Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Plant, L. J. Articles by Jonsson, A.-B. Search for Related Content PubMed PubMed Citation Articles by Plant, L. J. Articles by Jonsson, A.-B.

 Previous Article  |  Next Article 

Infection and Immunity, January 2006, p. 442-448, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.442-448.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Type IV Pili of Neisseria gonorrhoeae Influence the Activation of Human CD4⁺ T Cells

Laura J. Plant^* and Ann-Beth Jonsson

Department of Medical Biochemistry and Microbiology, Biomedical Centre, Uppsala University, PO Box 582, Uppsala, Sweden

Received 19 July 2005/ Returned for modification 9 September 2005/ Accepted 13 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea, and infection with this organism is typically associated with an intense inflammatory response. In many individuals, however, the infection is asymptomatic and can progress to serious secondary complications. The type IV pili of Neisseria gonorrhoeae mediate binding of the bacteria to host cells and are involved in cellular signal transduction. In these studies we have demonstrated that gonococcal pili influence human CD4⁺ T cells by using isogenic strains of N. gonorrhoeae with piliated and nonpiliated phenotypes. To determine the impact of piliation on the cellular status, we examined the expression of activation markers, cellular proliferation, and the production of cytokines after infection. The activation marker CD69 showed significantly increased expression on cells infected with the piliated strain, and this expression was dependent on costimulation of the T-cell receptor. Infection with piliated gonococci also altered T-cell proliferation and influenced the production of the regulatory cytokine interleukin-10. PilC, the putative pilus adhesin, was also observed to influence cellular activation but had no impact on the proliferation of cells further indicating that pilus-mediated adhesion is important in gonococcal stimulation of CD4⁺ T cells. These results show that the piliation status of gonococci influences CD4⁺ T-cell activation and that the adhesion mediated by pilus components aids in the regulation of the T-cell response to N. gonorrhoeae.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neisseria gonorrhoeae causes millions of new cases of gonorrhea each year. The infection is typically characterized by an intense inflammatory response, but in a large percentage of the infected population the disease remains asymptomatic. Infection correlates with a transient reduction in T lymphocyte numbers in the blood, which resolves when the infection is cleared, suggesting an immune suppressive effect (3). The specific mechanisms by which the organism evades the host immune responses are only beginning to be elucidated.

Variation in gonococcal epitopes assists in immune evasion and specific inhibitory cellular interactions have also been identified. Boulton and Gray-Owen reported that binding of gonococcal opacity proteins (Opa₅₂) to CEACAM1 on primary CD4⁺ T lymphocytes results in the suppression of cellular activation and proliferation via the immunoreceptor tyrosine inhibitory motif (5). In contrast, other neisserial factors have been shown upregulate the proliferation and activation of immune cells. Gonococcal porin induces the proliferation of T lymphocytes from patients with urogenital gonococcal disease, without inducing proliferation of cells from noninfected patients, and a significant increase in porin-specific interleukin-4 (IL-4)-producing CD4⁺ and CD8⁺ T cells have been observed in patients with mucosal gonococcal disease (28). It has also been demonstrated by Levine et al. that the number of CD4⁺ T cells in vaginal washings from women with acute gonococcal disease is greater than that of uninfected women (16).

Infection by pathogenic Neisseria spp. is initiated by interaction between the type IV pili and cellular receptors including CD46 (14), the complement regulatory protein 3 (8), and I-domain containing integrins (7). Adherence of Neisseria to CD46 is correlated with the expression of the PilC protein, a pilus-associated 110-kDa protein, located at the tip of the pilus fiber, and in the cell membrane (22). However, numerous studies have also shown that the major pilus subunit, PilE, is involved in receptor recognition, with antigenic variant forms of PilE influencing epithelial cell adherence and tissue tropism (11, 20, 23, 32). CD46, which is also a receptor for pathogens other than Neisseria (6, 14, 21, 25, 26), has been observed to impact lymphocyte activation and has the capacity to modulate the activation and proliferation of T lymphocytes in response to T-cell receptor (TCR) cross-linking in vitro (25). Recently, it has also been shown that CD46 is involved in the induction the T-regulatory 1 (Tr1) phenotype of human CD4⁺ T cells (15).

In the present study we examined the capacity of gonococcal strains with mutations in pilus components to modulate the activity of primary human CD4⁺ T lymphocytes. We show that bacterial interaction induces the activation and proliferation of cells and that proliferation can be observed with purified pili alone. Gonococcal infection of T cells also stimulates the production of IL-10. These findings provide further evidence that gonococcal pili is an important cellular component modulating T-cell activity in vivo and show that the gonococcal pilus is involved in the induction of signaling events leading to modulation of the CD4⁺ T-cell response.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions. Opacity protein (Opa)-nonexpressing derivatives of N. gonorrhoeae MS11mk (MS11P⁺), P^–n (nonpiliated; MS11P^–) (29), and the variants MW4 (PilT_ind, PilC⁺) and MW7 (PilT_ind, PilC^–) (33) were selected and grown on GCB agar (Difco) with Kellogg's supplement in a 5% CO₂ atmosphere. Pili from bacterial strains were purified by using previously described procedures (12). Briefly, pili were sheared from the bacterial surface and subjected to five rounds of precipitation and resuspension to remove impurities. Precipitation was performed by dialysis against 0.05 M Tris-HCl-0.15 M NaCl (pH 8.0) and centrifugation at 23,000 x g for 60 min at 4°C: resuspension was performed in 0.15 M ethanolamine (pH 10.5). The final pilus preparation was resuspended in 0.1 M phosphate-buffered saline.

Cell lines and primary T-cell purification. Human peripheral blood mononuclear cells were purified from buffy coats (Bloodcentralen, Karolinska Hospital) by using Ficoll-Hypaque gradient centrifugation (400 x g, 20 min). CD4⁺ T cells were isolated by negative selection with a MACS CD4⁺ T-cell isolation kit (Miltenyi Biotech). The purification efficiency of CD4⁺ T cells was assessed by examining coexpression of CD3 and CD4 with fluorescein isothiocyanate anti-CD4 and allophycocyanin anti-CD3 (BD Pharmingen). Purified lymphocytes were routinely >90% CD3⁺ CD4⁺ as determined by flow cytometry. Cells were maintained in RPMI 1640 containing 10% fetal calf serum and 2 mM L-glutamine at 37°C in 5% CO₂ and humidified air.

Adhesion of Neisseria to T cells. Isolated CD4⁺ T lymphocytes and bacteria were resuspended in cell media, and the cells were infected with bacterial strains for 1 h (37°C, 5% CO₂). After incubation the cells were centrifuged at 280 x g, washed five times with media, and lysed with saponin (1%) for 5 min. Serial dilutions were spread on GCB plates and grown overnight, and the CFU were enumerated. The assay was repeated three times with different blood donors.

Lymphocyte activation assay. Cells were stimulated with recombinant human IL-2 (200 U/ml; Pharmingen) for 48 h before infection or antibody challenge. The cells were washed three times prior to use in the assay to remove the influence of IL-2 in stimulation. TCR stimulation was induced by treatment with 5 µg of mouse anti-human CD3 immunoglobulin G (clone UCHT1; Pharmingen)/ml, and costimulation controls were induced with 5 µg of anti-CD28 (clone CD28.2 Pharmingen) or anti-CD46 Ab-2 (169-1. E4.3; Neomarker)/ml. Bacterial stimulation was induced by infection with a multiplicity of infection (MOI) of 20 of bacteria grown for 18 h on GC agar plates, and gentamicin (50 µg/ml) was added at 2 h postinfection to prevent bacterial overgrowth. CD69 expression was examined by staining 5 x 10⁵ lymphocytes with phycoerythrin-labeled anti-CD69 (BD Pharmingen). A minimum of 10,000 cells from each sample was analyzed by flow cytometry with CellQuest software (Becton Dickinson). Activation conditions were performed and assayed in duplicate, and the data represented are from five separate experiments.

Lymphocyte proliferation assay. Cells were stimulated with IL-2 and washed as described above. In vitro primary stimulation was carried out in 96-well culture plates with monoclonal antibodies to CD3 and costimulation with antibodies to CD28 or CD46. Purified lymphocytes (5 x 10⁴ cells per well) were incubated with antibodies at 5 µg/ml, bacterial strains at an MOI of 20 or purified pili from all strains at 1 µg/ml, and MS11P⁺ pili at various concentrations. Two hours after infection gentamicin (50 µg/ml) was added to all wells to prevent bacterial overgrowth. After 3 days of primary stimulation proliferation was monitored by using the XTT Cell Proliferation Kit II (Roche). Each activation condition was performed in triplicate, and the data represented are from three independent experiments.

Cytokine assay. Cells (2 x 10⁵/well) were incubated for 5 days in 96-well plates coated with monoclonal antibodies to CD3 (clone HIT3a; Pharmingen), CD46 and CD28. The antibodies were used at 10 µg/ml. Cells were infected with neisserial strains at an MOI of 20. IL-2 was incubated with cells at a concentration of 200 U/ml. Two hours after infection gentamicin was added to all wells at a final concentration of 50 µg/ml. The secretion of IL-10 and tumor necrosis factor (TNF) in the supernatants was assessed by enzyme linked immunosorbent assays (ELISAs) with kits from Diaclone. Assays were repeated twice in independent experiments with different donors.

Western blotting and silver staining of pili preparations. Bacterial pilus extracts (500 ng) were separated by sodium dodecyl sulfate-12% polyacrylamide electrophoresis and transferred to polyvinylidene difluoride membranes or silver stained by using standard protocols. The membranes were blocked overnight with 5% nonfat dried milk in Tris-buffered saline (0.05 M Tris and 2 M NaCl [pH 7.4]) containing 0.05% Tween 20. PilC was identified by incubation with the K3 antibody that recognizes both PilC1 and PilC2 (polyclonal rabbit antibody diluted 1:5,000). PilE was identified with an antigonococcal PilE antibody (polyclonal rabbit antibody diluted 1:1,000). The Opa proteins were detected with the 4B12/C11 antibody that recognizes all Opa proteins (1) (monoclonal mouse antibody diluted 1:5,000). Incubation with primary antibodies was followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad; diluted 1:10,000) or by horseradish peroxidase-conjugated goat anti-mouse antibody (Santa Cruz Biotechnologies; diluted 1:10,000). A chemiluminescence kit from Perkin-Elmer Life Sciences was used for detection.

Statistical analysis. Statistical variations were determined by using Student t tests for the adhesion assays, or Kruskal-Wallis tests for nonparametric data in all other tests, with significance accepted at P < 0.05. Blood from numerous donors was analyzed in each assay.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adhesion to T lymphocytes is dependent on pilus expression. The interaction between N. gonorrhoeae and T cells was assessed by examination of the adhesion of nonopacity (Opa) protein expressing piliated and nonpiliated gonococcal strains to primary human CD4⁺ T cells (Fig. 1). The lack of Opa expression was confirmed with Western blotting (data not shown). After a 1 h of incubation the wild-type piliated strain adhered in significantly higher numbers (P = 0.001) to cells compared to the nonpiliated mutant, indicating that pili have a significant role in the interaction of gonococci (GC) with human CD4⁺ T cells.

View larger version (15K): [in this window] [in a new window]

FIG. 1. The adherence of wild-type piliated N. gonorrhoeae MS11P+ and the nonpiliated isogenic mutant MS11P– to primary human CD4+ T cells. GC were allowed to adhere to cells for 60 min, and unbound bacteria were removed by washing the cells in multiple centrifugation steps to pellet cells. Infected cells were treated with saponin, serially diluted, and spread on GCB plates. CFU were counted, and the attachment is represented as the percentage of applied bacteria associated with cells.

Expression of the CD69 activation marker by T lymphocytes infected with Neisseria. CD69 is expressed early on the surface of activated lymphocytes and correlates with antigen-specific proliferative responses (2, 17). We evaluated the effect of piliated and nonpiliated gonococcal strains on the CD4⁺ T-cell expression of CD69 (Fig. 2). Due to variation in donor T-cell responses it was necessary to group donors according to their responsiveness, and the data shown reflect only assays where CD69 was expressed on 5% or more untreated cells. The trends in the results observed have also been seen with blood isolated from other donors. The mean expression on untreated cells was 9%, which increased to 18% upon ligation of the TCR receptor with an antibody to CD3. This was significantly increased (P = 0.016) upon costimulation with antibodies to CD28 (>48% cells). Surprisingly, a nonsignificant increase in the CD69⁺ population was noted after costimulation with anti-CD46 (24% cells), which has been shown in other studies to influence cell activation (4). The piliated GC strain induced a significantly enhanced expression of CD69 (32.5% cells; P = 0.03), whereas the nonpiliated strain did not significantly enhance expression of CD69 upon TCR costimulation (22.5% cells). In the absence of TCR ligation both strains induced similar levels of CD69 expression in the CD4⁺ T-cell population, which were not significantly enhanced compared to uninfected cells, suggesting that the increase observed with the piliated strain is due to specific signaling via a TCR coreceptor. These data indicate that stimulation mediated by the piliated strain is likely to occur independently of CD46 signaling.

View larger version (16K): [in this window] [in a new window]

FIG. 2. Expression of the activation marker CD69 by primary human CD4+ T cells after infection with GC. Purified cells were left unstimulated () or stimulated with antibodies to CD3 () in the presence or absence of antibodies to the known costimulatory molecules CD28 and CD46 or the piliated and nonpiliated MS11 gonococcal strains. Bacteria were added at an MOI of 20, and gentamicin was added at 2 h postinfection to prevent bacterial overgrowth. The percentage of CD69+ cells was determined by staining with phycoerythrin-labeled anti-CD69 antibody and analysis by flow cytometry. Significant increases (P < 0.05) in CD69 levels compared to anti-CD3 treated cells are indicated by a double asterisk. Significance was determined by using a Kruskal-Wallis test.

Neisserial pili promote the proliferation of human T lymphocytes. The interaction of piliated gonococci with T lymphocytes has been reported to promote cellular proliferation (5). We have used the described piliated strain and the nonpiliated mutant strain to examine the interaction between pili and CD4⁺ T cells (Fig. 3). An MOI of 20 was used in this assay since higher MOIs resulted in cell stimulation irrelevant of piliation status, showing the complexity in the determination of cellular interactions with whole bacterial strains. TCR ligation induced T-cell proliferation, although this was not significantly different (P = 0.219) from untreated cells. However, the positive controls used to costimulate cells, antibodies to CD28 and CD46, significantly enhanced proliferation (P = 0.021 and P = 0.03, respectively). This was noted in three independent experiments with different blood donors, and the data represented in Fig. 3 are an average of the three donors. TCR costimulation with the piliated strain induced T-cell proliferation significantly compared to the nonpiliated strain (P = 0.0495), indicating that pili can induce signaling in T cells leading to an increased proliferative response.

View larger version (15K): [in this window] [in a new window]

FIG. 3. Proliferation of human T cells after infection with the piliated and nonpiliated GC. Purified human cells were incubated in 96-well plates with antibodies to CD46 and CD28, as well as with the gonococcal strains in the absence () or presence () of TCR ligation for 3 days. Bacteria were added at an MOI of 20, and gentamicin was added at 2 h postinfection to prevent bacterial overgrowth. Proliferation was determined by using the XTT Cell Proliferation Kit. Significant increases (P < 0.05) in proliferation compared to anti-CD3 treated cells are indicated by a double asterisk. Significance was determined by using a Kruskal-Wallis test.

Piliated gonococci induce IL-10 production by CD4⁺ T lymphocytes. Purified human CD4⁺ T cells were stimulated with immobilized monoclonal antibodies and gonococcal strains, and the production of the regulatory anti-inflammatory cytokine IL-10 (Fig. 4A) and the proinflammatory cytokine TNF (Fig. 4B) were assessed. Unstimulated T cells in the absence of TCR ligation produced high levels of IL-10 that decreased significantly upon stimulation with all factors, including IL-2, indicating that proliferating cells change their IL-10 production upon stimulation. The addition of antibody to CD46 and the piliated GC strain in the presence of IL-2 induced similar levels of IL-10 to that seen with nonstimulated cells, suggesting that these treatments induce signaling resulting in the production of IL-10 by T cells. Engagement of the TCR also decreased the production of IL-10. In cells treated with anti-CD3 the induction of IL-10 by all treatments was greatly enhanced in the absence of IL-2, although this was more substantially increased in cells treated with anti-CD28 and piliated GC. These results indicate that in the absence of TCR stimulation anti-CD46 and piliated GC can induce IL-10 in a similar manner. However, in the presence of TCR stimulation the induction occurs via a different mechanism, with the stimulation induced by all treatments. The pili are likely to be the modulator in TCR-dependent signaling, since the amount of IL-10 induced by the piliated strain was greater than that of the nonpiliated strain (P = 0.045) in both the absence and the presence of IL-2. As a cytokine control, TNF production was measured. This cytokine was induced by anti-CD28 stimulation in the presence or absence of TCR costimulation. It was also noted that nonpiliated GC induced significantly higher levels of TNF in the absence of TCR costimulation. The data represented in this figure were also observed for another donor (not shown).

View larger version (25K): [in this window] [in a new window]

FIG. 4. T cells stimulated with antibodies to CD3, CD28, and CD46, and/or infected with GC were assayed for IL-10 (A) and TNF (B) production. Assays were conducted both in the presence () or absence () of recombinant IL-2. Purified human cells were incubated with antibodies and GC strains for 5 days, with gentamicin added at 2 h postinfection to prevent bacterial overgrowth, and cytokines in the supernatant were measured by ELISA (Diaclone). Significant reductions (P < 0.05) in cytokine levels compared to control cell treatments are indicated by a single asterisk, while significant cytokine inductions are indicated by a double asterisks. Significance was determined by using a Kruskal-Wallis test.

PilC modulates T-cell activity. Isogenic derivatives of the wild-type piliated strain MS11 were used to examine the influence of the putative adhesin PilC on the T-cell status. The mutant strains MW4 (PilC⁺) and MW7 (PilC^–) both express pili; however, the MW7 strain does not express PilC (Fig. 5A). The MW4 strain showed significantly enhanced adhesion to T cells compared to the PilC^– strain MW7 (Fig. 5B; P = 0.001), and the adhesion of MW4 was comparable to that of the wild-type piliated strain (Fig. 1), further supporting the role of PilC as an adhesive pilus component. Although there was no significant difference in the proliferation of T cells, the T-cell activation marker CD69 was significantly upregulated (P = 0.0062) after treatment with the PilC-expressing MW4 strain (Fig. 5C). This indicates that PilC mediated adhesion has a role in the activation of T cells to gonococcal infection.

View larger version (31K): [in this window] [in a new window]

FIG. 5. The role of the putative adhesive pilus component PilC on the CD4+ T-cell status was determined with the isogenic mutant strains of GC, MW4 (PilC+), and MW7 (PilC–). (A) Western blotting of these strains confirmed the expected phenotypes after staining with antibodies to PilE and PilC. A Coomassie blue-stained gel of the two bacterial preparations shows equal loading. (B) The adhesion to T cells was determined and is expressed as the percentage of applied bacteria that associate with cells. (C) The number of activated cells was determined by examination of expression of CD69 after treatment with bacterial strains. Significant increases (P < 0.05) compared to untreated cells are indicated by a single asterisk, and significant increases compared to anti-CD3 treated cells are indicated by a double asterisks. Significance was determined by using a Student's t test for the adhesion assay and a Kruskal-Wallis test for the activation assay. Assays conducted in the absence of TCR ligation are by indicated with black bars and in the presence of TCR ligation are indicated by white bars.

Purified pili activate T lymphocytes. Pili were prepared from the gonococcal strains and did not contain detectable levels of contaminants, as determined by silver staining (Fig. 6A). The pili preparations were assayed for their ability to stimulate T cells (Fig. 6B). Only pili from GC (P⁺) were able to significantly stimulate T-cell proliferation (P = 0.0495) compared to stimulation with anti-CD3 alone. To confirm the effect of pili on the T-cell status, the pili purified from the wild-type strain were used to stimulate cells at various concentrations (Fig. 6C). The pili induced proliferation of T cells in a dose-dependent manner, and this proliferation was increased with TCR costimulation. The proliferation was only significantly enhanced with the pili added at 1 and 0.1 µg/ml (P = 0.0196 and P = 0.0323, respectively). Tests were conducted in two independent experiments.

View larger version (30K): [in this window] [in a new window]

FIG. 6. (A) Pili were purified from the piliated wild-type gonococcal strain MS11P+ and the mutant strains MS11P–, MW4 (PilC+), and MW7 (PilC–) and analyzed by silver staining of SDS-PAGE. (B) Pili were used to stimulate T cells in the absence (black bars) and presence (white bars) or TCR ligation. (C) The proliferation of cells following infection with various concentrations of pili from the wild-type strain was monitored at 3 days postinfection by using the XTT Cell Proliferation Kit. Significant increases (P < 0.05) in proliferation were determined by using a Kruskal-Wallis test, and significant changes are indicated by an asterisk.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adherence of microbes to host tissue represents a crucial initial step in the pathogenesis of infections. Type IV pili promote the adherence of N. gonorrhoeae to cells, aiding colonization of the human host (9), as well as contributing to other phenotypes such as autoagglutination, competence for natural transformation, and twitching motility (30). There is increasing evidence that pili are involved in host cell signaling. Purified pili of N. gonorrhoeae have been shown to induce a calcium release in target epithelial cells (13), and pilus-mediated adhesion of Neisseria to host cells triggers a rapid, localized formation of cortical plaques in host epithelial cells (19). Recently, it has been shown that the mechanical forces exerted by pilus retraction also have a role in cell signaling (10).

Several studies have indicated that CD46 is an important signaling factor in T cells. It is able to transduce signals and aggregation on human T cells induces phosphorylation of the adaptor proteins p120^CBL, ZAP-70 and linker for activation of T cells (LAT) which regulate TCR signaling, as well as activating ERK, JNK, and p38 (4, 24). Also, TCR-CD46 costimulation strongly promotes T-cell activation and proliferation, and the production of Th1 cytokines, indicating further that CD46 is a potent costimulatory molecule for human T cells (4, 24). Other Neisseria receptors are also likely to have a role in T-cell modulation. It is well known that in addition to mediating adhesion to cell surface and extracellular matrix ligands, integrins generate a diverse array of intracellular signals. In T cells, LFA-1 and several ß1 integrins can provide costimulatory signals for TCR-induced T-cell activation (27, 31), indicating that molecules other than CD46 which act as receptors for gonococci could also be involved in the stimulation of T cells noted in the present study.

We show that binding of Neisseria to T cells induces the activation and proliferation of these cells. The piliated GC strain strongly promoted the activation of cells, and infection also increased the cell culture density by 50% compared to the nonstimulated control. The PilC-expressing strain, MW4, showed a similar effect to the piliated strain, whereas the nonpiliated strain and the PilC deletion mutant, MW7, did not induce activation of T cells, probably due to the lack of interaction with cells since both PilE and PilC are considered important in mediating adhesion to host cells, including T cells (Fig. 1 and 5B).

The importance of the pili in inducing the stimulatory effect is evident from the data showing a dose-dependent proliferative effect of purified MS11P⁺ pili on cells, as seen in Fig. 6C. This effect was also enhanced by TCR costimulation. At a higher MOI all strains stimulated the activation and proliferation of T cells (data not shown), which is most likely due to the presence of saturating amounts of stimulatory components in the bacterial cell wall, such as lipopolysaccharide and porin. The data shown in Fig. 6 indicate that the effect is mediated by pili, since only the MS11P⁺ pilus preparation was able to mediate an increase in proliferation of cells. There were no contaminating proteins present in the pilus preparation, as determined by silver staining. However, despite this, it is also possible that other components that were not detectable could be mediating this effect seen with piliated gonococci. Nonetheless, we have shown here that piliation enhances T-cell activation and proliferation, regardless of whether this is mediated by the pilus itself or is due to the act of binding. It is likely that Opa₅₀ also mediates such an effect since Boulton and Gray-Owen previously showed that an Opa₅₀ expressing gonococcal strain also induced T-cell proliferation in the absence of piliation (5).

The costimulation of human CD4⁺ T cells with antibodies to CD3 and CD46 has been shown to induce a Tr1 cell phenotype in the presence of IL-2 (15). The poor response to gonococcal infection may be due to the induction of Tr1 cells upon CD46 ligation, suppressing the activation of bystander T cells via the induction of IL-10 and other inhibitory cytokines. The finding that piliated gonococci induce IL-10 in the cell culture supernatant suggests a T-cell suppressive effect. This effect is pilus dependent, particularly since treatment with other neisserial components such as gonococcal porin do not induce significant levels of IL-10 (28). The effect however may not necessarily be linked to CD46 binding.

It would be interesting to inhibit binding and cellular activation and proliferation by using antibodies to CD46 and other known adhesins to determine the specificity for this interaction. However, due to the response to CD46 costimulation, it is not possible to use these antibodies in the cell reaction, since addition alone alters cellular responses. Attempts to inhibit the binding of the wild-type GC to epithelial cells using antibodies to CD46 have been unsuccessful and the specificity of an association between pili and CD46 on T cells is therefore difficult to prove.

The pili of gonococci stimulate CD4⁺ T cells, inducing their activation and proliferation. The expression of pili is also involved in the production of IL-10 by T cells, and the possible induction of cells with a regulatory phenotype may provide further explanation for asymptomatic gonococcal infection. It is likely that a pilus receptor, such as CD46 or integrins, may have a role in this process, although such an involvement has been impossible to elucidate since it is difficult to block interactions using specific antibodies, which modulate T-cell responses themselves. Nevertheless, pili have an important role in the modulation of the T-cell response, and the results shown here indicate that pilus-induced activation and proliferation of T cells overrides the effects of other gonococcal components, excluding Opa, which were not examined in the present study.

 
This study was supported by grants from the Swedish Medical Research Council (Dnr 10846), the Swedish Cancer Society, Magnus Bergvalls Stiflelse, and the Wenner-Gren Foundation.

The gonococcal mutants were kindly provided by Michael Koomey from the University of Oslo, Oslo, Norway.

 
* Corresponding author. Mailing address: Department for Medical Biochemistry and Microbiology, Biomedical Centrum, Uppsala University, PO Box 582, Uppsala, Sweden. Phone: 46 18 471 46 05. Fax: 46 18 50 98 76. E-mail: Laura.Plant{at}imbim.uu.se.

Editor: V. J. DiRita

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Achtman, M., M. Neibert, B. A. Crowe, W. Strittmatter, B. Kusecek, E. Weyse, M. J. Walsh, B. Slawig, G. Morelli, A. Moll, et al. 1988. Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A. J. Exp. Med. 168:507-525.[Abstract/Free Full Text]
2
2. Angulo, R., and D. A. Fulcher. 1998. Measurement of Candida-specific blastogenesis: comparison of carboxyfluorescein succinimidyl ester labeling of T cells, thymidine incorporation, and CD69 expression. Cytometry 34:143-151.[CrossRef][Medline]
3
3. Anzala, A. O., J. N. Simonsen, J. Kimani, T. B. Ball, N. J. Nagelkerke, J. Rutherford, E. N. Ngugi, J. J. Bwayo, and F. A. Plummer. 2000. Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts. J. Infect. Dis. 182:459-466.[CrossRef][Medline]
4
4. Astier, A., M. C. Trescol-Biemont, O. Azocar, B. Lamouille, and C. Rabourdin-Combe. 2000. Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phosphorylation. J. Immunol. 164:6091-6095.[Abstract/Free Full Text]
5
5. Boulton, I. C., and S. D. Gray-Owen. 2002. Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes. Nat. Immunol. 3:229-236.[CrossRef][Medline]
6
6. Dorig, R. E., A. Marcil, A. Chopra, and C. D. Richardson. 1993. The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75:295-305.[CrossRef][Medline]
7
7. Edwards, J. L., and M. A. Apicella. 2005. I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells. Cell Microbiol. 7:1197-1211.[CrossRef][Medline]
8
8. Edwards, J. L., E. J. Brown, K. A. Ault, and M. A. Apicella. 2001. The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia. Cell Microbiol. 3:611-622.[CrossRef][Medline]
9
9. Heckels, J. E., J. N. Fletcher, and M. Virji. 1989. The potential protective effect of immunization with outer-membrane protein I from Neisseria gonorrhoeae. J. Gen. Microbiol. 135(Pt. 8):2269-2276.[Abstract/Free Full Text]
10
10. Howie, H. L., M. Glogauer, and M. So. 2005. The N. gonorrhoeae type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism. PLoS Biol. 3:e100.[CrossRef][Medline]
11
11. Jonsson, A. B., D. Ilver, P. Falk, J. Pepose, and S. Normark. 1994. Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue. Mol. Microbiol. 13:403-416.[Medline]
12
12. Jonsson, A. B., G. Nyberg, and S. Normark. 1991. Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly. EMBO J. 10:477-488.[Medline]
13
13. Kallstrom, H., and A. B. Jonsson. 1998. Characterization of the region downstream of the pilus biogenesis gene pilC1 in Neisseria gonorrhoeae. Biochim. Biophys. Acta 1397:137-140.[Medline]
14
14. Kallstrom, H., M. K. Liszewski, J. P. Atkinson, and A. B. Jonsson. 1997. Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria. Mol. Microbiol. 25:639-647.[CrossRef][Medline]
15
15. Kemper, C., A. C. Chan, J. M. Green, K. A. Brett, K. M. Murphy, and J. P. Atkinson. 2003. Activation of human CD4⁺ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421:388-392.[CrossRef][Medline]
16
16. Levine, W. C., V. Pope, A. Bhoomkar, P. Tambe, J. S. Lewis, A. A. Zaidi, C. E. Farshy, S. Mitchell, and D. F. Talkington. 1998. Increase in endocervical CD4 lymphocytes among women with nonulcerative sexually transmitted diseases. J. Infect. Dis. 177:167-174.[Medline]
17
17. Marzio, R., J. Mauel, and S. Betz-Corradin. 1999. CD69 and regulation of the immune function. Immunopharmacol. Immunotoxicol. 21:565-582.[Medline]
18
18. Merz, A. J., and M. So. 2000. Interactions of pathogenic neisseriae with epithelial cell membranes. Annu. Rev. Cell Dev. Biol. 16:423-457.[CrossRef][Medline]
19
19. Merz, A. J., M. So, and M. P. Sheetz. 2000. Pilus retraction powers bacterial twitching motility. Nature 407:98-102.[CrossRef][Medline]
20
20. Nassif, X., J. Lowy, P. Stenberg, P. O'Gaora, A. Ganji, and M. So. 1993. Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells. Mol. Microbiol. 8:719-725.[Medline]
21
21. Okada, N., M. K. Liszewski, J. P. Atkinson, and M. Caparon. 1995. Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus. Proc. Natl. Acad. Sci. USA 92:2489-2493.[Abstract/Free Full Text]
22
22. Rudel, T., I. Scheurerpflug, and T. F. Meyer. 1995. Neisseria PilC protein identified as type-4 pilus tip-located adhesin. Nature 373:357-359.[CrossRef][Medline]
23
23. Rudel, T., J. P. van Putten, C. P. Gibbs, R. Haas, and T. F. Meyer. 1992. Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells. Mol. Microbiol. 6:3439-3450.[Medline]
24
24. Sanchez, A., M. J. Feito, and J. M. Rojo. 2004. CD46-mediated costimulation induces a Th1-biased response and enhances early TCR/CD3 signaling in human CD4⁺ T lymphocytes. Eur. J. Immunol. 34:2439-2448.[CrossRef][Medline]
25
25. Santoro, F., H. L. Greenstone, A. Insinga, M. K. Liszewski, J. P. Atkinson, P. Lusso, and E. A. Berger. 2003. Interaction of the gH glycoprotein of human herpesvirus 6 with the cellular receptor CD46. J. Biol. Chem. 278:2596-2599.
26
26. Segerman, A., J. P. Atkinson, M. Marttila, V. Dennerquist, G. Wadell, and N. Arnberg. 2003. Adenovirus type 11 uses CD46 as a cellular receptor. J. Virol. 77:9183-9191.[Abstract/Free Full Text]
27
27. Shimizu, Y., G. van Seventer, K. Horgan, and S. Shaw. 1990. Costimulation of proliferative responses of resting CD4⁺ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin. J. Immunol. 145:59-67.[Abstract]
28
28. Simpson, S. D., Y. Ho, P. A. Rice, and L. M. Wetzler. 1999. T lymphocyte response to Neisseria gonorrhoeae porin in individuals with mucosal gonococcal infections. J. Infect. Dis. 180:762-773.[CrossRef][Medline]
29
29. Swanson, J., S. Bergstrom, K. Robbins, O. Barrera, D. Corwin, and J. M. Koomey. 1986. Gene conversion involving the pilin structural gene correlates with pilus⁺ in equilibrium with pilus^– changes in Neisseria gonorrhoeae. Cell 47:267-276.[CrossRef][Medline]
30
30. Tonjum, T., and M. Koomey. 1997. The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships: a review. Gene 192:155-163.[CrossRef][Medline]
31
31. van Seventer, G., Y. Shimizu, K. Horgan, and S. Shaw. 1990. The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T-cell receptor-mediated activation of resting T cells. J. Immunol. 144:4579-4586.[Abstract]
32
32. Virji, M., and J. E. Heckels. 1984. The role of common and type-specific pilus antigenic domains in adhesion and virulence of gonococci for human epithelial cells. J. Gen. Microbiol. 130(Pt. 5):1089-1095.[Abstract/Free Full Text]
33
33. Wolfgang, M., H. S. Park, S. F. Hayes, J. P. van Putten, and M. Koomey. 1998. Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. USA 95:14973-14978.[Abstract/Free Full Text]

---

Infection and Immunity, January 2006, p. 442-448, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.442-448.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Youssef, A.-R., van der Flier, M., Estevao, S., Hartwig, N. G., van der Ley, P., Virji, M. (2009). Opa+ and Opa- Isolates of Neisseria meningitidis and Neisseria gonorrhoeae Induce Sustained Proliferative Responses in Human CD4+ T Cells. Infect. Immun. 77: 5170-5180 [Abstract] [Full Text]  
• Imarai, M., Candia, E., Rodriguez-Tirado, C., Tognarelli, J., Pardo, M., Perez, T., Valdes, D., Reyes-Cerpa, S., Nelson, P., Acuna-Castillo, C., Maisey, K. (2008). Regulatory T Cells Are Locally Induced during Intravaginal Infection of Mice with Neisseria gonorrhoeae. Infect. Immun. 76: 5456-5465 [Abstract] [Full Text]  
• Hansen, J. K., Demick, K. P., Mansfield, J. M., Forest, K. T. (2007). Conserved Regions from Neisseria gonorrhoeae Pilin Are Immunosilent and Not Immunosuppressive. Infect. Immun. 75: 4138-4147 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Plant, L. J. Articles by Jonsson, A.-B. Search for Related Content PubMed PubMed Citation Articles by Plant, L. J. Articles by Jonsson, A.-B.