Simon Peters     email
Institute for Hygiene and Microbiology

Supervisor:
Prof. Dr. Alexandra Unkmeir (Würzburg)
Promotion Committee:
Prof. Dr. Alexandra Unkmeir (Würzburg)
Prof. Dr. Markus Sauer (Würzburg)
Dr. Kai Johswich (Würzburg)

Analysis of the functional relevance of sphingomyelinases and ceramide in meningococcal pathogenesis

Neisseria meningitidis (Nm) is a gram-negative bacterium and a mostly commensal colonizer of the human nasopharynx. Approximately 10% of the human population carries Nm without showing any symptoms. But in some cases, Nm is able to cross the nasopharynx epithelium and proliferate in the bloodstream where it could cause septicemia or, when crossing the blood brain barrier (BBB), meningitis.

A critical step in the pathogenesis of Nm is the interaction and manipulation of the endothelial cells forming the BBB. The first interaction requires adherence related pathogenicity factors like the Type IV Pili, or the outer membrane proteins Opc and Opa. Preliminary experiments show that the adherence of Nm leads to the translocation of intracellular acid sphingomyelinases to the membrane of the host cell where it degrades sphingosine, a naturally compound of the lipid bilayer, to ceramide. Accumulation of ceramides in the membrane leads to the formation of ceramide enriched platforms in which receptors, necessary for Nm uptake into endothelia cells, could cluster.

In my work, I will focus on the pilus-mediated ceramide platform formation and on the role of Ca2+ release from the endoplasmic reticulum.  I will use human brain microvascular endothelial cells (HBMEC) as an established in vitro cell culture model and treat these cells either with a highly piliated Nm strain 8013/12 or purified Pili. The changes of ceramide surface level will be detected by flow cytometry analysis and visualized by immunfluorescence including high-resolution imaging techniques (e.g. dSTORM). Ca2+ release after infection or treatment with purified Pili will be determined, using an adapted protocol based on the Fluo-8 calcium indicator dye, by live cell imaging and flow cytometry analysis.