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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

As an exclusive human pathogen, Neisseria gonorrhoeae (the gonococcus) has developed an impressive array of adherence factors that allow it to colonize the diverse microenvironments encountered within its sole human host. Pili (or fimbriae) are long, filamentous polymers of pilin protein subunits that extend from the bacterial cell surface. N. gonorrhoeae express the type IV-A class of bacterial pili, which are characterized by the presence of a N-methyl-phenylalanine amino acid residue at the first position in the mature pilin protein (Patel et al., 1991). Expression of type IV pili by the pathogenic Neisseria is subject to phase and antigenic variation, thereby, contributing to immune avoidance and the ability of these bacteria to initiate disease. Several researchers have demonstrated the importance of these polymeric adhesins to N. gonorrhoeae colonization of epithelial cells, and it is generally accepted that pili promote adherence by allowing the gonococcus to overcome electrostatic repulsion that occurs with the host cell.

Pili of the pathogenic Neisseria are post-translationally modified with phosphorylcholine (ChoP) (Weiser et al., 1998). The Neisseria meningitidis pptA gene is involved in the addition, and thus the phase-variable expression, of ChoP on pili (Warren and Jennings, 2003). Structural data suggested that (in pilV null background) gonococcal pili undergo phosphoethanolamine (PEtN) or ChoP modification at serine 68 (Hegge et al., 2004) and at serine 156 (Aas et al., 2006) residues. In contrast, when present, the ChoP modification on pili of N. meningitidis is covalently linked to serine 157 and to serine 160 (M.P. Jennings, submitted). Additionally, gonococcal pilus is covalently modified with an O-linked galactose (α1-3)-2,4-diacetimido-2,4,6-trideoxyhexose (Gal-DATDH) disaccharide (Hegge et al., 2004); a trisaccharide (Gal(β1-4)-Gal-DATDH) (Stimson et al., 1995), is present in this same position (Marceau et al., 1998) (i.e. serine 63) on meningococcal pilin (Fig. 1). Pilin glycosylation involves multiple pilin glycosylation (i.e. pgl) genes. For example,PglD contributes to the biosynthesis of the basal DATDH sugar after which PglA adds the (first) hexose to the basal monosaccharide (Jennings et al., 1998). Whereas pglA is subject to phase variation (Jennings et al., 1998; Banerjee et al., 2002), pglD expression does not appear to be phase-variable (Power et al., 2000). Hence, in vivo, the mature gonococcal pilin possesses either a mono- or a disaccharide. However, data indicate that a Gal-Gal-DATDH, or an alternative glycan structure, is displayed on the pili of at least some gonococcal strains (Craig et al., 2006; M.P. Jennings, unpubl. data).

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Figure 1. Diagram denoting the contribution of PglA and PglD to the expression of the pilin glycan, [Gal (β1-4)] Gal (α1-3)-DATDH glycan. PglD, in conjunction with other pgl gene products, functions in the biosynthesis of DATDH, which is O-linked to the pilin monomer. PglA, functioning as a galactosyl transferase, then adds a galactose to the DATDH core sugar. An additional galactosyl transferase, PglE, may add a terminal galactose to Gal-DATDH to form a trisaccharide. However, in the majority of gonococcal strains, PglE is switched off (Power et al., 2007).

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Among the host receptors described for gonococcal pili (Källstrom et al., 1997; Kirchner et al., 2005), we show previously that pili bind to I-domain-containing integrins on primary cells derived from cervical (Edwards et al., 2002) and male urethral (Edwards and Apicella, 2005) epithelia. In this regard, complement receptor 3 (CR3; integrin αMβ2 or CD11b/CD18) serves as the key receptor mediating gonococcal adherence to cervical epithelia, in vivo, as well as ex vivo, on primary human cervical epithelial (pex) cells (Edwards et al., 2001). It is well accepted that the I-domain region modulates ligand binding for the few integrin heterodimers that possess an I-domain (Diamond et al., 1993; Zhou et al., 1994; Zhang and Plow, 1996a). However, it is also accepted that high-affinity integrin function may require engagement of multiple binding sites within the integrin receptor (Obara et al., 1988; Kimizuka et al., 1991; Bowditch et al., 1991a,b; Aota et al., 1994; Miyamoto et al., 1995; Loftus and Liddington, 1997; Humphries and Newman, 1998; Mesri et al., 1998). Thus, although we have shown that the interaction of gonococci with CR3 on pex cells requires a pilus–I-domain interaction, it is currently not clear if multiple sites within gonococcal pilus mediate this interaction. Similarly, the biological consequences of N. gonorrhoeae pilin glycan phase variation are not known. Therefore, we initiated studies to determine whether the pilin glycan affects the interaction of gonococci with pex cells. Taken together, our data suggested that the pilin glycan was required to mediate binding to the CR3 I-domain when it was presented in a closed, low-affinity conformation and, in this way, pili contributes to the activation state of CR3 during N. gonorrhoeae challenge of pex cells.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Differences exist in the ability of wild-type and pgl mutant gonococci to associate with pex cells

Scanning electron microscopy analysis of uninfected pex cells (Fig. 2A) or of cells that were challenged with N. gonorrhoeae 1291 wild-type (Fig. 2B), 1291pglA (Fig. 2C) or 1291pglD (Fig. 2D) revealed relatively few bacteria bound to the pex cell surface at 30 min post-infection. However, ‘blemishes’ (i.e. lightened circular patches) were readily visible across the surface of 1291pglA and 1291pglD mutant-infected, but not wild-type-infected or uninfected, pex cells. In that these blemishes were observed proximal to adherent (mutant) bacteria, and in that they were of a size and shape that was reminiscent of gonococci, we wanted to quantify the ability of 1291 wild-type and mutant gonococci to associate (adherence plus invasion) with and to invade pex cells over a 90 min challenge (Fig. 3).

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Figure 2. Scanning electron microscopic analysis of pex cells. Scanning electron microscopy analysis of uninfected pex cells (A) or pex cells that were challenged for 30 min with (B) 1291 wild-type (C) 1291pglA or (D) 1291pglD was performed as indicated in the text. Lightened areas/blemishes (encircled) are readily evident across the surface of pgl mutant-infected cells that are not present on the surface of wild-type-infected cells and hint at a role for the pilin glycan in mediating a hyper-adhesive and/or hyper-invasive phenotype during cervical infection. Magnification: (A) ×1 K, (B) ×9 K, (C) ×7 K and (D) ×7 K.

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Figure 3. Glycan-deficient gonococci exhibit an early, pilus-dependent, hyper-adhesive phenotype during pex cell challenge. Pex cells were challenged with gonococci for various times as noted (A and B) or infections were limited to 30 min (C and D). The ability of wild-type and mutant bacteria to associate with (A, C, D) or to invade (B) pex cells was quantified as a percentage of the original inocula, as described in the Experimental procedures. (C) Comparative assays were performed using pgl mutant bacteria in the presence (+) and absence (−) of their respective (isogenic) PglA or PglD pili competimer (10 ng ml−1) or (D) by using wild-type bacteria without (i.e. none) or with (10 ng ml−1) wild-type, PglA or PglD pili competimers, as noted. *P ≥ 0.66 for comparisons of 1291pglA or 1291pglD bacteria with 1291 wild-type. †P = 0.052 for the comparison of 1291 bacteria in the presence of wild-type isogenic pili with pili isolated from 1291pglA.

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When compared with infections performed using wild-type gonococci, pex cell association by the 1291pglA and 1291pglD mutants was increased (3.8-fold and 7.7-fold respectively) at 15 min post-challenge, after which the association of each mutant with pex cells progressively decreased. Similar data were obtained from parallel gentamicin-survival (i.e. invasion) assays (Fig. 3B), as well as from confirmatory adherence and invasion assays performed using wild-type N. gonorrhoeae strain MS11 and the (independent) MS11pglA and MS11pglD mutants (Fig. S1).

Additional quantitative association assays also demonstrated that at 15 min post-challenge an approximate 80% decrease in the adherence of 1291pglA and 1291pglD gonococci had occurred in the presence of exogenously added, isogenic PglA or PglD pili respectively (Fig. 3C). Assays were then performed in which pex cells were challenged for 30 min (a time in which pex cell association for wild-type and mutant bacteria was expected to be approximately equivalent; Fig. 3A) with wild-type N. gonorrhoeae in the presence or absence of isolated wild-type, pglA or pglD pili competimer (Fig. 3D). Although pili isolated from each bacterial strain was capable of significantly inhibiting the association of 1291 wild-type gonococci with pex cells, the degree to which inhibition occurred was variable. The inclusion of isolated, wild-type pili to 1291 wild-type pex cell association assays resulted in approximately 80.9% inhibition of association; 74.7% inhibition occurred in the presence of isolated PglA pili (P ≤ 0.053 vs. the use of wild-type pili), and 58.9% inhibition occurred in the presence of PglD mutant pili (P ≤ 0.003 vs. the use of wild-type pili).

Thereby, these data collectively suggested that pili, and not another surface constituent (Ku et al., 2009), mediated the initial association of gonococci with pex cells. Additionally, these data demonstrated that modest differences existed among these pilin competimers in their ability to inhibit the interaction of wild-type gonococci with CR3 on the pex cell surface.

ChoP is located proximally to the glycan (Hegge et al., 2004; Aas et al., 2006). Some mucosal pathogens are shown to bind to the platelet activating factor receptor (PAFr) through an interaction mediated by ChoP present on surface-exposed bacterial constituents (Cundell et al., 1995a,b; Swords et al., 2000; Barbier et al., 2008). We have also provided evidence to demonstrate that the adherence of N. meningitidis to a human bronchial epithelial cell line is mediated by an interaction between the PAFr and ChoP on meningococcal pili (M.P. Jennings, submitted). Loss of the pilin glycan might make ChoP more accessible to participate in pex cell adhesion, and in this way contribute to a hyper-adhesive phenotype. However, after an exhaustive line of experimentation aimed at blocking a potential PAFr–gonococcus/ChoP interaction, we found no evidence to support a role for the PAFr in mediating adherence to the pex cells (data not shown). Taken together we conclude from the above data that gonococcal adherence to pex cells occurs by a distinct mechanism from the PAFr dependent mechanism used by the meningococcus. The above data also suggested that pili, and not another surface constituent (Ku et al., 2009), mediated the initial association of gonococci with pex cells. Importantly, these data provided the first clues that modest differences in the pilin glycan structure might potentially influence the ability of wild-type gonococci to interact with CR3 on the pex cell surface.

pgl mutants co-immunoprecipitate with the CR3 alpha subunit, CD11b

Our previous works show that a pilus–CD11b (i.e. the CR3 alpha subunit) interaction is critical for the association of wild-type gonococci with pex cells. Therefore, we immuno-captured CD11b from uninfected and N. gonorrhoeae-challenged pex cells and asked whether the pilin glycan played a role in mediating the pilus–CD11b interaction. Gonococcal pilin was readily evident in immuno-complexes captured from 1291-, 1219pglA- and 1291pglD-infected pex cells upon Western blotting using anti-pilin antibody, IE8G8 or anti-pilin rabbit immune serum, 2533 (Fig. 4). Pilin was not observed in uninfected pex cells, nor was it observed upon the omission of the primary, anti-CD11b (Fig. 4) or the secondary agarose-conjugated (not shown) antibodies from the initial immuno-capture step, demonstrating the specificity of the assay. Thus, these data demonstrated that 1291pglA and 1291pglD pili are still capable of mediating adherence to CR3 on pex cells.

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Figure 4. Glycan-deficient gonococci co-immunoprecitate with CR3. Pex cells were challenged with N. gonorrhoeae strains 1291 (A and B), 1291pglA (A) or 1291pglD (B) for various lengths of time, as indicated across the top of each Western blot image. The CR3 alpha subunit, CD11b, was immuno-captured from pex cell lysates using (rabbit) antibody, H-61. The presence of a single band of a size equivalent with gonococcal pili, after immuno-blotting with the (mouse) IE8G8 anti-pilin monoclonal antibody, is consistent with a CR3–gonococcal pilin interaction occurring during the course of pex cell challenge. UI, uninfected pex cells; Ab, the anti-CD11b capture antibody was omitted from the initial capture step; Pil, purified PglA (A) or PglD (B).

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The pilus glycan is required for the ability of gonococci to interact with the CR3 I-domain when it is in a closed conformation

Within CR3, the I-domain dynamically equilibrates between two, open (high-affinity) or closed (low-affinity), conformational states that modulate ligand binding (Xiong et al., 2000; Zhang and Plow, 1996b; Dickeson and Santoro, 1998; Li et al., 1998; Mesri et al., 1998). To evaluate the role of the pilin glycan in modulating the pilin–I-domain interaction, we performed Far-Western blot analyses in which immobilized 1291 wild-type and mutant pili were incubated with recombinant (r)I-domain in a wild-type, a locked-open or a locked-closed conformation. Subsequent immunoblotting with an anti-I-domain-specific antibody demonstrated that wild-type and locked-open rI-domain bound to pili independently of the pilin glycan structure present (Fig. 5A). Conversely, whereas rI-domain in a locked-closed conformation bound pili isolated from wild-type (disaccharide) and pglA (monosaccharide) mutant gonococci, it did not bind pili isolated from the pglD mutant (no glycan) (Fig. 5A).

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Figure 5. pglD mutant gonococci cannot bind to the closed conformation of the CR3 I-domain. A. Purified gonococcal pili that were transferred to a solid support medium were subjected to Far-Western blot analysis using wild-type (left panel), locked-open (middle panel) or locked-closed (right panel) rI-domain, as outlined in the text. The presence of an approximate 18–20 kDa band, after immuno-blotting with the (mouse) LM2/1 I-domain-specific antibody, is indicative of rI-domain bound to immobilized gonococcal pilin. B. Microtitre plates were coated with purified 1291, 1291pglA or 1291pglD gonococcal pilus, as noted; incubated with wild-type, locked-open or locked-closed rI-domain (also as indicated); and subjected to ELISA analysis using the I-domain-specific (mouse) antibody, Bear 1, as described in the text. *P ≥ 0.35 when comparing the use of wild-type rI-domain with the use of locked-open rI-domain.

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Support for these data were obtained by semi-quantitative enzyme-linked immunosorbent assay (ELISA) analyses of the pilus–rI-domain interaction. Microtitre plates coated with 1291 wild-type, 1291pglA or 1291pglD pili were incubated with wild-type, locked-open or locked-closed rI-domain (Fig. 5B). There was no significant difference in the ability of wild-type or locked-open rI-domain to adhere to 1291 or 1291pglA pili (Fig. 5B). Similarly, no significant difference was observed in the ability of locked-closed rI-domain to adhere to either 1291 or 1291pglA pili, albeit less locked-closed rI-domain bound to either pili than that observed for rI-domain in a wild-type or locked-closed conformation (Fig. 5B). Conversely, a significant decrease in the comparative (vs. wild-type rI-domain) ability of locked-open and locked-closed rI-domain to adhere to 1291pglD (glycan-deficient) pili was observed (Fig. 5B). Furthermore, only background levels of adherence to 1291pglD pili were recorded for rI-domain present in a locked-closed conformation (Fig. 5B). Thus, these data are highly suggestive of a role for pilin glycosylation in aiding the association of gonococci with the CR3 I-domain when it is in a low-affinity (i.e. closed) conformation.

To confirm the above data, as well as to determine, ex vivo, the biological significance of pili glycosylation in mediating adherence to the I-domain in a low-affinity conformation, two methods then were used to quantify gonococcal adherence to pex cells. (i) Competitive association assays were performed in the presence and absence of rI-domain competimers (Fig. 6A) and (ii) modified ELISAs were performed in which gonococci pre-incubated with rI-domain were used to challenge pex cells (Fig. 6B). Data obtained by the use of either assay indicated that adherence of wild-type or 1291pglA gonococci to pex cells was significantly (P ≤ 0.001) decreased by the presence of rI-domain competimer regardless of its conformational state. Conversely, the addition of locked-closed rI-domain competimer to association and ELISA assays had no significant (P ≥ 0.36) effect on the ability of 1291pglD mutant bacteria to associate with pex cells. Although wild-type and locked-open rI-domain competimers did result in a significant decrease in the association of 1291pglD with pex cells, the degree to which pex cell association was inhibited was significantly (P ≤ 0.001) less than that observed for parallel assays performed using wild-type or 1291pglA gonococci. Collectively, these data indicated that pilin glycosylation may direct the interaction of the gonococcus with CR3, with the gal-DATDH or DATDH glycan being required for an interaction with the I-domain when it is in a low-affinity conformation.

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Figure 6. The pilin glycan is required to bind to CR3 in a low-affinity conformation on pex cells. A. Pex cells were challenged with 1291, 1291pglA or 1291pglD gonococci for 30 min in the presence or absence of wild-type, locked-open or locked-closed rI-domain, as noted. B. Alternatively, gonococci pre-opsonized with rI-domain were used to challenge pex cell monolayers. The ability of gonococci to adhere to pex cells in the presence or absence of the I-domain competimer was measured by an ELISA in which (mouse) antibody 2C3, which is specific for the conserved H.8 outer membrane gonococcal protein was used. *P ≥ 0.36 for assays performed in the presence of the rI-domain competimer when compared with assays in which the rI-domain competimer was omitted.

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The pilus glycan is required to confer a high-affinity conformation to CR3 during pex cell challenge

Integrins (e.g. CR3) rapidly oscillate between active and inactive conformational states in a dynamic equilibrium in which the inactive conformation is prevalent under normal physiological conditions (reviewed in Takagi and Springer, 2002). Upon ligand binding and/or cell stimulation, high-affinity receptor function is obtained. As our data indicated that pilus glycosylation was required to mediate adherence to the I-domain in a closed conformation, we hypothesized that pili glycosylation may similarly be required to trigger an active high-affinity state to CR3 during N. gonorrhoeae challenge of pex cells. To test this hypothesis, we performed a modified ELISA in which the ratio of active CR3 to total CR3 was measured on uninfected pex cells and on cells challenged with gonococci, gonococcal pilin, or with exogenous ChoP or PEtN (Fig. 7). These assays demonstrated that approximately 30% of the total amount of CR3 on uninfected pex cells was present in an active conformation. These data are slightly greater than those recorded for the total amount of active CR3 for resting neutrophils in blood (i.e. 10–30%) (Diamond and Springer, 1993; Takagi and Springer, 2002), and they may be reflective of the different immunological function, and/or the resident microenvironment, observed for neutrophil and cervical epithelial cells. Following a 30 min challenge with wild-type or pglA mutant bacteria or pili, a significant (P ≤ 0.001) increase in active CR3 was observed on the surface of pex cells. However, we also observed a modest, but significant (P ≤ 0.03), difference in the ability of wild-type or pglA mutant bacteria or pili to elicit an active conformation to CR3. Conversely, experiments performed using pglD mutant bacteria, isolated PglD pili, ChoP or PEtN did not significantly (P ≥ 0.15) alter the ratio of active CR3 on pex cells, when compared with unchallenged cells (Fig. 7A). Comparable data were obtained in separate experiments performed using wild-type and mutant strain MS11 bacteria or isolated pili as well as when using alternative anti-CR3 antibodies (Fig. 7B and C). Thus, these data provide strong evidence that, during pex cell challenge, the pilin glycan is required to confer a high-affinity active conformation to CR3.

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Figure 7. The pilin glycan is required to confer an active state to CR3 during pex cell challenge. Pex cell monolayers in 96-well plates were challenged for 30 min with wild-type (WT) and mutant N. gonorrhoeae, isolated gonococcal pili, ChoP or PEtN, or they were left unchallenged (UI), as indicated. The ability of each biological agent to induce an active, high-affinity, conformation to CR3 was determined using the CR3 active state-specific antibodies CBRM1/5 (A) or MEM48 (B and C) and the anti-CD11b (i.e. total CR3) antibodies H-61 (A) Ox42 (B) or LM2/1 (C) by a fluorometric ELISA, as described in the Experimental procedures. Data shown are the percentage of the amount of active CR3, relative to the amount of total CR3, present on the pex cell surface following challenge with (A) 1291 wild-type and pgl mutant bacteria and isolated pili, (B) 1291 and MS11 wild-type and pgl mutant bacteria or (C) pili isolated from 1291 or MS11 wild-type or pgl mutant bacteria. *P ≥ 0.15 for pex cells challenged as indicated when compared with unchallenged cells.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

For several years, it has been increasingly realized that many bacterial proteins are glycosylated. However, evidence to indicate how protein glycosylation may mediate virulence has, generally, remained elusive. In this regard, functions proposed for microbial glycoproteins include increased antigenic variation and protection from immunological or proteolytic attack. Several studies further indicate that bacterial glycoproteins may mediate host cell adhesion as well as those processes promoting colonization in animal models; although, in most cases, the precise role of the glycan in mediating these events is unclear. An understanding of the role of the neisserial pilin glycan in promoting meningococcal or gonococcal disease may be further complicated in that glycan expression has the potential to be phase-variable. Early investigations suggested that phase variation to a pglA‘off’ state was potentially associated with disseminated gonococcal infection (Banerjee et al., 2002). However, no such correlation was found upon the comprehensive analysis of a large panel of clinical isolates (Power et al., 2007). Similarly, when compared with wild-type N. meningitidis, no significant difference is observed in the ability of pilin glycan-deficient meningococci to associate with immortal epithelial cell lines or with human umbilical vein endothelial cells (Virji et al., 1993; Jennings et al., 1998; Marceau et al., 1998). In contrast to these earlier works, we have now presented data to indicate that the gonococcal pilin glycan ameliorates the interaction of these bacteria with CR3 on the cervical cell surface. In this regard, we have demonstrated that whereas pglA and pglD exhibit an early hyper-adhesive phenotype during pex cell challenge, their ability to invade/survive within these cells is severely compromised when compared with wild-type gonococci. The pglD mutant was further impaired in its ability to interact with the CR3 I-domain when present in a closed conformation and to trigger an active state to this host molecule, suggesting that the pilin glycan is required for an interaction with the I-domain when it is in a low-affinity conformation. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to infection/disease.

It is generally accepted that integrins (e.g. CR3) rapidly oscillate between active and inactive conformational states in a dynamic equilibrium in which the inactive conformation is prevalent under normal physiological conditions (reviewed in Takagi and Springer, 2002). It is also well accepted that the configuration of the I-domain, within the integrin alpha subunit, modulates ligand binding. The open conformation of the I-domain is distinguished from the closed conformation by the presence of an exposed cation co-ordination site (i.e. the metal ion-dependent adhesion site or MIDAS) in the open conformation, although structural differences in those I-domain regions located proximal to the MIDAS also exist. A negatively charged residue donated by the ligand completes the MIDAS co-ordination sphere, thereby triggering conformational changes within the integrin heterodimer and a high-affinity receptor state. However, high-affinity integrin function may require engagement of multiple binding sites within the integrin receptor that are intimately linked, that act synergistically and that are under allosteric control (Bowditch et al., 1991a; Aota et al., 1994; Miyamoto et al., 1995; Loftus and Liddington, 1997; Humphries and Newman, 1998; Mesri et al., 1998). For example, binding of fibronectin to integrin receptors involves a hierarchy of multiple domains within the fibronectin molecule. Proper folding, as well as spatial orientation, allows these fibronectin domains to function independently, but co-operatively, to mediate integrin binding (Humphries et al., 1987; Obara et al., 1988; Aota et al., 1991; Kimizuka et al., 1991; Bowditch et al., 1991b).

We show previously that successful infection of the cervical epithelium requires the concerted actions of gonococcal pilin, porin and iC3b bound to the gonococcus surface (Edwards et al., 2002). Our data now indicate that, with regard to the pilus–I-domain interaction, the pilin glycan is required to bind to the I-domain when it is in a closed conformation and to confer an active state to CR3. Thus, these data are reminiscent of the interaction of ICAM-1 with the I-domain of integrin αLβ2 (i.e. LFA-1; CD11a/CD18) in which an initial, specific ICAM-1–LFA-1 interaction triggers a conformational change within LFA-1 that then allows a subsequent, high-affinity interaction (Cabañas and Hogg, 1993). Therefore, by analogy, it is within reason to propose that an initial, specific, interaction of the pilin glycan occurs with regions of the I-domain residing proximal to the MIDAS. This, in turn, may spatially favour the subsequent interaction of the MIDAS with one of the several glutamate or aspartate residues that lie adjacent to the glycan on the pilin protein (Parge et al., 1995; Craig et al., 2006). In this scenario, binding of the pilin glycan to the closed I-domain would trigger a conformational change within the integrin heterodimer, resulting in an open I-domain conformation, with a glutamate or aspartate on the pilin molecule then completing the MIDAS co-ordination sphere. These ideas are consistent with our present data demonstrating that the pilin mono- or disaccharide is required for gonococcal adherence to the I-domain when present in a closed conformation as well as the inability of the pglD mutant, which would not be observed in vivo, to confer an active state to CR3 on pex cells. However, studies to further define the specific interaction of the pilin glycan with CR3 are required and are currently on-going in our lab.

CR3 is known to serve as an important signal transducer in professional phagocytic cells, where the modular structure of CR3 allows differential cellular effects based on the recognition of endogenous (e.g. iC3b-opsonized particles) or exogenous (e.g. LPS) ligands. On professional phagocytic cells, I-domain-mediated adherence to CR3 occurs independently of a proinflammatory response, whereas interactions occurring outside the I-domain (e.g. the lectin-binding domain, LBD) are thought to stimulate a respiratory burst through the generation of a second signal within the eukaryotic (host) cell (Becherer et al., 1989; Zhou and Brown, 1994; Petty et al., 1997; Yefenof, 2000). Consequently, it is generally believed that a CR3 association mediated through the LBD, e.g. of this receptor may result in the decreased survival of the invasive organism, whereas I-domain engagement confers a survival advantage to invasive organisms within professional phagocytic cells. Although similar data are not available for primary epithelial cells, the interaction of (wild-type) gonococci with CR3 occurs exclusively through the I-domain, and our unpublished data indicate this results in the decreased expression of proinflammatory cytokines by these cells.

The biosynthesis/expression of DATDH appears to be limited only to bacteria. The ability of the CR3 LBD to specifically recognize and to bind unusual sugars, e.g. DATDH, has not been directly examined; however, (bacterial) saccharides comprised of mannose and glucopyranose moieties are known to readily bind to the CR3 LBD. In this regard, CR3 plays an important role in mediating the host immune response to infection. Our data support the hypothesis that the absence of the terminal Gal on the pilin glycan of the 1291pglA and MS11pglA mutants resulted in these gonococci residing within an intracellular environment that was unfavourable for bacterial survival. Conversely, the absence of the terminal Gal residue on the pilin glycan had only a modest effect on the ability of pglA mutant bacteria to induce an active CR3 configuration. The activity state of CR3 is linked to its phagocytic function and to cellular cytotoxicity. Thus, if an interaction occurs between the CR3 LBD and an exposed DATDH (or like sugar, e.g. pseudaminic or legioaminic acid) on a bacterial glycoprotein, it would also be expected to lead to increased phagocytic uptake of these organisms and enhanced cytotoxicity of that host cell. In addition to the lower female genital tract, CR3 is highly prevalent on cells of monocytic lineage (e.g. macrophages, neutrophils, dendritic cells) and may be expressed by the intestinal epithelium (Hussain et al., 1995). Thus, our data may have implications to the study of other microbial pathogens in that diacetamido sugars are prevalent among the bacterial glycoproteins described to date (reviewed in Power and Jennings, 2003; Schmidt et al., 2003).

To better understand the interplay occurring between the gonococcus and the cervical epithelium, we sought to define the role of the gonococcal pilin glycan in potentially mediating the interaction with CR3 on pex cells. To this end, we have provided evidence to indicate that the presence of the disaccharide on the pilin adhesin plays an important role in mediating the interaction of gonococci with CR3 on pex cells. In this regard, the pilin glycan was required for mediating adherence to the CR3 I-domain when it was in a low-affinity conformation and for conferring an active state to this receptor. In the presence of a pilin monosaccharide, as well as in the absence of pilin glycosylation, bacterial survival was dramatically reduced. Greater than 92% of gonococci associate with this receptor on the epithelium of cervical tissue obtained from women with culture-documented gonococcal cervicitis (Edwards et al., 2001). However, whether a pglA‘on’ phenotype confers a survival advantage to resident gonococci within the lower female genital tract, as is suggested by our data, remains to be determined. Similarly, studies are on-going in our lab to further define the pilin glycan–CR3 interaction. Nevertheless, data presented herein further our understanding of the pathological processes contributing to cervical gonorrhoea as well as to the function of CR3 within the lower female genital tract. Importantly, we believe these to be the first data to directly demonstrate a role for a bacterial protein glycan in modulating bacterial infection. Whether pilin glycosylation similarly mediates binding of gonococci to the I-domain region of the α1- and α2-β1 integrins present on the male urethral epithelium is, currently, not known (Edwards and Apicella, 2005). However, invasion of male urethral epithelial cells is triggered by the interaction of lipooligosaccharide with the asialoglycoprotein receptor (ASGP-R) (Harvey et al., 2001), and present data indicate that internalization of gonococci in this scenario follows a pathway consistent with that observed for the ASGP-R (Harvey et al., 1997; 2002; Giardina et al., 1998). Thus, the ability of gonococci to survive within the male urethra following integrin-mediated adherence and ASGP-R-mediated invasion likely would be similar between those gonococci in which pglA is in an ‘on’ state or in an ‘off’ state. The role of the pilin glycan in modulating the adherence of N. meningitidis to human epithelial cells is discussed in a separate manuscript (M.P. Jennings, submitted).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cell culture and bacteria

Pex cells were procured from surgical cervical tissue and maintained as described previously (Edwards et al., 2000). Cervical tissue was obtained from pre-menopausal women undergoing hysterectomy at The Ohio State University Medical Center for medically indicated reasons not related to our study and was provided by the Cooperative Human Tissue Network (The Research Institute at Nationwide Children's Hospital, Columbus, OH). In accordance with NIH guidelines, these tissues do not constitute human subjects. Pharmacological agents used were not cytotoxic (viability greater than 95%) at the indicated concentrations as determined by trypan blue exclusion (pex cells) or by counting colony-forming units of gonococci incubated in the presence of, compared with the absence of, each reagent. Infection studies were performed as previously described using a multiplicity of infection of 100 (Edwards et al., 2000). Pex cells were challenged with gonococci for variable lengths of time (as noted); uninfected, control, cell monolayers were simultaneously processed with challenged cell monolayers, where applicable.

Neisseria gonorrhoeae strains 1291 (Apicella, 1974), 1291pglA and 1291pglD were used in the infection studies described herein. Confirmatory assays were performed using wild-type and pgl mutant strains of N. gonorrhoeae MS11 (Schoolnik et al., 1984). Strain 1291 was originally isolated from a man with gonococcal urethritis; strain MS11 was obtained from a woman with pelvic inflammatory disease. These wild-type strains are commonly used to study gonococcal pathogenesis.

pglA and pglD genes were mutated in each strain as described previously by the insertion of a kanamycin-resistance cassette that lacks a promoter exhibiting functional activity in Neisseria. Furthermore, this cassette is shown to not cause polar effects in several genetic systems (Jennings et al., 1995; 1998; van der Ley et al., 1997); including, more specifically, within the neisserial pgl operon (Power et al., 2000). Putative pglA and pglD mutants were selected by their ability to grow on kanamycin-GC-IsoVitaleX agar plates and confirmed by PCR as well as by the comparison of each gene sequence to the Broad Institute, N. gonorrhoeae pilin sequences (NGAG_00671.1, NGAG_01337.1) (http://www.broadinstitute.org/annotation/genome/neisseria_gonorrhoeae/MultiHome.html). No differences occurred among the pilin gene sequences obtained for 1291 wild-type, 1291pglA and 1291pglD. Wild-type and pgl mutant gonococci were further analysed by Western blotting using anti-pili antibody, SM1 (Virji et al., 1983) (generously provided by M. Virji, University of Bristol, Bristol, UK), or anti-PglA serum (specific for N. meningitidis C311#3 monosaccharide, DATDH) (Power et al., 2000). DATDH was evident on 1291pglA but not on 1291pglD. ELISA analyses confirmed that the expression of LOS as well as pili, Opa and H.8 proteins was not significantly (P ≥ 0.962) variable among the parental wild-type strain or either pgl mutant strain (Fig. S2).

Scanning electron microscopy

Uninfected and N. gonorrhoeae-infected pex cells on placental collagen-coated coverslips were processed for scanning electron microscopy as previously described (Ketterer et al., 1999). Images were captured using the Hitachi S-4000 located at the University of Iowa Central Microscopy Research Facility (Iowa City, IA, USA).

Association and invasion assays

The ability of gonococci to adhere to and/or invade pex cells was quantitatively determined using standard gentamicin-resistance (i.e. invasion) assays, performed as described previously (Edwards et al., 2000). Briefly, pex cells were challenged with gonococci for various periods, as noted. Potential inhibitors of the gonococcus–pex cell interaction were included in or excluded from these assays and they included: 10 ng ml−1 gonococcal pili; 100 nM PAFr antagonist (1-O-hexadecyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine; Calbiochem, San Diego, CA, USA); 10 µg ml−1 anti-PAFr antibody, H-300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); 10 µg ml−1 anti-ChoP antibody, TEPC-15 (Sigma-Aldrich, St Louis, MO, USA); 10 µg ml−1 ChoP (Sigma); 100 ng ml−1 PEtN (Sigma); or 10 ng ml−1 rI-domain (generously provided by E.J. Brown, University of California, San Francisco). At the indicated time post-challenge, gentamicin was then omitted from (association, adherence plus invasion, assays) or added to (invasion assays) infected pex cell monolayers to kill extracellular cell-associated bacteria. Pex cell monolayers were subsequently washed, then lysed, and serial dilutions of the pex cell lysate were plated to allow gonococcal enumeration by counting colony-forming units. Per cent association and/or invasion of N. gonorrhoeae when in the presence or absence of experimental additives were determined as a function of the inoculum and the number of colonies formed. A Kruskal–Wallis non-parametric analysis of variance was used to determine the statistical significance of the invasion assays described above. Each assay was performed in triplicate on at least three separate occasions.

Pili isolation

Pili were sheered from an overnight culture of gonococci by vortexing at high speed and pulling the cell suspension through a 21G syringe. Following centrifugation, the supernatant was transferred to a fresh tube, heated to 56°C for 1 h, and then sequentially filtered through Centricon centrifugal filter units (Millipore Corporation; Bedford, MA, USA) to yield a final filtrate. Electrophoretic separation of the final filtrates revealed a peptide band of the appropriate molecular mass for wild-type and glycan-deficient gonococcal pilin within each appropriate lane upon subsequent silver-staining of the gel. This was confirmed by Western blot analysis to be pilin.

Immunoprecipitation, Western and Far-Western blot analyses

Confluent pex cell monolayers in 35 mm tissue culture-treated dishes were challenged for various periods, as noted, with wild-type or mutant N. gonorrhoeae as described above, or they were left uninfected. Immunoprecipitation was performed as we have described previously (Wen et al., 2000) using the (rabbit) anti-CD11b antibody, H-61 (Santa Cruz), to capture immune complexes. The specificity of the assay was ensured by the omission of the primary or secondary antibody, which yielded a negative result upon Western blotting. Western blotting was performed according to standard protocols using the (mouse) anti-pilus antibody, IE8G8 (generously provided by M. Blake, Food and Drug Administration, Bethesda, MD, USA) or anti-pilin rabbit immune sera, 2533.

The ability of rI-domain to bind to gonococcal pili was determined by Far-Western blot analysis as we have described (Edwards et al., 2002). In this regard, an equal amount (25 ng) of immobilized gonococcal 1291, 1291pglA or 1291pglD pili was incubated overnight with 100 ng of wild-type, locked-open or locked-closed rI-domain before standard Western blotting using anti-CD11b, I-domain-specific, antibodies Bear 1 or LM2/1 (both from Santa Cruz). SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) was used to develop (Far-)Western blots. ELISAs were performed to confirm Far-Western blot analysis as outlined below.

ELISA analyses

Semi-quantitative analysis of the ability of wild-type, locked-open and locked-closed rI-domain to adhere to pili was determined, as we have described previously (Edwards et al., 2002). Briefly, 200 ng of isolated 1291, 1291pglA or 1291pglD pili were used to coat microtitre plates that were then rinsed, non-specific binding sites were blocked, incubated with 100 µl of a 20 ng ml−1 solution of rI-domain, and then blocked again. ELISAs were then performed using anti-CD11b (Bear 1) primary and peroxidase-conjugated secondary antibodies. Absorbance of the o-phenylenediamine dihydrochloride peroxidase substrate was determined spectrophotometrically at 490 nm using a Synergy HT Multimode plate reader (BioTek, Winooski, VT, USA). In separate experiments, pex cells in microtitre plates were challenged for 30 min with rI-domain ‘opsonized’ gonococci. Adherence of opsonized and non-opsonized gonococci to pex cells was determined using antibody, 2C3, which is specific for the conserved gonococcal outer membrane protein, H.8. Omission of the primary or secondary antibodies from select wells served as controls.

The percentage of active CR3 to total CR3 residing on the pex cell surface was determined in a similar manner. Pex cells in 96-well plates were left uninfected or they were challenged (30 min) with gonococci, gonococcal pilin, ChoP or PEtN, as outlined above. Following challenge, pex cell monolayers were fixed after which parallel wells were immuno-labelled using either the anti-CD11b antibodies; H-61, Ox42 or LM2/1; or the CR3 active conformation-specific antibodies CBRM1/5 or MEM48 (all from Santa Cruz). Antibody CBRM1/5 recognizes a neoepitope present within the (open) CD11b I-domain (Diamond and Springer, 1993; Takagi and Springer, 2002). Antibody MEM48 recognizes a neoepitope present within the CD11b-CD18 interface. This epitope is masked in the bent, low-affinity, conformation of the CR3 heterodimer but becomes accessible following CR3 activation upon extension of the CR3 head region to form the high-affinity, active, conformation (Takagi and Springer, 2002). Fluorescein-conjugated secondary antibodies were then applied to increase assay sensitivity. Arbitrary fluorescence units were recorded (485 nm excitation/530 nm emission) using a Synergy HT Multi-mode microplate reader. Blank wells, as well as the omission of the primary or the secondary antibody, served as controls for non-specific binding and for auto-fluorescence. Each of the above assays was performed in triplicate on three separate occasions. Statistical significance of data obtained was determined using the Student's t-test.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors are grateful to the Cooperative Human Tissue Network (The Research Institute at Nationwide Children's Hospital, Columbus, OH) for providing cervical tissue specimens. NIAID Grant 1R01AI076398 and a Young Investigator Award to J.L.E. from The Research Institute at Nationwide Children's Hospital and Program Grant 565526 from the National Health and Medical Research Council of Australia to M.P.J. provided support for this work.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Time-course of MS11 infection of pex cells. Whereas pglA and pglD gonococci exhibit an early, hyper-adhesive, phenotype, their ability to invade/survive within pex cells is attenuated.

Fig. S2. Expression of select outer membrane constituents by N. gonorrhoeae. No significant difference was observed in the expression levels of pili, outer membrane protein H.8, Opa proteins or LOS among wild-type or pgl mutant gonococci.

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CMI_1586_sm_supp_info.pdf223KSupporting info item
CMI_1586_sm_fig_legends.docx27KSupporting info item

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