BPS2026 – Determining the role of lipopolysaccharide in defining the binding specificity of Neisserial opacity-associated proteins and human carcinoembryonic antigen-related cell adhesion molecules

Neisseria gonorrhoeae ( Gc ) is a Gram-negative human pathogen that causes the sexually transmitted infection known as gonorrhea, which infects millions of people across the globe annually. Furthermore, the rapid rise of antibiotic resistant Gc strains has caused both the centers for disease control and World Health Organization to designate Gc a threat to global health. Opacity-associated (Opa) proteins are Neisserial outer membrane (OM) proteins that are integral to Gc pathogenesis. Opa proteins consist of an eight-stranded β-barrel with four highly dynamic extracellular loops that contain three regions of sequence variability. Only a select subset of Opa variants bind to specific members of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM) family, which are a part of the immunoglobulin (Ig) superfamily. Although the variable Opa loop regions play a critical role in determining Opa/CEACAM specificity, due to the high sequence variability of these loop regions, a consensus sequence for Opa/CEACAM specificity has not been identified. To assess the determinants of Opa/CEACAM specificity, we employed in vitro microscale thermophoresis (MST) binding assays using purified HaloTag-CEACAM (HTCEACAM) fusion proteins. We found that Opa expressed to and purified from isolated outer membranes (OM-Opa) co-purify with lipopolysaccharide (LPS). Furthermore, we found that OM-Opa preserves in vivo Opa/CEACAM binding specificity a binding affinity of K D = ∼ 2.0 ± 0.6 μM. Conversely, Opa denatured and refolded in vitro absent LPS (RF-Opa) did not preserve Opa/CEACAM binding specificity; with all RF-Opa proteins binding indiscriminately to all HTCEACAMs at a much weaker affinity of K D ≥ 7.3 ± 2.1 μM. Based on these results, we hypothesize that LPS is a necessary component to maintain Opa/CEACAM specificity by restricting the accessibility of the extracellular Opa loops.