Abstract
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.
Highlights
Sialic acids (Sias) are a family of 9-carbon sugars (nonoses, or nonulosonates (NulOs)) expressed in the tissues of every vertebrate and some “higher” invertebrates [1]
Gonococci have the capacity to scavenge CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac, a CMP-activated 9-carbon sugar that is a member of the ‘sialic acid family’) from the host to ‘cap’ its lipooligosaccharide with Neu5Ac, which renders gonococci resistant to complement, a key arm of innate immune defenses
We show that gonococci utilize derivatives of CMP-Neu5Ac, which fail to render the bacteria resistant to complement, and prevent complement inhibition mediated by the ‘physiologic’ human sialic acid donor, CMP-Neu5Ac
Summary
Sialic acids (Sias) are a family of 9-carbon sugars (nonoses, or nonulosonates (NulOs)) expressed in the tissues of every vertebrate and some “higher” invertebrates [1]. Cell surface-associated Sia regulates the immune system by inhibiting complement activation. Recent work has shown that FH C-terminal domains 19 and 20 bound simultaneously to C3b and glycosaminoglycans or Sias on host cells, which served to inhibit the alternative pathway [4]. This was further confirmed by structural data that suggest a trimolecular complex of the two C-terminal FH domains, Sia and C3b [5]. Neuraminidase treatment of sheep erythrocytes removes cell surface Sias and reduces the affinity of FH for C3b, which permits complement activation and promotes hemolysis. FH binds vertebrate cell surfaces via Sias to allow preferential protection of host cells (i.e., reduce complement-mediated damage)
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