Molecular Determinants of Neissserial Opa Protein Interactions with CEACAM Receptors

Neisseria gonorrhoeae is a unique pathogen in that it not only colonizes human tissue by interactions with host receptors, but these interactions can trigger engulfment of the bacteria even by typically non-phagocytic cells. N. gonorrhoeae possess a family of opacity-associated (Opa) proteins, which are 8-stranded β-barrel outer membrane proteins that bind to human host cell receptors. The majority of Opa proteins engage select human CEACAM receptors (carcino-embryonic antigen-like cellular adhesion molecules), and this interaction can trigger phagocytosis of the bacterium. To date, there are 345 distinct opa alleles sequenced; the most prominent differences are in the second and third extracellular loops (hypervariable regions, HV1 and HV2). In addition to contributing to the bacteria's ability to evade the human immune system, these HV regions determine receptor specificity. While the Opa protein family has conserved structural elements, the molecular determinants of receptor recognition and engagement remain unclear. We aim to compare the interactions of various Opa proteins in vitro (reconstituted into liposomes) in order to define how differences in Opa sequence and structure inform receptor binding. To investigate structural motifs involved in Opa-receptor interactions, we are conducting binding assays and protein footprinting experiments to determine how differences in sequence affect receptor engagement. Affinities for a selection of Opa proteins with a variety of CEACAM receptors were determined using biolayer interferometry, with KD values in the low nM range. Protease protection assays are currently being performed to determine which amino acid residues in the HV regions are involved in receptor recognition and engagement. Together, the results of these experiments provide insight into the molecular determinants of the Opa-CEACAM interaction, and provide a clearer understanding of the bacterial mechanism for engulfment into human host cells.