.: +49 931 31-81128
Tel.: +49 931 31-84472
Prof. Dr. med. Alexandra Schubert-Unkmeir
Tel.: +49 931 31-46721
Tel.: +49 931 31-85307
Endres LM, Jungblut M, Divyapicigil M, Sauer M, Stigloher C, Christodoulides M, Kim BJ, Schubert-Unkmeir A. Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection. Fluids Barriers CNS. 2022 Oct 26;19(1):81. doi: 10.1186/s12987-022-00379-z. PMID: 36289516; PMCID: PMC9597984. https://pubmed.ncbi.nlm.nih.gov/36289516/
Peters S, Fohmann I, Rudel T and Schubert-Unkmeir A, A comprehensive review on the interplay between Neisseria spp. and host sphingolipid metabolites, cells. Cells. 2021 Nov 17;10(11):3201. doi: 10.3390/cells10113201.
Krone M, Gütling J, Wagener J, Lâm TT, Schoen C, Vogel U, Stich A, Wedekink F, Wischhusen J, Kerkau T, Beyersdorf N, Klingler S, Backes S, Dölken L, Gasteiger G, Kurzai O, Schubert-Unkmeir A (2021) Performance of Three SARS-CoV-2 Immunoassays, Three Rapid Lateral Flow Tests, and a Novel Bead-Based Affinity Surrogate Test for the Detection of SARS-CoV-2 Antibodies in Human Serum. J Clin Microbiol 59(8):e0031921.
Peters S, Kaiser L, Fink J, Schumacher F, Perschin V, Schlegel J, Sauer M, Stigloher C, Kleuser B, Seibel J, Schubert-Unkmeir A. (2021) Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria. Sci Rep. 2021 Feb 22;11(1):4300. doi: 10.1038/s41598-021-83813-w.
Schlegel J, Peters S, Doose S, Schubert-Unkmeir A, Sauer M (2019) Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis invasion sites. Front Cell Dev Biol. 2019 Sept 7, 194.
Martins Gomes SF, Westermann AJ, Sauerwein T, Hertlein T, Förstner KU, Ohlsen K, Metzger M, Shusta EV, Kim BJ, Appelt-Menzel A, Schubert-Unkmeir A. Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection. Front Microbiol. 2019 May 29;10:1181
Peters S, Schlegel J, Becam J, Avota E, Sauer M, Schubert-Unkmeir A. Neisseria meningitidis type IV pili trigger Ca2+-dependent lysosomal trafficking of the acid sphingomyelinase to enhance surface ceramide levels. Infect Immun. 2019 Jun 3. pii: IAI.00410-19. doi: 10.1128/IAI.00410-19.
Herrmann JB, Muenstermann M, Strobel L, Schubert-Unkmeir A, Woodruff TM, Gray-Owen SD, Klos A, Johswich KO. Complement C5a Receptor 1 Exacerbates the Pathophysiology of N. meningitidis Sepsis and Is a Potential Target for Disease Treatment. MBio. 2018 Jan 23;9(1). pii: e01755-17. doi: 10.1128/mBio.01755-17.
Becam J, Walter T, Burgert A, Schlegel J, Sauer M, Seibel J, Schubert-Unkmeir A. Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria. Sci Rep. 2017 Dec 15;7(1):17627.
Burgert A, Schlegel J, Bécam J, Doose S, Bieberich E, Schubert-Unkmeir A, Sauer M. Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy. Angew Chem Int Ed Engl. 2017 Apr 5. doi: 10.1002/anie.201700570.
von Papen M, Oosthuysen WF, Becam J, Claus H, Schubert-Unkmeir A. Disease and Carrier Isolates of Neisseria meningitidis Cause G1 Cell Cycle Arrest in Human Epithelial Cells. Infect Immun. 2016 Sep 19;84(10):2758-70.
Oosthuysen WF, Mueller T, Dittrich MT, Schubert-Unkmeir A. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2. Cell Microbiol. 2016 Jan;18(1):46-65.
Simonis, A., Hebling, S., Gulbins, E., Schneider-Schaulies, S. and Schubert-Unkmeir, A. Differential activation of acid sphingomyelinase and ceramide release determines invasiveness of Neisseria meningitidis into brain endothelial cells. PLoS Pathog. 2014 Jun 12;10(6):e1004160.
Further publications of the research group on Pubmed