A novel sialylation site on Neisseria gonorrhoeae lipooligosaccharide links heptose II lactose expression with pathogenicity [preprint]

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 mAb 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)-containing media enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOS of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and a MS11 mutant with only lactose 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. 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 reiterate the candidacy of the 2C7 epitope as a vaccine antigen and mAb 2C7 as an immunotherapeutic antibody

IL-6 secretion assay - Fig.5A

Raw data and statistical analysis of IL-6 secretion ELISA assay of Neisseria gonorrhoeae challenged THP-1 cells expressing Siglec-5 or Siglec-14

Siglec-Fc Binding Assays - Data of Figure 2, 3B, S6B

Raw data and statistical analysis of Siglec-Fc binding assays with Neisseria gonorrhoeae strains

Molecular Mimicry of Host Sialome by Human Pathogens to exploit Immunoregulatory Siglecs

This thesis focuses on the characterization of two human-specific pathogens that mimic host sialic acid surface structures to engage human immunoregulatory Siglec receptors. Furthermore, it explores the use of sialic acid and sialic acid-like molecules as tools and therapeutics. The first chapter introduces the different types of sialic acids and their diverse functions in prokaryotes and eukaryotes. It reviews different viral, bacterial, fungal and parasitic pathogens that use sialic acid, in particular as molecular mimicry, to engage the host and subvert the host immune system. The second chapter characterizes the interactions between Escherichia coli K1, which mimics host polysialic acid, and the paired receptors Siglec-11 and Siglec-16. E. coli K1 engages the inhibiting receptor Siglec-11 to escapes killing. In contrast, binding to Siglec-16, an activating immunoregulatory receptor, increases inflammatory responses and phagocytosis. Chapter two also introduces a murine model to study the interaction of pathogens with paired receptors. The third chapter describes interactions between Neisseria gonorrhoeae, the causative agent of gonorrhea, and human Siglecs as well as their potential impact on pathogenesis. The interactions are mediated by sialylated lipooligosaccharide (LOS) structures that mimic human glycosphingolipids. Additionally, gonococcal porins engage Siglecs in a sialic acid-independent manner. In many individuals, a fusion event between SIGLEC5 and SIGLEC14 genes and a polymorphism in the SIGLEC16 allele lead to the loss of functional Siglec-14 and Siglec-16, respectively. The impact of the loss of these activating receptors on the infection rate is explored in a remote population of Namibian pastoralists, which have a high burden of gonorrhea. The increasing number of infections and antibiotic resistance of N. gonorrhoeae pose a risk to the future of gonorrhea treatment. Chapter 3 introduces a novel therapeutic strategy to that may treat and prevent gonococcal infections. N. gonorrhoeae incorporates host sialic acid into its LOS to inhibit the classical and alternative pathway of the host complement system. N. gonorrhoeae also incorporates sialic acid analogs (e.g. legionaminic acid) into its LOS, which are usually not available to the pathogen. Consequently, it is not able to inhibit the classical pathway anymore. Administration of legionaminic acid to a gonorrhea mouse model leads to a reduced clearance time and infection burden. The 9-O-acetyl modification of sialic acid is very common in humans and other animals. However, it is very difficult to study since this modification is very unstable. The addendum chapter suggests a chemical solution to this problem. The oxygen atom in the 9-O-acetyl group is substituted by a nitrogen atom, which leads to a chemically and biologically stable 9-N-acetyl group. This simple approach opens up many new opportunities to study 9-O-acetylated sialic acid, for example its role in host-pathogen interactions

Siglec-Fc Binding Assays with gonococcal porins - Fig.4

Raw data and statistical analysis of binding assay with Siglec-Fcs and gonococcal porins

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

Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates

Abstract Background Siglecs-11 and -16 are members of the sialic acid recognizing Ig-like lectin family, and expressed in same cells. Siglec-11 functions as an inhibitory receptor, whereas Siglec-16 exhibits activating properties. In humans, SIGLEC11 and SIGLEC16 gene sequences are extremely similar in the region encoding the extracellular domain due to gene conversions. Human SIGLEC11 was converted by the nonfunctional SIGLEC16P allele, and the converted SIGLEC11 allele became fixed in humans, possibly because it provides novel neuroprotective functions in brain microglia. However, the detailed evolutionary history of SIGLEC11 and SIGLEC16 in other primates remains unclear. Results We analyzed SIGLEC11 and SIGLEC16 gene sequences of multiple primate species, and examined glycan binding profiles of these Siglecs. The phylogenetic tree demonstrated that gene conversions between SIGLEC11 and SIGLEC16 occurred in the region including the exon encoding the sialic acid binding domain in every primate examined. Functional assays showed that glycan binding preference is similar between Siglec-11 and Siglec-16 in all analyzed hominid species. Taken together with the fact that Siglec-11 and Siglec-16 are expressed in the same cells, Siglec-11 and Siglec-16 are regarded as paired receptors that have maintained similar ligand binding preferences via gene conversions. Relaxed functional constraints were detected on the SIGLEC11 and SIGLEC16 exons that underwent gene conversions, possibly contributing to the evolutionary acceptance of repeated gene conversions. The frequency of nonfunctional SIGLEC16P alleles is much higher than that of SIGLEC16 alleles in every human population. Conclusions Our findings indicate that Siglec-11 and Siglec-16 have been maintained as paired receptors by repeated gene conversions under relaxed functional constraints in the primate lineage. The high prevalence of the nonfunctional SIGLEC16P allele and the fixation of the converted SIGLEC11 imply that the loss of Siglec-16 and the gain of Siglec-11 in microglia might have been favored during the evolution of human lineage

Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates.

BackgroundSiglecs-11 and -16 are members of the sialic acid recognizing Ig-like lectin family, and expressed in same cells. Siglec-11 functions as an inhibitory receptor, whereas Siglec-16 exhibits activating properties. In humans, SIGLEC11 and SIGLEC16 gene sequences are extremely similar in the region encoding the extracellular domain due to gene conversions. Human SIGLEC11 was converted by the nonfunctional SIGLEC16P allele, and the converted SIGLEC11 allele became fixed in humans, possibly because it provides novel neuroprotective functions in brain microglia. However, the detailed evolutionary history of SIGLEC11 and SIGLEC16 in other primates remains unclear.ResultsWe analyzed SIGLEC11 and SIGLEC16 gene sequences of multiple primate species, and examined glycan binding profiles of these Siglecs. The phylogenetic tree demonstrated that gene conversions between SIGLEC11 and SIGLEC16 occurred in the region including the exon encoding the sialic acid binding domain in every primate examined. Functional assays showed that glycan binding preference is similar between Siglec-11 and Siglec-16 in all analyzed hominid species. Taken together with the fact that Siglec-11 and Siglec-16 are expressed in the same cells, Siglec-11 and Siglec-16 are regarded as paired receptors that have maintained similar ligand binding preferences via gene conversions. Relaxed functional constraints were detected on the SIGLEC11 and SIGLEC16 exons that underwent gene conversions, possibly contributing to the evolutionary acceptance of repeated gene conversions. The frequency of nonfunctional SIGLEC16P alleles is much higher than that of SIGLEC16 alleles in every human population.ConclusionsOur findings indicate that Siglec-11 and Siglec-16 have been maintained as paired receptors by repeated gene conversions under relaxed functional constraints in the primate lineage. The high prevalence of the nonfunctional SIGLEC16P allele and the fixation of the converted SIGLEC11 imply that the loss of Siglec-16 and the gain of Siglec-11 in microglia might have been favored during the evolution of human lineage