The Characterization of the Binding of Opacity-Associated Proteins to Human Host Cell Receptors

Abstract

Opacity-associated (Opa) proteins are eight-stranded β-barreled monomeric outer membrane proteins found in the bacterial pathogens Neisseria meningitides and N. gonorrhoeae. In vivo, these proteins interact with specific human host cell receptors to induce phagocytosis of the bacterium, allowing Neisseria to breach the plasma membrane and gain entry to targeted human cells. There are at least 26 characterized Opa proteins, nearly identical in sequence except for three extracellular loops. Host-receptor specificity is determined by the variable extracellular loops; however, the specific molecular determinants of the interaction have not been identified. Opa proteins are classified into two families based on their host receptor selectivity, the larger class, OpaCEA, bind to carcinoembryonic antigen-like cellular adhesion molecules (CEACAMs) and the smaller class, OpaHS, bind to heparansulfate proteoglycan receptors (HSPGs) or to integrin receptors via an HSPG-mediated interaction with fibronectin or vitronectin. Though the Opa-host receptor interaction is directly related to the invasion efficiency of Neisseria, the thermodynamics (e. g. affinities) of Opa proteins interactions with host receptors is not known. We present a study investigating the binding of two Opa protein variants, OpaI (an OpaCEA) and OpaA (an OpaHS) with their cognate host receptors. A centrifugal “pull-down” assay is used with fluorescence spectroscopy to determine the specificity and affinity of the Opa - receptor interactions. Although attempts have been made to study Opa specificity in vivo, this study demonstrates Opa protein selective binding to the respective receptors in vitro, thereby facilitating investigations of protein determinants relating both to binding specificity and affinity.