Neisseria meningitidis Type IV Pili Trigger Ca ²⁺ -Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels

Abstract

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. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we 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, we show that the overall number of CRPs with a size of ϳ80 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 Ca 2ϩ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca 2ϩ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

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

“…Still, the slight changes in mobility observed may indicate cytoskeletal rearrangements of the plasma membrane sphingolipid organization. Indeed a recent study revealed an increase in ceramide-rich platforms upon treatment of HBMEC with type IV pili (Peters et al, 2019). Therefore, we investigated the distribution and localization of the native glycosphingolipids GM1 and Gb3 by super-resolution microscopy.…”

“…In general, the affinity of the PilE and PilV monomers to CD147 is low and the need for multimeric organization as type IV pili seems to play an important role in mediating adherence (Bernard et al, 2014). Since our pili preparation contains mainly monomeric pilin subunits, as shown by Peters et al (2019), incubation with our PeF preparation might not resemble the native condition where in addition to the multimeric assembly as pilus fibers whole micrometer-sized bacteria are attached to CD147. It seems thus more likely, that binding of the competitive M6/6 antibody reflects the native interaction of type IV pili with CD147 although this has to be verified in future experiments.…”

“…Pilus enriched fractions (PeF) were prepared as described previously (Peters et al, 2019). The bacterial content of 50 blood agar plates was harvested in 40 ml of 0.15 M ethanolamine (in PBS) with a pH of 10.5.…”

Meningococcal disease: A paradigm of type‐IV pilus dependent pathogenesis

“…In addition, Opc binding to heparan sulfate proteoglycans causes transient activation of acid sphingomyelinase ASM, which triggers the formation of ceramide‐enriched membrane platforms, also promoting enrichment of ErbB2 receptor (Simonis, Hebling, Gulbins, Schneider‐Schaulies, & Schubert‐Unkmeir, ). Organisation of these platforms requires prior pilus‐mediated adhesion that triggers Ca 2+ flux and subsequent lysosomal exocytosis and exposition of ASM at the outer leaflet of the plasma membrane (Peters et al, ). Although not required for adhesion, these platforms facilitate invasion of Opc expressing N. meningitidis into human brain endothelial cells (Simonis et al, ).…”

Diverse Facets of Sphingolipid Involvement in Bacterial Infections

“…N. meningitidis likewise causes the activation of ASM and ceramide release that are essential for the internalization of meningococci into brain endothelial cells, which is connected to the expression of the outer membrane protein OpcA and binding to cell surface heparan sulfate proteoglycans (HSPGs) (Simonis et al, 2014). Recently, a role of meningococcal pilus in the translocation of ASM to the surface of infected cells has been described (Peters et al, 2019). In all cases, invasion of the host cell contributes to immune evasion and spreading of these pathogenic bacteria from the site of the initial contact to other tissues.…”