Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics

Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-n-neotetraose (lnnt) structure of lipooligosaccharide (los) is capped by the bacterial sialyltransferase,using host cytidine-5’-monophosphate (cmp)-activated forms of the nine-carbon nonulosonate (nulo) sugar n-acetyl-neuraminic acid (neu5ac),a sialic acid (sia) abundant in humans. this allows evasion of complement-mediated killing by recruiting factor h (fh),an inhibitor of the alternative complement pathway,and by limiting classical pathway activation (“serum-resistance”). we utilized cmp salts of six additional natural or synthetic nulos,neu5gc,neu5gc8me,neu5ac9ac,neu5ac9az,legionaminic acid (leg5ac7ac) and pseudaminic acid (pse5ac7ac),to define structural requirements of sia-mediated serum-resistance. while all nulos except pse5ac7ac were incorporated into the lnnt-los,only neu5gc incorporation yielded high-level serum-resistance and fh binding that was comparable to neu5ac,whereas neu5ac9az and leg5ac7ac incorporation left bacteria fully serum-sensitive and did not enhance fh binding. neu5ac9ac and neu5gc8me rendered bacteria resistant only to low serum concentrations. while serum-resistance mediated by neu5ac was associated with classical pathway inhibition (decreased igg binding and c4 deposition),leg5ac7ac and neu5ac9az incorporation did not inhibit the classical pathway. remarkably,cmp-neu5ac9az and cmp-leg5ac7ac each prevented serum-resistance despite a 100-fold molar excess of cmp-neu5ac in growth media. the concomitant presence of leg5ac7ac and neu5ac on los resulted in uninhibited classical pathway activation. surprisingly,despite near-maximal fh binding in this instance,the alternative pathway was not regulated and factor bb remained associated with bacteria. intravaginal administration of cmp-leg5ac7ac to balb/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. bacteria recovered from cmp-leg5ac7ac-treated mice were sensitive to human complement ex vivo,simulating in vitro findings. these data reveal critical roles for the sia exocyclic side-chain in gonococcal serum-resistance. such cmp-nulo analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea. © 2015 gulati et al.