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.
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
Bacteria can hijack checkpoints of the cell cycle to establish infection and a growing family of bacterial toxins and effectors have been described that interfere with the host cell cycle.
In the current study, we analyzed the effect of the human pathogen Neisseria meningitidis on the cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N. meningitidis causes an accumulation of cells in the S-phase early at 3 hrs and at 24 hrs post-infection. Importantly, we could show that the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S-phase. In collaboration with the colleagues from the department of bioinformatics, Tobias Müller and Marcus T. Dittrich, a focused cell cycle RT-qPCR based array and integrated network analysis was carried out. These analyses revealed changes in the abundances of several cell-cycle regulatory mRNAs, including the cell cycle inhibitors p21WAF1/CIP1 and cyclin G2, of which the contribution to S-phase accumulation during meningococcal infection was characterized.
Oosthuysen, W. F., Mueller, T., Dittrich, M. T. and 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. 2015 Jul 7. doi: 10.1111/cmi.12482.
Sphingomyelin is a major component of the outer plasma membrane layer where, under certain conditions of stress or the induction of inflammatory cytokines, it is metabolized into ceramide and phosphorylcholine by the activity of the acid sphingomyelinase (ASM). Once activated, this enzyme translocates from its lysosomal intracellular storage to outer cell membrane where it is co-displayed with released ceramides. Due to their biophysical properties, ceramide-enriched membrane domains fuse into extended ceramide-enriched platforms which span a few hundred nanometers to several micrometers. In addition to altering membrane fluidity and rigidity, ceramide-enriched platforms serve to sort and eventually concentrate membrane receptors and membrane proximal signaling components thereby amplifying cellular responses and signal transduction
In the current study, we unravel a differential activation of the ASM/ceramide system by the species N. meningitidis determining its invasiveness into brain endothelial cells. We show that N. meningitidis causes transient activation of ASM followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation.
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.Published: June 12, 2014, PLoS Pathog. 2014 Jun 12;10(6):e1004160.