Dataset Information

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

ABSTRACT: 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 hemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signaling 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 alpha-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 multi-molecular 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Microvascular Endothelial Cell

DISEASE(S): Meningococcal Meningitis

SUBMITTER: Sandrine BOURDOULOUS

LAB HEAD: Sandrine BOURDOULOUS

PROVIDER: PXD006212 | Pride | 2017-07-10

REPOSITORIES: Pride

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Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β<sub>2</sub>-adrenoceptor clusters assembled by alpha-actinin-4.

Maïssa Nawal N Covarelli Valentina V Janel Sébastien S Durel Beatrice B Simpson Nandi N Bernard Sandra C SC Pardo-Lopez Liliana L Bouzinba-Ségard Haniaa H Faure Camille C Scott Mark G H MGH Coureuil Mathieu M Morand Philippe C PC Lafont Frank F Nassif Xavier X Marullo Stefano S Bourdoulous Sandrine S

Nature communications 20170601

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 (β<sub>2</sub>AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination ...[more]

PMID: 28569760

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Project description:Background The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA pro-cessing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oli-go(dT) beads and the RNA binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. Results Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seed-lings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5´untranslated region. The efficiency of RNA capture was greatly enhanced by the use of locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. In total, we identified more than 300 proteins interacting with the AtGRP7 RNA. These were benchmarked against proteins pulled down by AtGRP7 in vitro transcripts from nuclear lysates. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation. The circadian expression of the AtGRP7 transcript was slightly changed in an alba loss-of-function mutant compared to wild-type, demonstrating the functional relevance of the interaction. Conclusion We adapted specific RNP capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up-scaling of the protocol should be applicable for less abundant transcripts. Continuous improvements of mass spectrometry sensitivity will also help to increase the range of proteins that can be identified.

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