Neisseria gonorrhoeae is a sexually transmitted Gram-negative high-priority pathogen, causing rising case rates and widespread antimicrobial resistance globally. Gonococcus utilises a family of variable adhesins, called Opacity proteins (Opa), to interact with critical host receptors, thus enhancing bacterial invasion. This project investigated two key host targets of Opa proteins: glycosaminoglycans (GAGs), a family of related polysaccharides, and vitronectin, a glycoprotein regulator of the complement system, to further understand their roles in pathogenesis.
Serum bactericidal assays revealed that both vitronectin and the GAG heparin independently enhanced serum resistance, with optimal protection observed following co-incubation, suggesting potential synergy in complement inhibition. The degree of protection provided by alternative GAGs correlated with their sulphation state, indicating an essential role for sulphate groups in promoting evasion of complement-mediated killing. To further investigate the impact of vitronectin and GAGs on complement inhibition, a dual approach of pull-down assays and proteomics was employed. This approach revealed differential surface deposition of complement proteins and inhibitors in the presence of these molecules, adding mechanistic detail to the serum resistance conferred by them on N. gonorrhoeae. Vitronectin-binding assays across a panel of clinical gonococcal isolates, sequenced using a hybrid approach, allowed comparison of Opa profiles between high- and low-binding isolates. Such binding assays revealed N. gonorrhoeae-vitronectin interactions are electrostatic in nature and required bridging saccharides. Finally, vitronectin-affinity chromatography identified several novel vitronectin-binding proteins within gonococcal outer membrane vesicles. Recombinant expression confirmed vitronectin binding by a selected previously uncharacterised protein, with homologues identified across clinical isolates. Antibodies raised against this protein inhibited interactions with vitronectin and confirmed its expression across several Neisserial spp. including Neisseria meningitidis.
Together, this work demonstrates an important role for both vitronectin and GAGs in enhancing gonococcal serum resistance. Given the high abundance of these host molecules in vivo, such findings likely have important implications for gonococcal pathogenesis. Characterisation of Opa-vitronectin interactions in combination with the identification of uncharacterised putative gonococcal vitronectin binding ligands adds important molecular detail regarding how N. gonorrhoea recruits this host regulatory glycoprotein.