Sébastien Janel

Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization

Neisseria meningitidis is a cause of meningitis epidemics worldwide and of rapidly progressing fatal septic shock. A crucial step in the pathogenesis of invasive meningococcal infections is the adhesion of bloodborne meningococci to both peripheral and brain endothelia, leading to major vascular dysfunction. Initial adhesion of pathogenic strains to endothelial cells relies on meningococcal type IV pili, but the endothelial receptor for bacterial adhesion remains unknown. Here, we report that the immunoglobulin superfamily member CD147 (also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin) is a critical host receptor for the meningococcal pilus components PilE and PilV. Interfering with this interaction potently inhibited the primary attachment of meningococci to human endothelial cells in vitro and prevented colonization of vessels in human brain tissue explants ex vivo and in humanized mice in vivo. These findings establish the molecular events by which meningococci target human endothelia, and they open new perspectives for treatment and prevention of meningococcus-induced vascular dysfunctions.

cAMP Signaling by Anthrax Edema Toxin Induces Transendothelial Cell Tunnels, which Are Resealed by MIM via Arp2/3-Driven Actin Polymerization

RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

Quantifying bacterial adhesion on antifouling polymer brushes via single-cell force spectroscopy

Bacterial adhesion poses serious problems in food safety and biomedical applications. Antifouling polymer brushes have been shown to be effective as surface modifications to prevent biofilm formation from pathogenic bacteria. In this work, the adhesion of Yersinia pseudotuberculosis on seven types of brushes is examined by single-cell force spectroscopy. The brushes, known to possess excellent resistance to protein adsorption, greatly reduced the maximum force and the work required to detach the bacterium.

Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure

AIMS Previous studies have reported that decreased serine 208 phosphorylation of troponin T (TnTpSer208) is associated with ischaemic heart failure (HF), but the molecular mechanisms and functional consequences of these changes are unknown. The aim of this study was to characterize the balance between serine phosphorylation and O-N-acetylglucosaminylation (O-GlcNAcylation) of TnT in HF, its mechanisms, and the consequences of modulating these post-translational modifications. METHODS AND RESULTS Decreased TnTpSer208 levels in the left ventricles of HF male Wistar rats were associated with reduced expression of PKCε but not of other cardiac PKC isoforms. In both isolated perfused rat hearts and cultured neonatal cardiomyocytes, the PKCε inhibitor εV1-2 decreased TnTpSer208 and simultaneously decreased cardiac contraction in isolated hearts and beating amplitude in neonatal cardiomyocytes (measured by atomic force microscopy). Down-regulating PKCε by silencing RNA (siRNA) also reduced TnTpSer208 in these cardiomyocytes, and PKCε-/- mice had lower TnTpSer208 levels than the wild-type. In parallel, HF increased TnT O-GlcNAcylation via both increased O-GlcNAc transferase and decreased O-GlcNAcase activity. Increasing O-GlcNAcylation (via O-GlcNAcase inhibition with Thiamet G) decreased TnTpSer208 in isolated hearts, while reducing O-GlcNAcylation (O-GlcNAc transferase siRNA) increased TnTpSer208 in neonatal cardiomyocytes. Mass spectrometry and NMR analysis identified O-GlcNAcylation of TnT on Ser190. CONCLUSION These data demonstrate interplay between Ser208 phosphorylation and Ser190 O-GlcNAcylation of TnT in ischaemic HF, linked to decreased activity of both PKCε and O-GlcNAcase and increased O-GlcNAc transferase activity. Modulation of these post-translational modifications of TnT may be a new therapeutic strategy in HF.

High potential of adhesion to biotic and abiotic surfaces by opportunistic Staphylococcus aureus strains isolated from orthodontic appliances.

Orthodontic and other oral appliances act as reservoir of opportunistic pathogens that can easily become resistant to antibiotics and cause systemic infections. The aim of this study was to investigate the ability of Staphylococcus aureus strains isolated from healthy patients with orthodontic appliances, to adhere to biotic (HeLa cells) and abiotic surfaces (polystyrene and dental alloy). Adhesive ability to polystyrene was tested by crystal violet staining and quantitative biofilm production on dental alloy surfaces was evaluated by MTT reduction assay. In addition, the presence of icaA and icaD genes was achieved by polymerase chain reaction (PCR). Qualitative biofilm production revealed that 70.6% of strains were slime producers. The metabolic activity of S. aureus biofilms on dental alloy surfaces was high and did not differ between tested strains. Moreover, all the isolates were adhesive to HeLa cells and 94% of them harbor icaA and icaD genes. Considerable adhesion and internalization capacity to the epithelial HeLa cells and strong biofilm production abilities together, with a high genotypic expression of icaA/icaD genes are an important equipment of S. aureus to colonize orthodontic appliances and eventually to disseminate towards other body areas.

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

CLAFEM: Correlative light atomic force electron microscopy

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β2-adrenoceptor clusters assembled by alpha-actinin-4

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction.