Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

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Abstract
The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates. Neisseria meningitidis is a human pathogen that can cross the natural cellular barriers to reach the blood and the brain, causing septicaemia and meningitis. One of its surface molecules, Opc, has the capacity to attach to human cells lining the blood vessels. In vitro, the bacterium can do this by coating itself with the human serum factor vitronectin; and, by mimicking as vitronectin, it can bind to human cellular vitronectin-binding proteins (receptors). In this study, we have investigated the structural features of vitronectin that N. meningitidis recognises; such knowledge could help develop future strategies to control bacterial spread. We describe two different aspects of bacterial binding to vitronectin and demonstrate that one of these is a novel method, which occurs directly through the sulphated tyrosines of vitronectin available only when the molecule presents itself in an unfolded form. Such unfolded or activated vitronectin levels may be elevated during bacterial presence in the blood. Thus our observations imply that when bacteria appear in the blood, their presence may help to generate the form of vitronectin that they require for binding to and invading the cells that line human blood vessels to spread throughout body tissues including the brain.

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