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.

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen.

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.

A Novel Sialylation Site on Neisseria gonorrhoeae Lipooligosaccharide Links Heptose II Lactose Expression with Pathogenicity.

Sialylation of lacto-N-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody.

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.

Evolution of the exclusively human pathogen Neisseria gonorrhoeae: Human-specific engagement of immunoregulatory Siglecs.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea exclusively in humans and uses multiple strategies to infect, including acquisition of host sialic acids that cap and mask lipooligosaccharide termini, while restricting complement activation. We hypothesized that gonococci selectively target human anti-inflammatory sialic acid-recognizing Siglec receptors on innate immune cells to blunt host responses and that pro-inflammatory Siglecs and SIGLEC pseudogene polymorphisms represent host evolutionary adaptations to counteract this interaction. N. gonorrhoeae can indeed engage multiple human but not chimpanzee CD33rSiglecs expressed on innate immune cells and in the genitourinary tract--including Siglec-11 (inhibitory) and Siglec-16 (activating), which we detected for the first time on human cervical epithelium. Surprisingly, in addition to LOS sialic acid, we found that gonococcal porin (PorB) mediated binding to multiple Siglecs. PorB also bound preferentially to human Siglecs and not chimpanzee orthologs, modulating host immune reactions in a human-specific manner. Lastly, we studied the distribution of null SIGLEC polymorphisms in a Namibian cohort with a high prevalence of gonorrhea and found that uninfected women preferentially harbor functional SIGLEC16 alleles encoding an activating immune receptor. These results contribute to the understanding of the human specificity of N. gonorrhoeae and how it evolved to evade the human immune defense.