Therapeutic CMP-Nonulosonates against Multidrug-Resistant Neisseria gonorrhoeae

Neisseria gonorrhoeae deploys a unique immune evasion strategy wherein the lacto-N-neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac₂ and CMP-Neu5Ac9N₃, are also substrates for Lst. Incorporation of Leg5,7Ac₂ and Neu5Ac9N₃ into LOS results in N. gonorrhoeae being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac₂ attenuated N. gonorrhoeae colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N₃, but not CMP-Neu4,5Ac₂, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto-N-neotetraose LOS capped with Kdn and Kdn7N₃ bound FH to levels ∼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N₃, and CMP-Leg5,7Ac₂ administered intravaginally (10 μg/d) to N. gonorrhoeae-colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac₂ was the most pH and temperature stable. In addition, Leg5,7Ac₂-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac₂ was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome (Cmah mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac₂ and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant N. gonorrhoeae.