Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites

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.

Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus

“…To test, whether the impaired fungal binding in alloSCT derived NK cells might be due to changes in CD56 mobility we performed 2D single-molecule tracking (SMT) experiments of CD56 ( Supplementary Video 1 ) as described previously ( 24 , 25 ). We could not observe significant differences in the diffusion coefficient ( Figure 9A ), anomalous diffusion constant ( Figure 9B ) and bound fraction (data not shown) ( 38 ) between control and alloSCT NK cells.…”

“…Single-molecule tracking was performed as described recently ( 24 , 25 ). In short, CD56 primary antibodies (clone HCD56, Biolegend) were conjugated to the bright and photostable fluorophore SeTau-647-NHS (SETA BioMedicals, #K9-4149) ( 26 ) via N-hydroxysuccinimide ester amine crosslinking.…”

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

“…Super-resolution methods have enabled the investigation of key bacterial proteins, 87 DNA organization and transcription, 88 − 90 or bacterial membrane organization. 91 , 92 Similarly, yeast biology could be investigated at unprecedented length scales. 93 − 95 When SMACM is combined with single-molecule tracking, further insights have been obtained about bacterial microdomains and their roles in regulation.…”

Neuronal Signaling Involved in Neuronal Polarization and Growth: Lipid Rafts and Phosphorylation

“…However, the development of super-resolution microscopy permitted the observed lipid rafts in various cell types, leading to the wider acceptance of this concept (Owen et al, 2012 ). There are several styles of super-resolution microscopy that use distinct probes; each of these methods has successfully permitted the visualization of lipid rafts (Tobin et al, 2014 ; Chen et al, 2015 ; Hartley et al, 2015 ; Stahley et al, 2016 ; Gao et al, 2017 ; Schlegel et al, 2019 ; Angelopoulou et al, 2020 ). These new results have contributed to models suggesting possible roles for lipid rafts in multiple cellular pathways (Raghunathan and Kenworthy, 2018 ).…”