Abstract
Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.
Highlights
The obligate human pathogenic Gram-negative bacterium Neisseria meningitidis can cause epidemic meningitis and in severe cases sepsis and progressing fatal shock (Stephens et al, 2007)
It has been shown that CD147 and β2-adrenergic receptor (ß2AR) are organized in pre-existing complexes at the plasma membrane of endothelial cells, which accumulate at sites of meningococcal adhesion (Maïssa et al, 2017)
Since accumulation of receptors at bacterial adhesion sites requires a high mobility in the plasma membrane we performed live-cell single-molecule tracking experiments of CD147 under various experimental conditions using an N-terminal binding monoclonal antibody (MEM-6/1) directly conjugated to the photostable fluorescent dye SeTau-647 (Tsunoyama et al, 2018)
Summary
The obligate human pathogenic Gram-negative bacterium Neisseria meningitidis can cause epidemic meningitis and in severe cases sepsis and progressing fatal shock (Stephens et al, 2007). Super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM) (Heilemann et al, 2008) demonstrated that N. meningitidis binding to endothelial cells requires CD147/β2-adrenergic receptor clustering at bacterial adhesion sites (Maïssa et al, 2017). Glycosphingolipids in general are important host cell targets for a plenitude of pathogens such as fungi, bacteria, and viruses (Nakayama et al, 2018). They are composed of complex, highly variable glycan moieties linked to a lipophilic ceramide backbone with extensive molecular heterogeneity (Lingwood, 2011). Super-resolution microscopy by structured illumination microscopy (SIM) (Gustafsson, 2000) and direct stochastic optical reconstruction microscopy (dSTORM) (Heilemann et al, 2008; van de Linde et al, 2011) shows GM1 accumulation around meningococci highlighting their significant importance for bacterial invasion
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
