Investigating the Role of Lipopolysaccharide as a Determinant for Binding Specificity Between NeisseriaOpacity-associated Proteins and Human Carcinoembryonic Antigen-related Cell Adhesion Molecules 27 views

Neisseria gonorrhoeae (Gc) is the human pathogen responsible for causing the sexually transmitted infection commonly known as gonorrhea, which infects millions of people every year and has demonstrated the ability to rapidly adapt mechanisms of antimicrobial resistance in response to new treatments. Therefore, investigating the mechanisms of Gc pathogenesis is critical to identifying new possible treatments and developing a viable vaccine. Gc are diderms with a cell envelope composed of a symmetrical inner membrane and asymmetrical outer membrane (OM) divided by a thin layer of peptidoglycan. The outer leaflet of the Gc OM is primarily composed of lipooligosaccharide, the type of lipopolysaccharide (LPS) synthesized by Gc. Outer membrane proteins (OMPs) known as Opacity-associated (Opa) proteins are critical for Gc infection of host neutrophils and epithelia through engaging select host cell surface receptors known as carcinoembryonic antigen-related cell adhesion molecules (CEACAM). However, the specific mechanisms that dictate Opa/CEACAM specificity are not known. Herein, we present a high-throughput system for recombinant OMP purification from the isolated OMs of Escherichia coli strains optimized for protein expression. We found that all the recombinants OMPs tested co-purified with E. coli. Moreover, this system was successfully applied across multiple E. coli strains optimized for protein expression. The recombinant OMPs extracted from the OM demonstrated greater β-barrel stability than when refolded in vitro and the degree of enhanced stability was impacted by the properties of LPS, indicating this method of high-throughput OMP purification yields a potentially more native-like β-barrel fold than in vitro methods of refolding. Microscale thermophoresis (MST) was used to assess the impact of LPS co-purification on Opa/CEACAM binding. We found that MST binding assays using Opa proteins purified from isolated OMs as a fluorescently labeled reporter and GST-N-CEACAM fusion proteins as the ligand preserved the in vivo Opa/CEACAM binding patterns; signifying that LPS may be a critical determinant for Opa/CEACAM specificity. Future avenues of this research could expand the OMP purification system to other recombinant OMPs or OM protein complexes, identify the determinants of the Opa/LPS interaction, and explore the role of CEACAM clustering during the Opa/CEACAM interaction. The results presented in this dissertation highlight LPS as likely determinants of Opa/CEACAM specificity and yield deeper insight into the influence of OM insertion and LPS on OMP β-barrel stability.

PHD (Doctor of Philosophy)

Host-Pathogen Interactions; Membrane Proteins; Membrane Biophysics

English

2026-07-29