Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Repurposing Carbamazepine To Treat Gonococcal Infection in Women: Oral Delivery for Control of Epilepsy Generates Therapeutically Effective Levels in Vaginal Secretions.

Neisseria gonorrhoeae has developed resistance to all previous antibiotics used for treatment. This highlights a crucial need for novel antimicrobials to treat gonococcal infections. We previously showed that carbamazepine (Cz), one of the most commonly prescribed antiepileptic drugs, can block the interaction between gonococcal pili and the I-domain region of human complement receptor 3 (CR3)-an interaction that is vital for infection of the female cervix. We also show that Cz can completely clear an established N. gonorrhoeae infection of primary human cervical cells. In this study, we quantified Cz in serum, saliva, and vaginal fluid collected from 16 women who were, or were not, regularly taking Cz. We detected Cz in lower reproductive tract mucosal secretions in the test group (women taking Cz) at potentially therapeutic levels using a competitive ELISA. Furthermore, we found that Cz concentrations present in vaginal fluid from women taking this drug were sufficient to result in a greater than 99% reduction (within 24 h) in the number of viable gonococci recovered from ex vivo, human, primary cervical cell infections. These data provide strong support for the further development of Cz as a novel, host-targeted therapy to treat gonococcal cervicitis.

Serum Neu5Gc biomarkers are elevated in primary cutaneous melanoma.

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.

How bacteria utilize sialic acid during interactions with the host: snip, snatch, dispatch, match and attach.

N -glycolylneuraminic acid (Neu5Gc), and its precursor N-acetylneuraminic acid (Neu5Ac), commonly referred to as sialic acids, are two of the most common glycans found in mammals. Humans carry a mutation in the enzyme that converts Neu5Ac into Neu5Gc, and as such, expression of Neu5Ac can be thought of as a 'human specific' trait. Bacteria can utilize sialic acids as a carbon and energy source and have evolved multiple ways to take up sialic acids. In order to generate free sialic acid, many bacteria produce sialidases that cleave sialic acid residues from complex glycan structures. In addition, sialidases allow escape from innate immune mechanisms, and can synergize with other virulence factors such as toxins. Human-adapted pathogens have evolved a preference for Neu5Ac, with many bacterial adhesins, and major classes of toxin, specifically recognizing Neu5Ac containing glycans as receptors. The preference of human-adapted pathogens for Neu5Ac also occurs during biosynthesis of surface structures such as lipo-oligosaccharide (LOS), lipo-polysaccharide (LPS) and polysaccharide capsules, subverting the human host immune system by mimicking the host. This review aims to provide an update on the advances made in understanding the role of sialic acid in bacteria-host interactions made in the last 5-10 years, and put these findings into context by highlighting key historical discoveries. We provide a particular focus on 'molecular mimicry' and incorporation of sialic acid onto the bacterial outer-surface, and the role of sialic acid as a receptor for bacterial adhesins and toxins.

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes.

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton-Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE-LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE's rim domain contribute to hemolysis, namely residues 57-75 (DR1) and residues 182-196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE-DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

Human Plasminogen Exacerbates Clostridioides difficile Enteric Disease and Alters the Spore Surface.

BACKGROUND & AIMS: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. METHODS: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. RESULTS: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. CONCLUSIONS: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.

MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

The mechanics of myeloid cells.

The effects of cell size, shape and deformability on cellular function have long been a topic of interest. Recently, mechanical phenotyping technologies capable of analysing large numbers of cells in real time have become available. This has important implications for biology and medicine, especially haemato-oncology and immunology, as immune cell mechanical phenotyping, immunologic function, and malignant cell transformation are closely linked and potentially exploitable to develop new diagnostics and therapeutics. In this review, we introduce the technologies used to analyse cellular mechanical properties and review emerging findings following the advent of high throughput deformability cytometry. We largely focus on cells from the myeloid lineage, which are derived from the bone marrow and include macrophages, granulocytes and erythrocytes. We highlight advances in mechanical phenotyping of cells in suspension that are revealing novel signatures of human blood diseases and providing new insights into pathogenesis of these diseases. The contributions of mechanical phenotyping of cells in suspension to our understanding of drug mechanisms, identification of novel therapeutics and monitoring of treatment efficacy particularly in instances of haematologic diseases are reviewed, and we suggest emerging topics of study to explore as high throughput deformability cytometers become prevalent in laboratories across the globe.

Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells.

The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

Specific blood group antibodies inhibit Shigella flexneri interaction with human cells in the absence of spinoculation.

The classical models of investigating Shigella flexneri adherence and invasion of tissue culture cells involve either bacterial centrifugation (spinoculation) or the use of AfaE adhesin to overcome the low infection rate observed in vitro. However clinically, S. flexneri clearly adheres and invades the human colon in the absence of 'spinoculation'. Additionally, certain S. flexneri tissue cell based assays (e.g. plaque assays and infection of T84 epithelial cells on Transwells®), do not require spinoculation. In the absence of spinoculation, we recently showed that glycan-glycan interactions play an important role in S. flexneri interaction with host cells, and that in particular the S. flexneri 2a lipopolysaccharide O antigen glycan has a high affinity for the blood group A glycan. During the investigation of the effect of blood group A antibodies on S. flexneri interaction with cells, we discovered that Panc-1 cells exhibited a high rate of infection in the absence of spinoculation. Select blood group A antibodies inhibited invasion of Panc-1 cells, and adherence to T84 cells. The use of Panc-1 cells represents a simplified model to study S. flexneri pathogenesis and does not require either spinoculation or exogenous adhesins.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Colonization of the oropharynx is the initial step in Group A Streptococcus (GAS) pharyngeal infection. We have previously reported that the highly virulent M1T1 GAS clone attaches to oral epithelial cells via M1 protein interaction with blood group antigen carbohydrate structures. Here, we have identified that colonization of human oral epithelial cells by GAS serotypes M3 and M12 is mediated by human blood group antigens [ABO(H)] and Lewis (Le) antigen expression. Removal of linkage-specific fucose, galactose, N-acetylgalactosamine, and sialic acid modulated GAS colonization, dependent on host ABO(H) blood group and Le expression profile. Furthermore, N-linked glycans from human salivary glycoproteins, when released and purified, were potent inhibitors of M1, M3, and M12 GAS colonization ex vivo. These data highlight the important role played by human protein glycosylation patterns in GAS attachment to oral epithelial cell surfaces.-De Oliveira, D. M. P., Everest-Dass, A., Hartley-Tassell, L., Day, C. J., Indraratna, A., Brouwer, S., Cleary, A., Kautto, L., Gorman, J., Packer, N. H., Jennings, M. P., Walker, M. J., Sanderson-Smith, M. L. Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Functional Microarray Platform with Self-Assembled Monolayers on 3C-Silicon Carbide.

Currently available bioplatforms such as microarrays and surface plasmon resonators are unable to combine high-throughput multiplexing with label-free detection. As such, emerging microelectromechanical systems (MEMS) and microplasmonics platforms offer the potential for high-resolution, high-throughput label-free sensing of biological and chemical analytes. Therefore, the search for materials capable of combining multiplexing and label-free quantitation is of great significance. Recently, interest in silicon carbide (SiC) as a suitable material in numerous biomedical applications has increased due to its well-explored chemical inertness, mechanical strength, bio- and hemocompatibility, and the presence of carbon that enables the transfer-free growth of graphene. SiC is also multifunctional as both a wide-band-gap semiconductor and an efficient low-loss plasmonics material and thus is ideal for augmenting current biotransducers in biosensors. Additionally, the cubic variant, 3C-SiC, is an extremely promising material for MEMS, being a suitable platform for the easy micromachining of microcantilevers, and as such capable of realizing the potential of real time miniaturized multiplexed assays. The generation of an appropriately functionalized and versatile organic monolayer suitable for the immobilization of biomolecules is therefore critical to explore label-free, multiplexed quantitation of biological interactions on SiC. Herein, we address the use of various silane self-assembled monolayers (SAMs) for the covalent functionalization of monocrystalline 3C-SiC films as a novel platform for the generation of functionalized microarray surfaces using high-throughput glycan arrays as the model system. We also demonstrate the ability to robotically print high throughput arrays on free-standing SiC microstructures. The implementation of a SiC-based label-free glycan array will provide a proof of principle that could be extended to the immobilization of other biomolecules in a similar SiC-based array format, thus making potentially significant advances to the way biological interactions are studied.

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile.

Clostridium difficile is a major cause of hospital-acquired antibiotic-associated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and ß-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and ß-linked galactose, including blood groups, Lewis antigens, N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues in vivo.

Lectin activity of Pseudomonas aeruginosa vaccine candidates PSE17-1, PSE41-5 and PSE54.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal ß-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal ß-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.

Specificity and utility of SubB2M, a new N-glycolylneuraminic acid lectin.

The B subunit of the subtilase cytotoxin (SubB) recognises N-glycolylneuraminic acid (Neu5Gc) containing glycans, the most prominent form of aberrant glycosylation in human cancers. We have previously engineered SubB by construction of a SubBΔS106/ΔT107 mutant (SubB2M) for greater specificity and enhanced recognition of Neu5Gc containing glycans. In this study, we further explore the utility of SubB2M as a Neu5Gc lectin by showing its improved specificity and recognition for Neu5Gc containing glycans over the wild-type SubB protein and an anti-Neu5Gc IgY antibody in a N-acetylneuraminic acid (Neu5Ac)/Neu5Gc glycan array and by surface plasmon resonance. Far-western blot analysis showed that SubB2M preferentially binds to bovine serum glycoproteins over human serum glycoproteins. SubB2M was also able to detect Neu5Gc containing bovine glycoproteins spiked into normal human serum with greater sensitivity than the wild-type SubB and the anti-Neu5Gc IgY antibody. These results suggest that SubB2M will be a useful tool for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.

The HMW2 adhesin of non-typeable Haemophilus influenzae is a human-adapted lectin that mediates high-affinity binding to 2-6 linked N-acetylneuraminic acid glycans.

Non-typeable Haemophilus influenzae (NTHi) is a human-adapted bacterial pathogen, responsible for infections of the human respiratory tract. This pathogen expresses a range of adhesins that mediate binding to host cells. Most NTHi strains can express the related adhesins HMW1 and HMW2. Expression of HMW proteins is phase-variable: changes in the length of simple-sequence repeats located in the encoding genes promoter regions results in changes in expression levels of these adhesins. HMW expression is also controlled by epigenetic regulation. HMW1 has been previously demonstrated to bind α 2-3 sialyl-lactosamine, but affinity of this interaction has not been investigated. The host receptor(s) for HMW2 is currently unknown. We hypothesized that host glycans may act as receptors for HMW2-mediated adherence. We examined the glycan-binding activity of HMW2 using glycan arrays and Surface Plasmon Resonance (SPR). These studies demonstrate that HMW2 binds 2-6 linked N-acetylneuraminic acid with high affinity. HMW2 did not bind glycan structures containing the non-human form of sialic acid, N-glycolylneuraminic acid. Thus, the specificity of HMW1 and HMW2 have complementary lectin activities that may allow NTHi distinct niches in the human host.

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems.

MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea.