The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

N-glycolylneuraminic acid as a carbohydrate cancer biomarker.

One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5'-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.

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.

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.

Complement Receptor 3 Mediates HIV-1 Transcytosis across an Intact Cervical Epithelial Cell Barrier: New Insight into HIV Transmission in Women.

Transmission of HIV across the mucosal surface of the female reproductive tract to engage subepithelial CD4-positive T cells is not fully understood. Cervical epithelial cells express complement receptor 3 (CR3) (integrin αMß2 or CD11b/CD18). In women, the bacterium Neisseria gonorrhoeae uses CR3 to invade the cervical epithelia to cause cervicitis. We hypothesized that HIV may also use CR3 to transcytose across the cervical epithelia. Here, we show that HIV-1 strains bound with high affinity to recombinant CR3 in biophysical assays. HIV-1 bound CR3 via the I-domain region of the CR3 alpha subunit, CD11b, and binding was dependent on HIV-1 N-linked glycans. Mannosylated glycans on the HIV surface were a high-affinity ligand for the I-domain. Man5 pentasaccharide, representative of HIV N-glycans, could compete with HIV-1 for CR3 binding. Using cellular assays, we show that HIV bound to CHO cells by a CR3-dependent mechanism. Antibodies to the CR3 I-domain or to the HIV-1 envelope glycoprotein blocked the binding of HIV-1 to primary human cervical epithelial (Pex) cells, indicating that CR3 was necessary and sufficient for HIV-1 adherence to Pex cells. Using Pex cells in a Transwell model system, we show that, following transcytosis across an intact Pex cell monolayer, HIV-1 is able to infect TZM-bl reporter cells. Targeting the HIV-CR3 interaction using antibodies, mannose-binding lectins, or CR3-binding small-molecule drugs blocked HIV transcytosis. These studies indicate that CR3/Pex may constitute an efficient pathway for HIV-1 transmission in women and also demonstrate strategies that may prevent transmission via this pathway. IMPORTANCE In women, the lower female reproductive tract is the primary site for HIV infection. How HIV traverses the epithelium to infect CD4 T cells in the submucosa is ill-defined. Cervical epithelial cells have a protein called CR3 on their surface. We show that HIV-1 binds to CR3 with high affinity and that this interaction is necessary and sufficient for HIV adherence to, and transcytosis across, polarized, human primary cervical epithelial cells. This suggests a unique role for CR3 on epithelial cells in dually facilitating HIV-1 attachment and entry. The HIV-CR3 interaction may constitute an efficient pathway for HIV delivery to subepithelial lymphocytes following virus transmission across an intact cervical epithelial barrier. Strategies with potential to prevent transmission via this pathway are presented.

The dCache Chemoreceptor TlpA of Helicobacter pylori Binds Multiple Attractant and Antagonistic Ligands via Distinct Sites.

The Helicobacter pylori chemoreceptor TlpA plays a role in dampening host inflammation during chronic stomach colonization. TlpA has a periplasmic dCache_1 domain, a structure that is capable of sensing many ligands; however, the only characterized TlpA signals are arginine, bicarbonate, and acid. To increase our understanding of TlpA's sensing profile, we screened for diverse TlpA ligands using ligand binding arrays. TlpA bound seven ligands with affinities in the low- to middle-micromolar ranges. Three of these ligands, arginine, fumarate, and cysteine, were TlpA-dependent chemoattractants, while the others elicited no response. Molecular docking experiments, site-directed point mutants, and competition surface plasmon resonance binding assays suggested that TlpA binds ligands via both the membrane-distal and -proximal dCache_1 binding pockets. Surprisingly, one of the nonactive ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands, and acted to block the binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest that TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. IMPORTANCE Numerous chemotactic bacterial pathogens depend on the ability to sense a diverse array of signals through chemoreceptors to achieve successful colonization and virulence within their host. The signals sensed by chemoreceptors, however, are not always fully understood. This is the case for TlpA, a dCache_1 chemoreceptor of H. pylori that enables the bacterium to induce less inflammation during chronic infections. H. pylori causes a significant global disease burden, which is driven by the development of gastric inflammation. Accordingly, it is essential to understand the processes by which H. pylori modulates host inflammation. This work uncovers the signals that TlpA can sense and highlights the underappreciated ability to regulate chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems.

The Lst Sialyltransferase of Neisseria gonorrhoeae Can Transfer Keto-Deoxyoctanoate as the Terminal Sugar of Lipooligosaccharide: a Glyco-Achilles Heel That Provides a New Strategy for Vaccines to Prevent Gonorrhea.

The lipooligosaccharide (LOS) of Neisseria gonorrhoeae plays key roles in pathogenesis and is composed of multiple possible glycoforms. These glycoforms are generated by the process of phase variation and by differences in the glycosyltransferase gene content of particular strains. LOS glycoforms of N. gonorrhoeae can be terminated with an N-acetylneuraminic acid (Neu5Ac), which imparts resistance to the bactericidal activity of serum. However, N. gonorrhoeae cannot synthesize the CMP-Neu5Ac required for LOS biosynthesis and must acquire it from the host. In contrast, Neisseria meningitidis can synthesize endogenous CMP-Neu5Ac, the donor molecule for Neu5Ac, which is a component of some meningococcal capsule structures. Both species have an almost identical LOS sialyltransferase, Lst, that transfers Neu5Ac from CMP-Neu5Ac to the terminus of LOS. Lst is homologous to the LsgB sialyltransferase of nontypeable Haemophilus influenzae (NTHi). Studies in NTHi have demonstrated that LsgB can transfer keto-deoxyoctanoate (KDO) from CMP-KDO to the terminus of LOS in place of Neu5Ac. Here, we show that Lst can also transfer KDO to LOS in place of Neu5Ac in both N. gonorrhoeae and N. meningitidis Consistent with access to the pool of CMP-KDO in the cytoplasm, we present data indicating that Lst is localized in the cytoplasm. Lst has previously been reported to be localized on the outer membrane. We also demonstrate that KDO is expressed as a terminal LOS structure in vivo in samples from infected women and further show that the anti-KDO monoclonal antibody 6E4 can mediate opsonophagocytic killing of N. gonorrhoeae Taken together, these studies indicate that KDO expressed on gonococcal LOS represents a new antigen for the development of vaccines against gonorrhea.IMPORTANCE The emergence of multidrug-resistant N. gonorrhoeae strains that are resistant to available antimicrobials is a current health emergency, and no vaccine is available to prevent gonococcal infection. Lipooligosaccharide (LOS) is one of the major virulence factors of N. gonorrhoeae The sialic acid N-acetylneuraminic acid (Neu5Ac) is present as the terminal glycan on LOS in N. gonorrhoeae In this study, we made an unexpected discovery that KDO can be incorporated as the terminal glycan on LOS of N. gonorrhoeae by the alpha-2,3-sialyltransferase Lst. We showed that N. gonorrhoeae express KDO on LOS in vivo and that the KDO-specific monoclonal antibody 6E4 can direct opsonophagocytic killing of N. gonorrhoeae These data support further development of KDO-LOS structures as vaccine antigens for the prevention of infection by N. gonorrhoeae.

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin.

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.

Shigella flexneri Targets Human Colonic Goblet Cells by O Antigen Binding to Sialyl-Tn and Tn Antigens via Glycan-Glycan Interactions.

Shigella flexneri targets colonic cells in humans to initiate invasive infection processes that lead to dysentery, and direct interactions between their lipopolysaccharide O antigens and blood group A related glycans are involved in the cell adherence interactions. Here, we show that treatment with Tn and sialyl-Tn glycans, monoclonal antibodies and lectins reactive to Tn/sialyl-Tn, and luteolin (a Tn antigen synthesis inhibitor) all significantly inhibited S. flexneri adherence and invasion of cells in vitro. Surface plasmon resonance analysis showed that lipopolysaccharide O antigen had a high affinity interaction with Tn/sialyl-Tn. Immunofluorescence probing of human colon tissue with antibodies detected expression of Tn/sialyl-Tn by MUC2 producing goblet cells (GCs), and S. flexneri incubated with human colon tissue colocalized with GCs. Our findings demonstrate that S. flexneri targets GCs in the human colonic crypts via glycan-glycan interactions, establishing new insight into the infection process in humans.

Repurposed Drugs That Block the Gonococcus-Complement Receptor 3 Interaction Can Prevent and Cure Gonococcal Infection of Primary Human Cervical Epithelial Cells.

In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal-I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.

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.

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.

Solution structure, glycan specificity and of phenol oxidase inhibitory activity of Anopheles C-type lectins CTL4 and CTLMA2.

Malaria, the world's most devastating parasitic disease, is transmitted between humans by mosquitoes of the Anopheles genus. An. gambiae is the principal malaria vector in Sub-Saharan Africa. The C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCXC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 form higher oligomeric states at physiological pH. Both lectins bind specific sugars, including glycosaminoglycan motifs with ß1-3/ß1-4 linkages between glucose, galactose and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is found to function in vivo, reversing the enhancement of phenol oxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection.

Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells.

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 µM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeaeIMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

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.

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.

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.

YesU from Bacillus subtilis preferentially binds fucosylated glycans.

The interaction of carbohydrate-binding proteins (CBPs) with their corresponding glycan ligands is challenging to study both experimentally and computationally. This is in part due to their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates, as exists in nucleic acids and proteins. We recently described a function-prediction technique called SPOT-Struc that identifies CBPs by global structural alignment and binding-affinity prediction. Here we experimentally determined the carbohydrate specificity and binding affinity of YesU (RCSB PDB ID: 1oq1), an uncharacterized protein from Bacillus subtilis that SPOT-Struc predicted would bind high mannose-type glycans. Glycan array analyses however revealed glycan binding patterns similar to those exhibited by fucose (Fuc)-binding lectins, with SPR analysis revealing high affinity binding to Lewisx and lacto-N-fucopentaose III. Structure based alignment of YesU revealed high similarity to the legume lectins UEA-I and GS-IV, and docking of Lewisx into YesU revealed a complex structure model with predicted binding affinity of -4.3 kcal/mol. Moreover the adherence of B. subtilis to intestinal cells was significantly inhibited by Lex and Ley but by not non-fucosylated glycans, suggesting the interaction of YesU to fucosylated glycans may be involved in the adhesion of B. subtilis to the gastrointestinal tract of mammals.