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The Journal of Immunology, 2007, 178: 4489-4497.
Copyright © 2007 by The American Association of Immunologists, Inc.

Factor H Binding and Function in Sialylated Pathogenic Neisseriae is Influenced by Gonococcal, but Not Meningococcal, Porin1

Guillermo Madico*, Jutamas Ngampasutadol{dagger}, Sunita Gulati{dagger}, Ulrich Vogel{ddagger}, Peter A. Rice{dagger} and Sanjay Ram2,{dagger}

* Evans Biomedical Research Center, Boston University Medical Center, Boston, MA 02118; {dagger} Division of Infectious Diseases and Immunology, University of Massachusetts Memorial Medical Center, Worcester, MA 01605; and {ddagger} Institute for Hygiene and Microbiology, Universität Würzburg, 97080 Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neisseria gonorrhoeae and Neisseria meningitidis both express the lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) molecule that can be sialylated. Although gonococcal LNT LOS sialylation enhances binding of the alternative pathway complement inhibitor factor H and renders otherwise serum-sensitive bacteria resistant to complement-dependent killing, the role of LOS sialylation in meningococcal serum resistance is less clear. We show that only gonococcal, but not meningococcal, LNT LOS sialylation enhanced factor H binding. Replacing the porin (Por) B molecule of a meningococcal strain (LOS sialylated) that did not bind factor H with gonococcal Por1B augmented factor H binding. Capsule expression did not alter factor H binding to meningococci that express gonococcal Por. Conversely, replacing gonococcal Por1B with meningococcal PorB abrogated factor H binding despite LNT LOS sialylation. Gonococcal Por1B introduced in the background of an unsialylated meningococcus itself bound small amounts of factor H, suggesting a direct factor H-Por1B interaction. Factor H binding to unsialylated meningococci transfected with gonococcal Por1B was similar to the sialylated counterpart only in the presence of higher (20 µg/ml) concentrations of factor H and decreased in a dose-responsive manner by ~80% at 1.25 µg/ml. Factor H binding to the sialylated strain remained unchanged over this factor H concentration range however, suggesting that LOS sialylation facilitated optimal factor H-Por1B interactions. The functional counterpart of factor H binding showed that sialylated meningococcal mutants that possessed gonococcal Por1B were resistant to complement-mediated killing by normal human serum. Our data highlight the different mechanisms used by these two related species to evade complement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The complement system forms an important arm of the innate immune defenses against the Neisseria meningitidis and Neisseria gonorrhoeae. Persons deficient in components of the terminal or alternative complement pathway are predisposed to recurrent disseminated neisserial infection (1, 2, 3). Pathogenic neisseriae have evolved several mechanisms to escape direct complement-mediated killing. The binding of soluble phase complement regulators such as the C4b-binding protein (C4bp; regulator of the classical pathway) and factor H (alternative pathway regulator) constitutes a mechanism of complement evasion (4, 5, 6, 7). Elegant studies by Smith and colleagues showed that the sialylation of gonococcal lacto-N-neotetraose (LNT)3 lipooligosaccharide (LOS; Galbeta1->4GlcNAcbeta1->3Galbeta1->4Glcbeta1->4HepI, where Gal is galactose, Glc is glucose, GlcNAc is N-acetylglucosamine, and Hep is heptose) resulted in the enhanced resistance of gonococci to direct killing by human serum (8, 9). This observation has been confirmed by other investigators (10, 11). The sialylation of gonococci occurs in vivo in humans (12, 13) and in the genital tract of experimentally infected mice (14). Mechanisms of serum resistance conferred by gonococcal LOS sialylation include the decreased binding of Ab (15) and enhanced factor H binding (7).

Both, N. gonorrhoeae and N. meningitidis may express two LOS glycoforms that can be sialylated. These include the LNT LOS species (described above) and the PK-like LOS structure (Gal{alpha}1->4Galbeta1->4Glcbeta1->4HepI). The latter structure defines the L1 immunotype of N. meningitidis. Sialic acid binds to the terminal Gal residues of LNT and PK-like LOS via {alpha}(2,3) and {alpha}(2,6) linkages, respectively. We have shown that sialylation of only the LNT, but not the PK-like LOS, in N. gonorrhoeae enhances factor H binding and serum resistance (16).

In contrast to gonococcal LOS sialylation, the full impact of LOS sialylation in mediating meningococcal serum resistance is uncertain. Although some reports suggest that serum resistance is not augmented by LOS sialylation (17), others have reported an enhancement of serum resistance when meningococcal LNT LOS is increasingly sialylated (18). In any event, meningococci that express the sialylated LNT LOS are recovered from the blood or cerebrospinal fluid of persons with invasive disease more frequently than isolates without LOS sialic acid (19), suggesting that LOS sialylation may be an important virulence factor. Nevertheless, serogroups B and C encapsulated meningococci genetically deficient in their ability to sialylate LOS have been found to be as virulent as wild-type strains in the infant rat model of meningococcal infection (20, 21).

In this report, we describe differences in the mechanism of complement regulation by LOS sialic acid on gonococci compared with meningococci. We demonstrate that neisserial LNT LOS sialylation augments factor H binding in either species only when the gonococcal, but not the meningococcal, porin (Por) molecule is present concomitantly. Such differences may contribute to differences in pathogenicity and to the clinical features caused by these two neisserial species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains

The neisserial strains used in this study are listed in Table I. All strains expressed the LNT LOS structure as the predominant LOS species. To avoid confounding by complement regulation that is mediated by meningococcal capsular polysaccharide and to allow a symmetric comparison between N. meningitidis and N. gonorrhoeae (the latter is unencapsulated), meningococcal strains were rendered unencapsulated by insertional inactivation of the polysialyltransferase (siaD) gene (or mynB of serogroup A strain 2594) as described previously (22, 23, 24). Strains of N. meningitidis serogroups B, C, W-135, and Y can endogenously sialylate their LOS, and the shunting of sialic acid to LOS is facilitated in the siaD mutants (25). The ability of the siaD mutants to sialylate their LOS was abrogated by the introduction of a kanamycin resistance marker to inactivate the LOS sialyltransferase (lst) gene as described previously (20), to yield siaD lst double mutants. LOS sialylation of A2594 mynB or gonococcal strains was achieved by adding 5'-CMP-N-acetylneuraminic acid (CMP-NANA) to a final concentration of 50 µg/ml in growth medium.


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  		Table I. Neisserial strains used in this study

 
Sera and complement reagents

Sera obtained fresh from 10 normal adults were pooled and stored at –80°C until used. Purified factor H was purchased from Advanced Research Technologies.

Antibodies

Affinity-isolated goat anti-human factor H was made by Bethyl Laboratories using purified factor H (Advanced Research Technologies) as the immunogen. Anti-LOS mAb 3F11 (26), which reacts with unsialylated LNT LOS (the LNT epitope), was provided by Dr. M. A. Apicella (University of Iowa, Iowa City, IA). LOS sialylation results in decreased mAb 3F11 binding. mAb 2C3, which binds to the H.8 lipoprotein Ag (12) present on all pathogenic neisserial strains (27), was used to monitor the capture of bacteria to microtiter wells. FITC-labeled anti-goat IgG and anti-mouse IgM, respectively, were used as secondary disclosing Abs for anti-factor H and 3F11 in flow cytometry assays, and anti-mouse IgM and anti-mouse IgG conjugated to alkaline phosphatase were used to disclose mAb 3F11 and mAb 2C3 binding in whole cell ELISA, respectively.

Allelic replacement of porins

We chose N. gonorrhoeae strain F62 and N. meningitidis strain Y2220 to perform allelic replacements of porB because the amount of factor H bound by these strains in the unsialylated state lies below the threshold of detection by flow cytometry. To replace Y2220 PorB2 with N. gonorrhoeae F62 Por1B we used overlap-extension PCR with primer pairs F62 por1B forward and F62 por1B-Erm reverse, Erm-por1B forward and Erm-Y down reverse, and Y porB2 down-Erm forward and Y porB2 down reverse (listed in Table II) to generate a hybrid amplicon (3055 bp) that contained (in 5' to 3' orientation) the F62 por1B, the erythromycin resistance cassette, and 1109-bp Y2220 DNA downstream of por1B. This hybrid amplicon was cloned into the TA cloning vector pCR2.1-TOPO 2.1 (Invitrogen) to yield pF62por1B-Erm. The sequence of the cloned DNA was confirmed, and pF62por1B-Erm was used to transform strains Y2220 siaD and Y2220 siaD lst. Erythromycin-resistant clones that yielded a PCR product using a primer specific for F62 por1B loop 1 (5'-GAAGGCAAAGTAGTTAGCGTGGG-3') and a reverse primer specific for F62 por1B loop 6 (5'-ATTAGCACGCCCTGTTCCATACAAT-3') were identified as those bearing the complete por1B. Clones that did not yield a PCR product with this primer pair possessed a hybrid Y2220/F62 Por molecule (because recombination occurred 3' to loop 1), and the por of such clones were fully sequenced to define the point of recombination. One such clone, which contained Y2220 loops 1 and 2 and F62 loops 3–8 (called Y2220 siaD por Y (1-2) F62 (3–8)) was studied further. A similar cloning strategy using primer pairs FA19 por1B forward and FA19 por1B-Erm reverse, Erm-por1B forward and Erm-Y down reverse, and Y porB2 down-Erm forward and Y porB2 down reverse was used to generate pFA19por1A-Erm, which was used to replace the PorB2 of Y2220 siaD and Y2220 siaD lst with the Por1A from N. gonorrhoeae strain FA19.


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  		Table II. Primers used for allelic replacement of porin

 
Efforts to create the "converse" mutant, where F62 Por1B was replaced with Y2220 PorB2, were not successful. The appropriate plasmid was created, but none of the gonococcal clones that we screened bore Y2220 porB2 and we hypothesize that this mutant was not viable. Therefore we then set out to replace the F62 Por with the PorB3 molecule of meningococcal strain H44/76. Although strain H44/76 itself binds to factor H, we have reported that this binding occurs via the GNA1870 molecule and not PorB3 (23). Again, using overlap extension (see Table II for primers) we constructed a 2572-bp hybrid amplicon that comprised (in 5' to 3' orientation) the H44/76 porB3, the erythromycin resistance cassette, and the 672-bp DNA 3' to F62 por1B. This hybrid amplicon was cloned into the TA cloning vector pCR2.1-TOPO to yield pH44/76-Erm and used to transform F62. Erythromycin-resistant clones in which por1B was completely replaced by porB3 yielded a PCR product using a forward primer specific for H44/76 porB3 loop 1 (5'-CGTAGACACTTCTCGCTCTGTATTT-3') with a reverse primer specific for H44/76 loop 7 (5'-CTATGTCTTCATCATCAACCCAAAC-3'). Clones that did not yield a PCR product with this primer pair were presumed to be hybrids and the por of these clones were fully sequenced to define the point of recombination. One such hybrid that possessed F62 Por loops 1 and 2 and H44/76 PorB3 loops 3–8 was used in this study.

As a control for factor H nonbinding in the background of a growth-retarded mutant (see Results), the PorB2 molecule of strain Y2220 siaD lst was replaced by the PorB3 molecule of strain H44/76 to yield Y2220 siaD lstPorB3H44/76+ (LOS not sialylated) as described previously (23). Chromosomal DNA from this mutant was used to transform Y2220 siaD to yield Y2220 siaDPorB3H44/76+ (the LOS-sialylated mutant); both mutant strains were used in flow cytometry experiments.

Flow cytometry

Factor H bound to the bacterial surface was quantified by flow cytometry using a BD Biosciences FACScan cytometer as described previously (7). Similarly, the extent of LOS sialylation in Por H44/76-transfected F62 was determined by the decreased expression of LNT LOS as measured by 3F11 binding.

Whole cell ELISA

LNT LOS expression and sialylation was assessed separately in the siaD and siaD lst mutant derivatives of the serogroup B, C, W-135, and Y strains and the mynB mutant of the serogroup A strain and gonococcal strains grown either in their native state or with CMP-NANA added to growth medium by the binding of mAb 3F11 in whole cell ELISA as described previously (7).

Serum bactericidal assay

Serum bactericidal testing was performed as described previously (28). Briefly ~2,000 CFU of bacteria grown to mid-log phase were incubated with normal human serum (NHS) (concentration of NHS is specified for each experiment) in a final reaction mixture volume of 150 µl. Duplicate aliquots of 25 µl were inoculated on to chocolate agar plates at 0 and 30 min. Survival was calculated as the percentage of the number of colonies that survived to min 30 relative to the baseline colony counts at min 0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Sialylation of gonococcal, but not meningococcal, LNT LOS results in increased factor H binding

We examined factor H binding to five meningococcal strains that represent each of the five major serogroups. Unencapsulated mutant derivatives that possessed either sialylated LOS (siaD mutants), or their unsialylated counterparts (siaD lst mutants) were examined (Fig. 1). As controls we used four diverse gonococcal strains: two that expressed a Por1B molecule (F62 and MS11) and two Por1A strains (FA19 and UU1). As expected, all gonococcal strains showed enhanced factor H binding when their LOS was sialylated by growth in CMP-NANA-containing medium (Fig. 1, top row). In contrast, no difference in factor H binding to each pair of meningococcal strains was seen (Fig. 1, bottom row). The baseline levels of factor H binding and the increment in factor H binding upon LOS sialylation differed among the gonococcal strains. The smallest increment in factor H binding was seen with Por1A strain UU1, which showed a ~2-fold increase in geometric mean fluorescence, while strains F62 and FA19 showed an ~1 to 2 log10 increment in fluorescence with LOS sialylation. There was no correlation between the amounts of LOS sialylation as evidenced by decreases in gonococcal LNT LOS expression with the degree of factor H binding achieved at the CMP-NANA concentration used in these assays (data not shown).


Figure 1
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  		FIGURE 1. The effect of LNT LOS sialylation on factor H binding to four N. gonorrhoeae (N.g.) strains (Por1B strains F62 and MS11 and Por1A strains FA19 and UU1) and five strains of N. meningitidis (N.m.). Bacteria were incubated with 5 µg/ml factor H and assessment of binding was performed by flow cytometry. The shaded histogram depicts factor H binding to strains with sialylated LOS, while the solid line shows factor H binding to strains with unsialylated LOS. The x-axis represents fluorescence on a log10 scale and the y-axis shows the number of events. Isotype controls (factor H omitted) in every instance lay within the first decade (<101) and, for the sake of simplicity, have not been shown. Each pair of strains was tested for factor H binding at least three times.

 
siaD meningococcal mutants of C2120, W171, and Y2220, the LOS molecules of which were sialylated, showed maximal (≥90%) decreases in mAb 3F11 binding in whole cell ELISA compared with their unsialylated siaD lst counterparts. Equal capture of each meningococcal strain pair to wells was demonstrated using mAb 2C3, which recognizes a common lipoprotein Ag called H.8 (12) that is expressed by all pathogenic neisseriae (27). A2594 mynB, grown in the presence of CMP-NANA, bound ~70% less mAb 3F11 than the native strain. A modest (~20%) decrease in 3F11 binding occurred with meningococcal strain H44/76 siaD compared with H44/76 siaD lst; the growth of H44/76 siaD in CMP-NANA-containing medium to achieve greater LOS sialylation further diminished 3F11 binding but did not enhance factor H binding (data not shown). Gonococcal strains had shown a 40–70% decrease in mAb 3F11 binding when their LOS molecules were sialylated. These data strongly suggest that the sialylation of LNT LOS of only N. gonorrhoeae, but not of N. meningitidis, results in increased factor H binding.

Allelic replacement of meningococcal PorB with gonococcal Por1B results in factor H binding

We have shown previously that several gonococcal Por1A-bearing strains bind factor H directly (6). We hypothesized that the selective ability of N. gonorrhoeae strains bearing the Por1B molecule (such as F62 and MS11) to bind to factor H when their LOS molecules were sialylated would require the presence of gonococcal Por1B. In contrast, meningococcal PorB in the context of intact bacteria did not bind factor H despite LNT LOS sialylation.

To demonstrate this in the context of intact bacteria, we replaced the PorB2 molecule of Y2220 with the corresponding Por1B molecule from F62 (Fig. 2A, upper panel). The Por replacement was made in the background of Y2220 siaD (LOS sialylated) and Y2220 siaD lst (LOS not sialylated). We noted a ~2 log10 increase in geometric mean fluorescence of factor H binding to Y2220 siaDPor1BF62+ (shaded histogram in Fig. 2A, upper panel) compared with control strain Y2220 siaD (thick solid line, Fig. 2A, upper panel). We also observed an increase in factor H binding to the unsialylated mutant Y2220 siaD lstPor1BF62+, (Fig. 2A, thin line) compared with Y2220 siaD, indicating that factor H could bind directly to F62 Por. Gonococcal strain F62 had not shown detectable binding of factor H in the unsialylated state and sialylation alone had resulted in increased factor H binding (Fig. 1). Similarly, we replaced PorB2 of strain Y2220 with Por1A from N. gonorrhoeae strain FA19. Gonococcal Por1A previously has been shown to bind to factor H even when LOS is not sialylated (6) and, as expected, Por1A in the meningococcal background also bound factor H even when LOS was not sialylated (Fig. 2A, lower panel, graph with solid line). LOS sialylation further increased factor H binding (Fig. 2A, lower panel, shaded area). To determine whether these results would be generalizable to encapsulated N. meningitidis of different serogroups, we first replaced the PorB2 of encapsulated Y2220 (LOS sialylated) and encapsulated Y2220 lst (LOS not sialylated) with F62 Por1B and observed that capsule expression did not affect factor H binding to these mutants (Fig. 2B, upper panel), simulating that seen with the corresponding unencapsulated mutants (Fig. 2A, upper panel). Similarly, when we examined a factor H nonbinding serogroup B (encapsulated) strain (strain 2996, LOS sialylated) and replaced the Por B molecule with Por1B from strain F62 we observed that factor H binding was further augmented by LOS sialylation in comparison with the lst mutant (LOS not sialylated) that contained the Por1B replacement alone (Fig. 2B, lower panel).


Figure 2
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  		FIGURE 2. Gonococcal Por is required for factor H binding to neisseriae-bearing sialylated LNT LOS. A, Replacing Y2220 siaD PorB2 with F62 Por1B (upper panels) or FA19 Por1A (lower panels) results in factor H binding. The binding of factor H to Y2220 siaDPor1BF62+ and Y2220 siaDPor1AFA19+ (LOS sialylated) is shown by the gray shaded histograms, the binding to Y2220 siaD lstPor1BF62+ and Y2220 siaD lstPor1AFA19+ (LOS not sialylated) is shown by the solid lines, and the control strain is shown Y2220 siaD by the broken lines. Bacteria were incubated with either 5 µg/ml (left panels) or 2.5 µg/ml (right panels) factor H. The isotype control (no factor H added) was identical to the graph seen with Y siaD (broken line) and has been omitted for easy visualization. B, The presence of the serogroup Y or the serogroup B capsular polysaccharide does not affect factor H binding to a neisserial surface bearing sialylated LNT LOS and gonococcal Por1B. The upper panels show factor H binding to Y2220Por1BF62+ (encapsulated and LOS sialylated; gray shaded area) and Y2220 lst (encapsulated and LOS not sialylated; solid line) in the presence of factor H at 5 µg/ml (left panels) or 2.5 µg/ml (right panels). In the lower panels the Por molecules of serogroup B strain 2996 (encapsulated and LOS sialylated; gray shaded area) and 2996 lst (encapsulated and LOS not sialylated; solid line) were each replaced with F62 Por1B and factor H binding was measured at each of the two concentrations indicated. Factor H binding to control strain 2996 is indicated by the broken line in both graphs. Isotype controls (factor H omitted from the reaction mixture) were identical with the broken line and have been omitted for easy visualization. C, A mutant with a hybrid Por (Y2220 loops 1 and 2 and F62 loops 3–8) incubated with 5 µg/ml factor H does not bind to factor H. Axes for all histograms are as described for Fig. 1. One representative experiment of three reproducible repeat experiments is shown.

 
In an effort to identify the Por1B loops involved in factor H binding to sialylated gonococci, we identified a mutant with a hybrid Por molecule that comprised Y2220 loops 1 and 2 and F62 loops 3–8 in the background of Y siaD (LOS sialylated). This mutant did not bind factor H (Fig. 2C, shaded histogram), suggesting that the two N-terminal loops of F62 Por1B were required for factor H binding.

We performed a dose-response assay of factor H binding to Y2220 siaDPor1BF62+ (LOS sialylated) and Y2220 siaD lstPor1BF62+ (LOS not sialylated) to assess the nature of factor H binding to F62 Por1B transfected into unsialylated and sialylated Y2220 siaD (Fig. 3). We found similar binding of factor H to the two strains at factor H with concentrations of 20 µg/ml. However, decreasing factor H concentrations resulted in a dose-dependent decrease in binding to the unsialylated strain but only marginally decreased binding to the sialylated strain. These data suggested that the binding capability of factor H for F62 Por1B bearing Neisseria in the context of whole organisms was maximal when LNT LOS was sialylated.


Figure 3
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  		FIGURE 3. Dose-response assay of factor H binding to F62 Por1B in a meningococcal background. A, Representative histogram plots of factor H binding to Y2220 siaDPor1BF62+ (LOS sialylated; gray shaded histogram), Y2220 siaD lstPor1BF62+ (LOS not sialylated; solid line), and control strain Y siaD (LOS sialylated; broken line) when strains were incubated with factor H at concentrations of 1.25, 5, or 20 µg/ml. B, Graphic representation of all the data points of the dose-response of factor H binding to Y2220 siaDPor1BF62+ and Y2220 siaD lstPor1BF62+. The means + SD of two independent experiments measuring factor H binding to Y2220 siaDPor1BF62+ (solid line) and Y2220 siaD lstPor1BF62+ (broken line) are shown.

 
Allelic replacement of gonococcal Por1B with meningococcal PorB abrogates factor H binding despite LOS sialylation

We were unable to completely replace the Por1B molecule of strain F62 with the PorB2 molecule of Y2220 and concluded that this mutation was lethal for F62. Therefore we replaced the F62 Por1B with the PorB3 from serogroup B meningococcal strain H44/76. This mutant (F62PorB3H44/76+), although viable, displayed retarded growth compared with the parent F62 strain. Sialylation of the LOS of F62PorB3H44/76+ did not result in increased factor H binding (Fig. 4A). We confirmed that F62PorB3H44/76+ expressed the LNT LOS species because it bound to mAb 3F11; sialylation of its LOS was evidenced by a decrease in 3F11 binding (Fig. 4B).


Figure 4
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  		FIGURE 4. Replacing gonococcal Por with meningococcal PorB does not result in factor H (5 µg/ml) binding when LNT LOS is sialylated. A, Replacing gonococcal Por1B with meningococcal PorB3 does not yield factor H binding upon LOS sialylation. B, Mutant strain F62PorB3H44/76+ expresses the LNT LOS and becomes sialylated. mAb 3F11 binds to unsialylated LNT LOS; binding decreases with LOS sialylation. C, A F62 mutant strain containing a hybrid Por molecule (F62 loops 1 and 2 and H44/76 loops 3–8) does not bind to factor H when LOS is sialylated. D, Replacing the PorB2 molecule with the PorB3 molecule in the background of a meningococcus with sialylated LNT LOS does not enhance factor H binding. Factor H binding to the PorB3-bearing strain H44/76 siaD lst (due to factor H binding to GNA 1870; Ref. 23 ) is shown with the broken line, while binding to the Y2220 siaDPorB3H44/76+ (LOS sialylated) and Y2220 siaD lstPorB3H44/76+ (LOS not sialylated) is shown by the shaded area and the solid line, respectively One representative experiment of two reproducible repeat experiments is shown in the figure.

 
As a control, we examined factor H binding to an F62 mutant with a hybrid Por molecule (F62 loops 1 and2, H44/76 loops 3–8). The hybrid Por molecule precluded factor H binding to F62 despite LOS sialylation (Fig. 4C). Taken together with the data in Fig. 2C (where a strain bearing a Por hybrid molecule comprising meningococcal Por B2 loops 1 and 2 and F62 Por1B loops 3–8 also did not bind factor H despite possessing sialylated LOS), we conclude that while F62 Por1B loops 1 and 2 are necessary for the binding of factor H to sialylated gonococci, they may not be sufficient to mediate factor H binding.

To confirm that the lack of factor H binding to the F62PorB3H44/76+ mutant strain did not result from retarded growth, we constructed a parallel meningococcal mutant where the PorB2 molecule of Y2220 siaD was replaced with H44/76 PorB3. Baseline binding of factor H to strain H44/76 siaD lst (Fig. 4D) is due to binding of factor H to an outer membrane lipoprotein called GNA1870 and not to PorB3; deleting GNA1870, but not PorB3, abrogates factor H binding to H44/76 siaD lst (23). The resulting mutant, Y2220 siaDPorB3H44/76+, displayed similar growth characteristics as the parent strain and did not bind factor H (Fig. 4D). These data confirmed that factor H binding to sialylated neisserial LOS was not mediated by meningococcal Por and required the presence of N. gonorrhoeae Por.

Enhanced factor H binding translates to increased serum resistance

We performed serum bactericidal assays to show that increased factor H binding correlated with increased serum resistance. As seen in Fig. 5, all strains that possessed a gonococcal Por molecule were fully resistant (100% survival) to killing by 5% NHS when LOS was concomitantly sialylated, independent of the strain background. Of note, strain Y2220 siaD lstPor1BF62+ (far right column in Fig. 5) was also serum sensitive despite its ability to bind moderate amounts of factor H (Fig. 2A, histogram represented by the thick line). Taken together, these data confirm that the effects of LNT LOS sialylation in enhancing factor H binding and serum resistance requires the concomitant presence of gonococcal Por.


Figure 5
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  		FIGURE 5. The presence of gonococcal Por is required in addition to sialylated LNT LOS for serum resistance. Gonococcal strain F62 in denoted by N.g and meningococcal strain Y2220 by N.m. Sialylation of the LOS of strains with F62 (N.g) background was achieved by growth in medium containing CMP-NANA to a final concentration of 50 µg/ml. siaD mutants in the meningococcal (N.m) background were used when LOS sialylation was required, and loss of sialylation in strains with the N.m background was achieved by using siaD lst mutants. In all bactericidal reaction mixtures the final concentration of NHS was 5% (v/v). The mean + SD of two representative experiments, each done in duplicate, is shown. The ability of each strain to bind factor H is indicated. Y2220 siaD lstPor1BF62+ (N.g Por in an N.m background, LOS not sialylated) binds intermediate levels of factor H (indicated as +/–) but remains sensitive to killing by NHS (see Discussion).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Sialic acid plays an important role in the pathogenesis of N. gonorrhoeae and N. meningitidis. Sialylation of the LOS of N. gonorrhoeae has been demonstrated on organisms recovered directly from the human genital tract (29) and on bacteria isolated from the vaginal fluid of experimentally infected mice (14). Sialylation of gonococcal LOS enhances serum resistance (9) and decreases the adherence to and phagocytic killing by neutrophils (30). Gonococci are unable to synthesize CMP-NANA (the donor molecule for sialic acid) and therefore must scavenge host CMP-NANA to sialylate their LOS. In contrast, most major serogroups of N. meningitidis (with the exception of serogroup A) can synthesize CMP-NANA and therefore can sialylate their LOS molecules endogenously. In addition to sialylating LOS, serogroups B, C, W-135, and Y possess capsular polysaccharides that also contain sialic acid in various linkages: serogroups B and C are homopolymers of {alpha}(2,8)- and {alpha} (2, 9)-linked sialic acid, respectively, while the capsular polysaccharides of serogroups W-135 and Y comprise repeating units of 6-D-Gal {alpha}(1,4)-NANA {alpha}(2,6)- and 6-D-Glc {alpha}(1,4)-NANA {alpha}(2,6)-, respectively (31, 32). Meningococcal capsular polysaccharide is perhaps the most important determinant of virulence and serum resistance. With rare exceptions (33, 34), almost all strains isolated from the blood or cerebrospinal fluid of infected patients possess a capsule. Capsular polysaccharide has also been shown to be necessary to cause invasive disease in the infant rat model (20, 21). The mechanism of complement regulation by capsular polysaccharide has not been fully elucidated, but we suggest that capsular polysaccharide regulates the classical pathway by a mechanism that is independent of the binding of complement regulatory molecules (our unpublished observations).

Small increments in factor H binding can significantly enhance the ability of a gonococcal stain to resist killing by NHS. As an example, even a 2-fold increase in factor H binding to the serum-sensitive strain UU1 (Fig. 1), which is >90% killed in 10% NHS, is sufficient to alter its phenotype to serum resistance (100% survival in 10% NHS) when grown in medium containing CMP-NANA (10). In contrast to the unequivocal role of LOS sialylation in enhancing gonococcal serum resistance, the role of LNT LOS sialylation in mediating meningococcal serum resistance is less clear. We addressed this question by first examining factor H binding to a set of diverse strains of N. meningitidis that all expressed a LNT substitution of HepI. Surprisingly, we found that the sialylation of LNT LOS in N. meningitidis did not enhance factor H binding (Fig. 1). This was most evident with derivatives of strain Y2220, where we saw barely any factor H binding above background levels by flow cytometry. We have identified the lipoprotein GNA1870 as the acceptor molecule for factor H binding on meningococci (23). Levels of GNA1870 expression correlated with the amount of factor H binding. Strain Y2220 expresses low amounts of GNA1870 and therefore bound minimal amounts of factor H (below the threshold of detection by flow cytometry). Furthermore, factor H binding to meningococci is not affected by capsule expression or by LOS sialylation (23, 35).

The LOS of strain Y2220 expresses phosphoethanolamine (PEA) substitutions simultaneously at the 3 and 6 positions of HepII, a feature that is not described in any of the standard meningococcal LOS immunotypes, L1 through L12 (36). However, the inability of sialylated Y2220 to bind factor H may not be related solely to this relatively unusual HepII PEA configuration, because the LOS of a gonococcal strain, called 398079, is structurally identical to Y2220 (LNT substitution of HepI and simultaneous 3- and 6-PEA on HepII); sialylation of this gonococcal strain results in enhanced factor H binding and serum resistance (16).

In the aggregate, these data provided strong circumstantial evidence that a molecule on the gonococcal surface distinct from LOS sialic acid was necessary for factor H binding to sialylated organisms. Because we have previously reported that certain gonococcal Por1A strains bind factor H directly (6), we hypothesized that Por1B may also be involved in the interaction of factor H with sialylated gonococci. Neisserial porins belong to the Gram-negative porin superfamily (37) and are the most represented outer membrane proteins in the pathogenic neisserial species (38). Meningococcal porins are designated as class 1 (Por A; ~45 kDa) and either class 2 or class 3 (PorB; ~33 kDa) and their genes are present in mutually exclusive alleles (39). N. gonorrhoeae express only single Por molecules that are alleles of the porB gene, either Por1A (~35 kDa) or Por1B (~37 kDa) (40); porA in N. gonorrhoeae is a pseudogene (41). Por comprises eight transmembrane loops (16 membrane-spanning segments) and its native configuration is a homogenous trimer that functions as a selective anion channel. Por1B gonococcal strains bind factor H weakly when unsialylated, but growth in CMP-NANA-containing medium increases factor H binding substantially (7, 16). Maximum factor H binding to sialylated gonococci is seen when bacteria are grown in relatively low concentrations of CM-NANA (1 µg/ml), which results in only partial LOS sialylation (16) and indicates the lack of a stoichiometric relationship between LOS sialylation and factor H binding. The requirement for Por1B in mediating factor H binding to sialylated neisseriae was confirmed when we replaced the meningococcal (strain Y2220) PorB2 with gonococcal (F62) Por1B, resulting in both factor H binding and serum resistance. However, in nonsialylated Y2220 harboring F62 Por1B, conversion to a serum resistance phenotype was not observed despite some factor H binding to the transformed strain (Fig. 5, far right column). This emphasizes that, in the case of Neisseria bearing the F62 Por 1B genotype, sialylation is also required to achieve serum resistance. We also showed that Por1A in the context of meningococci could bind factor H even when LOS was not sialylated (Fig. 2A, lower panel), consistent with our previous results with N. gonorrhoeae (6). LOS sialylation increased factor H binding to this mutant, consistent with observations made when Por1A was in its native background (Fig. 1). The FA19 Por1A genotype in N. meningitidis also transforms the phenotype to serum resistance; however, in addition to factor H binding this Por 1A also binds the C4b-binding protein, a classical pathway down-regulator, which also contributes to serum resistance (5). In the converse experiment, the replacement of gonococcal Por1B with meningococcal PorB3 (attempts to introduce PorB2 into the background of N. gonorrhoeae did not yield colonies) failed to enhance factor H binding when bacteria were sialylated.

We have shown previously that sialic acid is essential to maintain the binding of factor H to sialylated Por1B gonococci, because the neuraminidase treatment of bacteria (which desialylates LOS) with bound factor H on their surface results in the loss of factor H binding (7). The current model suggests that the presence of Por1B is an essential component of this interaction. Evidence in support of a factor H binding site on gonococcal Por1B was provided by the observation that F62 Por1B transformed into LOS unsialylated meningococcal strain Y2220 could bind factor H (Figs. 2, A and B, and 3). However, binding to F62 Por1B in the unsialylated meningococcal background was relatively weak (~1 log10 fluorescence less) compared with binding to the same strain when sialylated (Fig. 2A). This intermediate level of factor H binding was not sufficient to confer protection to Y2220 siaD lstPor1BF62+ against killing by 5% NHS (Fig. 5). We have shown that a high level of classical pathway activation occurs on Y2220 as a result of high levels of C4b binding to the 6-PEA expressed by the LOS of this strain (24) and believe that this could overcome the complement inhibitory effects of factor H.

In an attempt to identify the Por1B loop(s) involved in factor H binding to sialylated gonococci, we used synthetic peptides corresponding to the putative exposed regions of each of the eight loops of F62 Por1B to block factor H binding to sialylated F62. None of the peptides, either used individually or as a combination of all eight peptides simultaneously and each at a ~3,000-fold molar excess over factor H, inhibited factor H binding to sialylated F62 (data not shown). This suggests that the factor H binding sites on Por1B are not displayed by linear representations of the exposed regions of the Por 1B molecule and that factor H binding may only occur in the context of P1B-containing organisms when they are sialylated. Factor H binding to sialylated gonococci that harbor sialylated LNT LOS may result from alteration of the configuration of LOS configuration, which exposes a factor H binding region in Por1B (a site that is otherwise obscured by unsialylated LOS). A second possibility may be that LNT-linked sialic acid and Por each bind factor H independently, but their presence together on the bacterial surface, when it occurs, results in a more stable interaction with factor H. Another possibility may be that sialylated LOS and Por1B together form a "neo-epitope" that binds factor H. The effects of LNT LOS sialylation in augmenting factor H binding to gonococci were not restricted to Por1B but also occurred with the two Por1A strains that we tested (Fig. 1), where Por1A may bind more factor H than Por 1B (Fig. 2A).

It is noteworthy that factor H binding to PorB3 purified from strain H44/76 has been measured by ELISA (42). We have also shown the binding of factor H to H44/76 PorB3 on a Western blot (23), but H44/76 PorB3 may not bind to factor H directly in the context of the whole organism because introducing this molecule into the background of F62 (Fig. 4C) or Y2220 (Fig. 4D and Ref23) did not result in factor H binding. These data suggest that in intact bacteria the region in purified PorB3 that binds factor H is probably not accessible. The lack of specificity of factor H-Por interactions when Por is out of context underscores the importance of studying such interactions "in situ," which is likely to be more relevant in vivo.

Our findings highlight the differences in mechanisms used by two pathogenic neisseriae to evade complement. Meningococci possess a capsule, which is probably the most important determinant of serum resistance and virulence in this organism. The ability to bind factor H may enhance serum resistance further, as was demonstrated in a recent study where depleting the serum of factor H resulted in greater serum killing of N. meningitidis (35). Distinct mechanisms of factor H binding potentially could provide the redundancy needed to compensate for any disadvantage the organism may face because of the inability of its sialylated LOS to increase factor H binding. Therefore, dependence on LOS sialylation may be less critical for meningococcal survival in vivo. Complement regulation by meningococcal LOS may also result from blocking target(s) for C4b and C3b binding; neisserial LOS has been shown to bind both these molecules (24, 43).

In contrast to meningococci that cause invasive disease, gonococci do not possess a capsule and require other means to evade complement-dependent killing. Scavenging factor H from the host to thwart complement activation early in the cascade constitutes one important mechanism of immune evasion. In addition to possible shielding of targets for C3b and C4b (as may also occur with meningococci), factor H binding to sialylated gonococci results in efficient regulation of the alternative pathway. Acknowledging the subtle differences in complement activation by these two organisms may provide a better understanding of their diverse clinical features and insights into the pathogenic mechanisms of these two related bacterial species.


   Acknowledgments
 
We thank Maohua Lei and Gabrielle Heinze for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI32725 (to P.A.R.) and AI054544 (to S.R.). Back

2 Address correspondence and reprint requests to Dr. Sanjay Ram, Division of Infectious Diseases and Immunology, University of Massachusetts Memorial Medical Center, Lazare Research Building, Room 322, 364 Plantation Street, Worcester, MA 01605. E-mail address: sanjay.ram{at}umassmed.edu Back

3 Abbreviations used in this paper: LNT, lacto-N-neotetraose; LOS, lipooligosaccharide; lst, LOS sialyltransferase; NANA, N-acetylneuraminic acid; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; Hep, heptose; NHS, normal human serum; PEA, phosphoethanolamine; Por, porin. Back

Received for publication August 21, 2006. Accepted for publication January 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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