AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in N. gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization.

Neisseria meningitidis express numerous virulence factors that enable it to interact with diverse microenvironments within the host, during both asymptomatic nasopharyngeal colonization and invasive disease. Many of these interactions involve bacterial or host glycans. In order to characterise the meningococcal glycointeractome, glycan arrays representative of structures found on human cells, were used as a screening tool to investigate host glycans bound by N. meningitidis. Arrays probed with fluorescently labelled wild-type MC58 revealed binding to 223 glycans, including blood group antigens, mucins, gangliosides and glycosaminoglycans. Mutant strains lacking surface components, including capsule, lipooligosaccharide (LOS), Opc and pili, were investigated to identify the factors responsible for glycan binding. Surface plasmon resonance and isothermal calorimetry were used to confirm binding and determine affinities between surface components and host glycans. We observed that the L3 LOS immunotype (whole cells and purified LOS) bound 26 structures, while L8 only bound 5 structures. We further demonstrated a direct glycan-glycan interaction between purified L3 LOS and Thomsen–Friedenreich (TF) antigen, with a KD of 13 nM. This is the highest affinity glycan-glycan interaction reported to date. These findings highlight the diverse glycointeractions that may occur during different stages of meningococcal disease, which could be exploited for development of novel preventative and therapeutic strategies.

Glycomics is a rapidly growing field that focuses on the structure and function of carbohydrates (glycans) in biological systems. Glycan interactions play a major role in infectious disease, at all stages of colonisation and disease progression. Neisseria meningitidis, the cause of meningococcal sepsis and meningitis, and Neisseria gonorrhoeae, which causes the sexually transmitted infection gonorrhoea, are responsible for significant morbidity and mortality worldwide. Neisseria meningitidis displays a range of surface glycosylations including capsule polysaccharide, lipooligosaccharide and O-linked glycoproteins. While N. gonorrhoeae does not have a capsule, it does express both lipooligosaccharide and O-linked glycoproteins. Neisseria gonorrhoeae also has the ability to scavenge host sialic acids, while several N. meningitidis serogroups can synthesise sialic acid. Surface expressed sialic acid is key in serum resistance and survival in the host. On the host side, the pathogenic Neisseria protein adhesins such as Opc and NHBA bind to host glycans for adherence and colonisation of host cells. Essentially, from both the bacterial and host perspective, glycan interactions are fundamental in colonisation and disease of pathogenic Neisseria. The key aspects of glycobiology of the pathogenic Neisseria are reviewed herein.

Neisseria meningitidis is a Gram-negative bacterial pathogen that causes life threatening meningitis and septicemia. Neisseria Heparin Binding Antigen (NHBA) is an outer membrane protein that binds heparin and heparan sulfate and DNA. This protein is one of the four antigens in the meningococcal serogroup B vaccine Bexsero. In the current study, we sought to define the full glycan-binding repertoire of NHBA to better understand its role in meningococcal pathogenesis and vaccine efficacy. Glycan array analysis revealed binding to 28 structures by recombinant NHBA. Surface plasmon resonance was used to confirm the binding phenotype and to determine the affinity of the interactions. These studies revealed that the highest affinity binding of NHBA was with chondroitin sulfate (KD = 5.2 nM). This affinity is 10-fold higher than observed for heparin. Analysis of binding with well-defined disaccharides of the different chondroitin sulfate types demonstrated that the most preferred ligand has a sulfate at the 2 position of the GlcA/IdoA and 6 position of the GalNAc, which is an equivalent structure to chondroitin sulfate D. Chondroitin sulfate is widely expressed in human tissues, while chondroitin sulfate D is predominantly expressed in the brain and may constitute a new receptor structure for meningococci.