Abstract

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.

Highlights

  • The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis are the causative agents of gonorrhea and meningococcal meningitis, respectively [1, 2]

  • The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors

  • Models for the colonization of mucosal cells by Neisseria suggest that primary adherence occurs through pili but that this encounter is followed by a second, closer range contact between outer membrane proteins and host cell surface receptors [3,4,5]

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Summary

Introduction

The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis are the causative agents of gonorrhea and meningococcal meningitis, respectively [1, 2]. Stern-Volmer plots of Fo/F versus concentrations of ligand were non-linear for both the OpcA/sialic acid and OpaD/maltose data

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