The major focus of the group is studying the molecular interaction of Neisseria meningitidis with vascular endothelial cells, with a specific focus on the mechanism of brain endothelial barrier penetration. The human blood-brain/blood-cerebrospinal fluid (BB/B-CSF) barrier is one of the tightest barriers of the human body. Among invasive pathogens, few are capable of invading the subarachnoideal space, thus suggesting that they have developed specific attributes capable to circumvent the BBB. We investigate the strategies used by this microorganism to colonize the brain vasculature and to cross the BB/B-CSF barrier.
Within a newly DFG funded project we now seek to understand the significance of sphingolipid-enriched membrane microdomains in N. meningitidis pathogenesis.
To answer these main questions we are working with tissue culture based cell models including brain microvascular endothelial cells and employ a wide spectrum of innovative methods in molecular, biochemical and cell biological methods including siRNA/shRNA transfections, FACS analyses and microscopy.
Auch in diesem Jahr haben die Unterfränkische Gedenkjahrstiftung für Wissenschaft und die Universität Würzburg zum Stiftungsfest Promotionspreise vergeben. Das obligatorische Gruppenfoto kam wegen Corona allerdings nicht zustande. (Bild: iStock.com/skodonnell)
Mit 500 Euro sind die gemeinsamen Promotionspreise der Unterfränkischen Gedenkjahrstiftung für Wissenschaft und der Universität Würzburg dotiert. Sie werden jedes Jahr für herausragende Dissertationen verliehen. Voraussetzung: Die Arbeiten müssen sich mit Unterfranken befassen und/oder von Personen geschrieben sein, die in der Region aufgewachsen sind oder seit längerer Zeit hier leben.
Eigentlich werden diese Preise traditionellerweise im Rahmen des Stiftungsfests der Universität Würzburg von Unterfrankens Regierungspräsident Eugen Ehmann, in seiner Funktion als Vorsitzender des Vorstands der Unterfränkischen Gedenkjahrstiftung für Wissenschaft, und dem Präsidenten der JMU, Alfred Forchel, verliehen. Aufgrund der Einschränkungen zum Infektionsschutz konnte eine persönliche Übergabe in diesem Jahr jedoch nicht erfolgen. Immerhin erhielten die Ausgezeichneten ihre Urkunde per Post.
As Neisseria meningitidis is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.
Endres, L. M., Schubert-Unkmeir, A., Kim, B. J. Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells. J. Vis. Exp. (161), e61400, doi:10.3791/61400 (2020).
Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined.
In his recent work Simon Peters documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells. He found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, Simon Peters showed that the overall number of CRPs with a size of 80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.
Peters S, Schlegel J, Becam J, Avota E, Sauer M, Schubert-Unkmeir A. 2019. Neisseria meningitidis type IV pili trigger Ca2+-dependent lysosomal trafficking of the acid sphingomyelinase to enhance surface ceramide levels. Infection and Immunity doi:10.1128/iai.00410-19
The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking.
In a current collaboration project with the group of Prof. M Sauer (http://www.super-resolution.biozentrum.uni-wuerzburg.de/startseite/) we demonstrated that independent of the cell-type, membrane ceramides form nanodomains (CRPs) with a diameter of about 75 nm containing 50–60% of all ceramides. Treatment with sphingomyelinase increases the quantity of ceramides, the size and density of CRPs as well as the ceramide concentration in CRPs as shown for brain endothelial cells.
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.
Like many viruses that perturb the cell cycle machinery to adapt their own replication, bacteria can hijack checkpoints of the cell cycle to establish infection. During microbial pathogenesis, the particular function to induce cell cycle arrest might represent a strategy to prevent maturation and exfoliation of epithelial cells to support prolonged bacterial persistence and survival in the epithelial cells.
In the current study we demonstrated the ability of N. meningitidis to modulate the cell cycle in epithelial cells, thereby arresting infected cells in the G1 phase. Importantly, by including two pathogenic and two genetically related carrier isolates, we demonstrate that both carrier and disease isolates are capable to induce cell cycle arrest. Using propidium iodide staining and 5-ethynyl-2'-deoxyuridine pulse-labeling, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content, we provide evidence that meningococcal infection arrested cells in the G1 phase of the cell cycle, followed by a significant decrease of cells in the S phase. Mechanistically, we demonstrate differential regulation of cell cycle regulatory genes, including the cell cycle inhibitors p21WAF1/CIP1 and p27CIP1. Their alterations were reflected in changes in protein expression levels (p21WAF1/CIP1) and/or relocalization (both p21WAF1/CIP1 and p27CIP1) in N. meningitidis- infected cells.
Michael von Papen, Wilhelm F. Oosthuysen, Jérôme Becam, Heike Claus and Alexandra Schubert-Unkmeir Disease and Carrier Isolates of Neisseria meningitidis cause a G1 cell cycle arrest in human epithelial cells Accepted manuscript posted online 18 July 2016, doi: 10.1128/IAI.00296-16
Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. This review highlights recent progress made in our understanding of host–pathogen interactions in bacterial meningitis, exemplified by four of the most common pathogens, Streptococcus pneumoniae, N. meningitidis, GBS, E. coli K1, and S. suis.
Kelly S. Doran, Marcus Fulde, Nina Gratz, Brandon J. Kim, Roland Nau, Nemani Prasadarao, Alexandra Schubert-Unkmeir, Elaine I. Tuomanen, Peter Valentin-Weigand
Host–pathogen interactions in bacterial meningitis
Review Acta Neuropathologica
February 2016, Volume 131, Issue 2, pp 185-209