Evgeny A. Semchenko

Characterisation of a Multi-ligand Binding Chemoreceptor CcmL (Tlp3) of Campylobacter jejuni

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.

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

Significance Pathogens use cell surface carbohydrates as a means of attachment to host tissues. In several pathogenic bacteria, truncation of surface carbohydrates, lipooligosaccharide, or lipopolysaccharide have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the lipooligosaccharide/lipopolysaccharide of four distinct bacterial pathogens bind directly to a range of host glycans. Surface plasmon resonance data confirmed binding among 66 different host–glycan:bacterial–glycan pairs. We also demonstrated that bacterial adherence can be competitively inhibited by either host cell or bacterial glycans. Our discovery of high-affinity glycan:glycan interactions in infectious disease may provide new approaches for therapy and prevention. The discovery of the existence of extensive, high-affinity interactions between glycans will alter the perception of the importance of these macromolecular interactions in all biological systems. 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 (KD) 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.

Glycoconjugates play a key role in Campylobacter jejuni Infection: interactions between host and pathogen.

Glycan based interactions between host and pathogen are critical in many bacterial and viral diseases. Glycan interactions range from initial receptor based adherence to protecting the infective agent from the host’s immune response through molecular mimicry. Campylobacter jejuni is an ideal model for studying the role of glycans in host–pathogen interactions, as well as the role of bacterial surface glycoconjugates in infection. Using glycan array analysis, C. jejuni has been shown to interact with a wide range of host glycoconjugates. Mannose and sialic acid residues appear to play a role in initial interactions between host and pathogen following environmental exposure, whereas fucose and galactose based interactions are likely to be required for prolonged colonization. Other studies have highlighted potential decoy receptor type interactions between host’s intestinal mucins and C. jejuni, demonstrating the importance of host glycoproteins as defense against C. jejuni infection as well as the role for glycoconjugates found in human breast milk in protection of breast feeding infants from infection with C. jejuni. C. jejuni can produce N- and O-linked glycoproteins, capsular polysaccharide (CPS) and/or lipooligosaccharide (LOS) which results in C. jejuni presenting its own diverse sugar coated displays on the cell surface. Bacterial glycans play an important and versatile role in infection and disease. Of these, the best understood is the molecular mimicry of human gangliosides presented by C. jejuni’s LOS and its link to the onset of autoimmune neuropathies such as the Guillain Barrè syndrome (GBS). However, the role of glycoconjugates presented by C. jejuni extends beyond expression of sialylated ganglioside structures involved in initiation of GBS. Expression of surface glycans by C. jejuni may also relate to the ability of this organism to interact with the glycoproteins for initial host–pathogen interactions and continued infectivity.

Temperature-dependent phenotypic variation of Campylobacter jejuni lipooligosaccharides

BackgroundCampylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry.ResultsHerein we describe the existence and heterogeneity of multiple LOS forms in C. jejuni strains of human and chicken origin grown at 37°C and 42°C, respectively, as determined on sodium dodecyl sulphate-polyacrylamide electrophoresis gels with carbohydrate-specific silver staining and blotting with anti-ganglioside ligands, and confirmed by nuclear magnetic resonance (NMR) spectroscopy. The C. jejuni NCTC 11168 original isolate (11168-O) was compared to its genome-sequenced variant (11168-GS), and both were found to have a lower-Mr LOS form, which was different in size and structure to the previously characterized higher-Mr form bearing GM1 mimicry. The lower-Mr form production was found to be dependent on the growth temperature as the production of this form increased from ~5%, observed at 37°C to ~35% at 42°C. The structure of the lower-Mr form contained a β-D-Gal-(1→3)-β-D-GalNAc disaccharide moiety which is consistent with the termini of the GM1, asialo-GM1, GD1, GT1 and GQ1 gangliosides, however, it did not display GM1 mimicry as assessed in blotting studies but was shown in NMR to resemble asialo-GM1. The production of multiple LOS forms and lack of GM1 mimicry was not a result of phase variation in the genes tested of NCTC 11168 and was also observed in most of the human and chicken isolates of C. jejuni tested.ConclusionThe presence of differing amounts of LOS forms at 37 and 42°C, and the variety of forms observed in different strains, indicate that LOS form variation may play a role in an adaptive mechanism or a stress response of the bacterium during the colonization of different hosts.

Variation of chemosensory receptor content of Campylobacter jejuni strains and modulation of receptor gene expression under different in vivo and in vitro growth conditions

BackgroundChemotaxis is crucial for the colonisation/infection of hosts with Campylobacter jejuni. Central to chemotaxis are the group A chemotaxis genes that are responsible for sensing the external environment. The distribution of group A chemoreceptor genes, as found in the C. jejuni sequenced strains, tlp1-4, 7, 10 and 11 were determined in 33 clinical human and avian isolates.ResultsGroup A tlp gene content varied among the strains with genes encoding tlp1 (aspartate receptor, ccaA) and tlp7 present in all strains tested, where as tlp11 was present in only one of our international collection clinical isolates, C. jejuni 520, but was more prevalent (9/13) in the freshly isolated clinical stains from patients who required hospitalisation due to C. jejuni infection (GCH1-17). Relative expression levels of the group A tlp genes were also determined in C. jejuni reference strains NCTC 11168-GS, 11168-O and 81116 using cells grown in vitro at 37°C, 42°C and maintained at room temperature and with cells isolated directly from murine and avian hosts by immune magnetic separation without subsequent culture. Gene expression of tlp genes was varied based on strain, growth conditions and in vivo isolation source. Tlp1, although the most conserved, showed the lowest and most varied mRNA expression and protein production under laboratory conditions. Tlp7 was highly expressed at most conditions tested, and gene expression was not influenced by the tlp7 gene encoding a full length protein or one expressed as separate periplasmic and cytoplasmic domains.ConclusionWe have shown that chemosensory receptor set variation exists among C. jejuni strains, but is not dependent on the isolation source.

Structural Heterogeneity of Terminal Glycans in Campylobacter jejuni Lipooligosaccharides

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM1, asialo GM2 and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM2-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides.

The glycointeractome of serogroup B Neisseria meningitidis strain MC58

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.

Lectin Array Analysis of Purified Lipooligosaccharide: A Method for the Determination of Molecular Mimicry

Surface glycosylation of bacteria is involved in many critical host-microbe interactions. Lectin arrays consisting of diverse carbohydrate binding proteins have proven to be an important tool for evaluating a wide variety of glycosylation, including that present on whole bacteria. However, assessing glycosylation on whole bacteria using lectin arrays may not reflect bacterial glycosylation, but interactions between bacteria and the glycosylation present on lectins. The lipooligosaccharide of Campylobacter jejuni NCTC 11168 and 81-176 are known to mimic the human monosialylated gangliosides. This molecular mimicry by C. jejuni can result in the post infection sequelae Guillain–Barre syndrome. Using C. jejuni as a model system and a discreet lectin and antibody array, a method, applicable to many organisms has been developed and validated by to screening of the purified lipooligosaccharide of C. jejuni for molecular mimicry to monosialylated gangliodises. In case of C. jejuni, knowing whether clinically important bacterial strains are capable of inducing severe autoimmune responses may aid in prevention and/or early diagnosis of debilitating post infection conditions.

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

ABSTRACT 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.

The sweet side of the pathogenic Neisseria: the role of glycan interactions in colonisation and disease

&NA; 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. &NA; Graphical Abstract Figure. One sentence summary: Glycosylation of the surface of meningococci and gonococci, and interactions with glycan structures of the host, are essential for pathogenic Neisseria colonisation and disease.