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High Affinity Biomolecular Interactions That Can Mediate Binding of Pathogenic Bacteria to Host Cells

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High Affinity Biomolecular Interactions That Can Mediate Binding of Pathogenic Bacteria to Host Cells Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells Christopher J. Daya,1, Elizabeth N. Tranb, Evgeny A. Semchenkoa, Greg Trama, Lauren E. Hartley-Tassella, Preston S. K. Nga, Rebecca M. Kinga, Rachel Ulanovskya, Sarah McAtamneya, Michael A. Apicellac, Joe Tiralongoa, Renato Moronab,2, Victoria Korolika,2, and Michael P. Jenningsa,1,2 aInstitute for Glycomics, Griffith University Gold Coast Campus, Gold Coast, QLD 4222, Australia; bSchool of Biological Sciences, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide, SA 5005, Australia; and cDepartment of Microbiology, University of Iowa, Iowa City, IA 52242 Edited by Rino Rappuoli, GSK Vaccines, Siena, Italy, and approved November 10, 2015 (received for review November 3, 2014) Cells from all domains of life express glycan structures attached to Interestingly, there are specific reports of several bacteria lipids and proteins on their surface, called glycoconjugates. Cell-to- expressing truncated surface polysaccharides and oligosaccharides cell contact mediated by glycan:glycan interactions have been that are significantly less adherent than wild-type equivalents considered to be low-affinity interactions that precede high- (10, 11), or that their adherence can be blocked by extracted affinity protein–glycan or protein–protein interactions. In several LOS/LPS (10), indicating a role for bacterial surface glycans in pathogenic bacteria, truncation of surface glycans, lipooligosac- adherence to host cells. This decreased adherence of rough strains charide (LOS), or lipopolysaccharide (LPS) have been reported to or blocking of adherence using the free lipooligosaccharide (LOS)/ significantly reduce bacterial adherence to host cells. Here, we lipopolysaccharide (LPS) in both cell-based and animal infection show that the saccharide component of LOS/LPS have direct, models has been noted in a range of Gram-negative bacteria in- high-affinity interactions with host glycans. Glycan microarrays cluding Campylobacter jejuni, Haemophilus influenzae, Salmonella reveal that LOS/LPS of four distinct bacterial pathogens bind to typhi, Salmonella enterica serovar Typhimurium, E. coli, Shigella numerous host glycan structures. Surface plasmon resonance was flexneri, Pseudomonas aeruginosa,andSerratia marcescens (10, 12– used to determine the affinity of these interactions and revealed 20). Blocking of surface glycans with antibodies has also been shown 66 high-affinity host–glycan:bacterial–glycan pairs with equilib- to inhibit adherence and invasion of cell layers in a range of bac- rium dissociation constants (K ) ranging between 100 nM and D teria, including S. flexneri (21–23). The cellular receptors for ad- 50 μM. These glycan:glycan affinity values are similar to those re- herence via these bacterial surface glycans have not been identified. ported for lectins or antibodies with glycans. Cell assays demon- strated that glycan:glycan interaction-mediated bacterial adherence To address the hypothesis that there may be direct interactions could be competitively inhibited by either host cell or bacterial gly- between bacterial and host glycans that mediate adherence, we cans. This is the first report to our knowledge of high affinity conducted glycan microarray screening of four different species glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions Significance between a diverse range of structures suggests that these inter- actions may be important in all biological systems. Pathogens use cell surface carbohydrates as a means of attach- ment to host tissues. In several pathogenic bacteria, truncation of lipooligosaccharide | lipopolysccharide | glycoconjugates | adherence surface carbohydrates, lipooligosaccharide, or lipopolysaccharide have been reported to significantly reduce bacterial adherence ost surface glycosylation is ubiquitous and is targeted by to host cells. Here, we show that the lipooligosaccharide/lipo- Hpathogenic bacteria, viruses, fungi and parasites for adher- polysaccharide of four distinct bacterial pathogens bind di- ence and toxin binding and by glycosidases (1). Escherichia coli rectly to a range of host glycans. Surface plasmon resonance type 1 fimbriae, FimH, is one of the most widely studied glycan- data confirmed binding among 66 different host–glycan: recognizing protein adhesins, with specificity for monomannose to bacterial–glycan pairs. We also demonstrated that bacterial oligomannose structures with the variability of the mannose adherence can be competitively inhibited by either host cell or structure bound leading to different tissue tropism (2). Other bacterial glycans. Our discovery of high-affinity glycan:glycan glycan-recognizing adhesins expressed by bacteria include the interactions in infectious disease may provide new approaches following: Pseudomonas aeruginosa lectins 1 and 2 (PA-IL and for therapy and prevention. The discovery of the existence PA-IIL) that have specificity for galactose and fucose, re- of extensive, high-affinity interactions between glycans spectively (3); Helicobacter pylori SabA, specific for sialic acid will alter the perception of the importance of these containing glycoconjugates including sialyLewis X; and BabA- macromolecular interactions in all biological systems. specific for fucosylated glycoconjugates including Lewis B (4, 5). Although there are numerous known glycan binding adhesins, the Author contributions: C.J.D., E.N.T., M.A.A., J.T., R.M., V.K., and M.P.J. designed research; C.J.D., E.N.T., E.A.S., G.T., L.E.H.-T., P.S.K.N., R.M.K., R.U., S.M., and M.P.J. performed re- adhesins of some bacteria that interact with host surface glycans search; M.A.A. and R.M. contributed new reagents/analytic tools; C.J.D., E.N.T., E.A.S., remain unknown. G.T., L.E.H.-T., P.S.K.N., R.M.K., J.T., R.M., V.K., and M.P.J. analyzed data; and C.J.D., E.N.T., Direct interactions between surface glycans (glycan:glycan in- R.M., V.K., and M.P.J. wrote the paper. teractions) have been reported in sea sponges as heterogenous The authors declare no conflict of interest. glycan interactions, and in mouse embryo development and cancer This article is a PNAS Direct Submission. where homodimers of Lewis X (LeX) or ganglioside structures play Freely available online through the PNAS open access option. a role in cell adhesion and growth factor receptor interactions (6, 7). 1To whom correspondence may be addressed. Email: [email protected] or m.jennings@ Outside of these reports, glycan:glycan interactions, when noted, griffith.edu.au. have generally been considered to be low-affinity, weak interactions 2R.M., V.K., and M.P.J. contributed equally to this work. (8) that precede high-affinity protein:glycan or protein:protein in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. teractions(1,2,5,9). 1073/pnas.1421082112/-/DCSupplemental. E7266–E7275 | PNAS | Published online December 16, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1421082112 Downloaded by guest on October 2, 2021 of pathogenic bacteria with well-characterized surface glycan glycan pairs identified in the glycan microarray studies. Of the PNAS PLUS structures: C. jejuni, H. influenzae, S. typhimurium, and S. flexneri. interactions tested, 31 had affinities (equilibrium dissociation These studies included whole live bacteria expressing wild-type constant; KD) in the range of 140 nM–50 μM(Fig.3,Tables S1–S4, and LOS/LPS truncation mutants, as well as purified LOS/LPS and Dataset S2). Interactions with KD values less than 50 μM are from the same set of bacteria. comparable to typical values observed for binding between lec- tins or antibodies and glycans (2, 3, 24). For S. flexneri, wild-type Results 2a LPS, the highest affinity interaction was observed with the A Bacterial LOS/LPS Recognize Host Surface Glycans and Truncations of blood group antigen with a KD of 0.81 μM, whereas C. jejuni These Structures Reduce/Alter Binding. Fluorescently labeled whole 11168 LOS bound with the highest affinity to the B blood group bacterial cells from all four species bound to many structures on antigen (KD = 0.14 μM; Fig. 3 and Tables S1 and S2). S. typhi- the glycan microarray, including blood group and Lewis antigens murium was found to have 1.89–2.59 μM affinity for four dif- and glycosaminoglycans (Figs. 1 and 2 and Dataset S1). The total ferent structures including all three ABO blood groups and number and diversity of glycans bound by these bacteria were LewisX (Fig. 3 and Table S3), whereas H. influenzae showed reduced when the surface glycans (LOS/LPS) were truncated by higher affinity binding to Lewis A antigen (8 μM) than ABO mutation. The truncated LOS/LPS mutant bacteria of S. flexneri blood group antigens (42–80 μM; Fig. 2 and Table S4). In- RMA2161 (containing an ΔrmlD mutation; 44 structures bound terestingly, for S. flexneri, the increasing numbers of repeat units by wild-type reduced to 13 for ΔrmlD), C. jejuni (ΔwaaF; 104 of the O-antigen subunits in LPS corresponded to increased structures bound by C. jejuni wild-type grown at 42 °C to 2 binding constant, with 2–6 repeats or fewer displaying reduced structures bound by ΔwaaF), and S. typhimurium bound fewer affinity for all structures tested (Table S2). C. jejuni LOS dis- than half the structures observed for wild-type bacteria (180- played the highest affinity interaction observed in this study with Δ galE;
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