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Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

ABSTRACT: Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.

SUBMITTER: Doulet N

PROVIDER: S-EPMC2063870 | biostudies-other | 2006 May

REPOSITORIES: biostudies-other

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Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

Doulet Nicolas N Donnadieu Emmanuel E Laran-Chich Marie-Pierre MP Niedergang Florence F Nassif Xavier X Couraud Pierre Olivier PO Bourdoulous Sandrine S

The Journal of cell biology 20060501 4

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures r ...[more]

PMID: 16717131

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Project description:The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Project description:To extend our knowledge of target proteins in endothelial cells infected with the meningitis-causing pathogen Neisseria meningitidis, we characterized the interaction between the bacterial and human brain microvascular endothelial cell (HBMEC) monolayers. By use of human cDNA microarrays, transcriptional analysis revealed distinct responses to 4 and 8 h of infection. We also addressed the question of whether the major virulence factor of meningococci, i.e., the capsule, influences the host cell response. Of the 1,493 (at 4 h postinfection) and 1,246 (at 8 h postinfection) genes with altered expression upon bacterial contact, about 49.4% and 45%, respectively, depended on capsule expression. In particular, we identified an increase of expression for genes encoding proteins involved in bacterial adhesion and invasion. High levels of apoptosis-related gene (bad, bak, asp, and immediate-early response gene 1) expression could also be detected in infected cells. Further analyses confirmed that HBMECs displayed several hallmarks of apoptosis in response to N. meningitidis infection, namely, phosphatidylserine translocation and activation of caspase 3 and AMP-activated protein kinase alpha. Moreover, several differentially regulated genes not previously known to respond to meningococcal infection were identified. Of these, genes encoding cell adhesion proteins (CD44, CD98, and CD99), genes involved in downstream signaling of integrins (integrin-linked kinase, mitogen-activated protein kinase kinase 1, and mitogen-activated protein kinase kinase kinase 10) as well as negative regulators of these pathways (dual-specificity phosphatases 1, 5, and 14 and G protein pathway suppressor 2), and genes involved in cytoskeleton reorganization (those encoding Arp2/3, p34-arc, actinin alpha 1, vasodilatator-stimulated protein, and Wiskott-Aldrich syndrome protein) were the most prominent. This global transcriptional analysis creates a new platform for further molecular and cellular analysis of the interaction between N. meningitidis and target cells.

Dataset Information

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

Publications

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

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