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Intern
    Graduiertenkolleg 2157

    Meningococcal ligands and molecular targets required for adhesion and penetration of the blood-cerebrospinal fluid barrier under shear stress


    State of the art

    N. meningitidis (the meningococcus) colonizes the nasopharynx mainly as a commensal, being carried asymptomatically by 5–10% of the healthy population in non-endemic times. In rare cases, N. meningitidis can cross the epithelium of the nasopharynx, gain access to the bloodstream and cause severe septicemia and/or meningitis. One of the main factors affecting the pathogenicity of N. meningitidis is the ability to penetrate the vascular endothelial cell layer and infect the meninges. Although the role of adhesins and invasins in the virulence of N. meningitidis has been demonstrated, the mechanisms that govern meningococcal penetration are still not fully understood. Various models have been established to study the N. meningitidis-host interaction using mostly in vitro cell culture models based on immortalized human cell lines. However, none of these established cell lines provides an appropriate experimental resource to study N. meningitidis colonization and subsequent invasion and penetration of human endothelial surfaces.


    Previous Work


    We have shown that the interaction of N. meningitidis with cells of the endothelial vasculature requires binding of the outer membrane protein Opc to fibronectin linking meningococci to alpha5beta1-integrins [1]. To study the mechanisms of adhesion and invasion we used an immortalized cell line of human brain derived microvascular endothelial cells (HBMEC) [2]. Using this in vitro cell culture model, we have also shown that binding to integrins triggers phosphotyrosine signaling, involving the non-receptor tyrosine kinases (NRTKs) c-Src and the focal adhesion kinase as well as RTKs such as the epidermal growth factor receptor [3-5]. These particular activations are highly important for cytoskeleton rearrangement resulting in endothelial uptake of meningococci [6].

    Moreover, we could show that Opc-expressing meningococci activate the acid sphingomyelinase (ASM) in brain endothelial cells, which hydrolyses sphingomyelin to cause ceramide release and formation of extended ceramide-enriched membrane platforms wherein an important receptor involved in bacterial uptake clusters [7].

    In addition to bacterial uptake the penetration of the blood-cerebrospinal fluid (B-CSF) barrier can be facilitated by tight junction modulation. This relies on the recruitment of the polarity complex underneath meningococcal microcolonies and/or degradation of tight junctions proteins, such as occludin [8,9]. An in vitro circulatory 2D N. meningitidis-endothelium interaction model has been developed and shows that the adhesion to host cells is shear stress dependent and is therefore thought to be related to blood flow rates in the microvasculature [10]. We have now performed preliminary experiments to develop a human in vitro 3D model of the B-CSF-barrier (with H. Walles). Therefore, a biological vascularized scaffold (BioVaSc) of collagen I/III and decellularized jejunal segments was effectively reseeded with HBMEC and cultured under static and dynamic conditions.

    Figure: HE staining of HBMEC seeded on a BioVaSc with a cell concentration of
    3 x 105 /cm2 and cultured under static conditions for 7 days.


    Work Plan

    In the case of invasive meningococcal diseases (septicemia and/or meningitis) endothelial adhesion events must occur during circulation under conditions of physiological blood pressure. To investigate the bacterial and host factors, which contribute to this essential step of invasive meningococcal disease, we aim (Part 1) to develop an in vitro circulatory 2D N. meningitidis-endothelium interaction model. We will implement disposable laminar-flow chambers (ibidi) and video microscopy (Nikon Eclipse Ti) to study the meningococcal infection in this environment. Host cells will be seeded and grown in the flow chamber and fluorescent bacteria will be introduced in the flow controlled by a syringe pump. Such a model has been already set up to analyse the interaction of N. meningitidis with endothelial cells [10], however, only bacterial adhesion has been determined.

    Questions to be addressed will include the (1) analysis of the mechanisms of adhesion, invasion and transcytosis under dynamic conditions as well as (2) receptor recruitment and formation of ‘cortical plaques’ with recruitment of the polarity complex and (3) activation of the ASM/ceramide system. The infected cell culture will be used to analyse the (4) cell viability (LDH release), (5) pattern of cytokine secretion (ELISA) as well as (6) profiling the expression of genes related to endothelial cell activation and/or injury (Human Endothelial Cell Biology RT² Profiler™ PCR Array). Moreover, (7) histology processing and immunofluorescence microscopy will be performed to visualize invasion and transcytosis. Infection of brain endothelial cells will include the use of immortalized and primary HBMEC (already established in the lab) as well as hypervirulent Neisseria meningitidis strains and apathogenic commensal strains.

    Part 2 of the project addresses the application of 3D cell culture techniques and associated analytical tools for infection biology research. We plan to continue to develop a human in vitro 3D model of the B-CSF barrier in cooperation with H. Walles. A protocol to effectively reseed a BioVaSc of collagen I/III and SIS (SIS = small intestinal sub mucosa) with HBMEC has been established and will be extended using electro spun matrices and different human brain microvascular endothelial cell lines (BB19, HBMEC/ciβ) and primary HBMEC (commercial source). Questions addressed in the circulatory 2D model will be transferred to the 3D cell culture model.


    References


    1. Unkmeir et al. (2002) Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells. Mol Microbiol 46: 933-946. PubMed
    2. Stins et al. (1997) Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 76: 81-90. PubMed
    3. Slanina et al. (2012) Cell invasion by Neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin. PLoS One 7: e39613. PubMed
    4. Slanina et al. (2010) Entry of Neisseria meningitidis into mammalian cells requires the Src family protein tyrosine kinases. Infect Immun 78: 1905-1914. PubMed
    5. Slanina et al. (2014) Role of Epidermal Growth Factor Receptor Signaling in the Interaction of Neisseria meningitidis with Endothelial Cells. Infect Immun 82: 1243-1255. PubMed
    6. Sokolova et al. (2004) Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release. Cell Microbiol 6: 1153-1166. PubMed
    7. Simonis et al. (2014) Differential activation of acid sphingomyelinase and ceramide release determines invasiveness of Neisseria meningitidis into brain endothelial cells. PLoS Pathog 10: e1004160. PubMed
    8. Coureuil et al. (2009) Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium. Science 325: 83-87. PubMed
    9. Schubert-Unkmeir et al. (2010) Neisseria meningitidis induces brain microvascular endothelial cell detachment from the matrix and cleavage of occludin: a role for MMP-8. PLoS Pathog 6: e1000874. PubMed
    10. Mairey et al. (2006) Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier. J Exp Med 203: 1939-1950. PubMed