Neisseria gonorrhoeae (Ng) is a major public health threat due to its rapid development of antibiotic resistance and the absence of a vaccine. Glycan interactions between the Ng surface and host cells are important for bacterial adherence and pathogenesis, offering potential targets for novel therapeutics. Our previous studies identified several putative Ng mannose-binding proteins, including the TonB-dependent transporters TbpA, TdfH and TdfJ, suggesting that these nutrient-acquisition proteins may also contribute to other host interactions.
In this study, we investigated the glycan-binding properties of TbpA, TdfH and TdfJ and examined their potential roles in nutrient acquisition and other putative functions, including interactions with host cells. Glycan array and surface plasmon resonance (SPR) analyses showed that recombinant TbpA, TdfH and TdfJ bind multiple host-associated glycans, including mannosylated glycans and glycans containing N-acetylneuraminic acid, N-acetylgalactosamine, N-acetylglucosamine and galactose, with high affinity (0.53–570.43 nM). Using in vitro cell binding with recombinant proteins, together with cell-adherence assays with wild-type and knock-out isogenic Ng strains, we demonstrated that TbpA and TdfH are involved in adherence to cervical and urethral epithelial cells.
To investigate the druggability of TbpA and TdfH, we screened both recombinant proteins against a panel of 10 mannosides and a library of 2,400 open‑access/FDA‑approved compounds using SPR. We identified 8 compounds that bind TbpA and TdfH with high affinity and inhibit protein-associated functions, including transferrin binding, protein mediated adherence, and Ng growth under metal-limited conditions. To obtain molecular insights into the mechanisms of inhibition, we performed molecular docking and molecular dynamics simulations on selected inhibitors. These analyses suggested that selected compounds preferentially interact with a functionally relevant plug-domain-associated region proximal to the surface-exposed loops, where binding was more stable over time, supporting a possible mechanism for interference with transferrin binding and adherence-associated functions.
Together, these findings highlight that TonB-dependent transporters may have additional roles beyond nutrient acquisition and support their potential as therapeutic targets for the development of new strategies to treat gonorrhoea.