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* Center for Immunochemistry and Veterans Affairs Medical Center, San Francisco, CA 94121; Departments of
Pediatrics and
Laboratory Medicine, University of California, San Francisco, CA 94143;
Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, United Kingdom; and
¶ Institute of Child Health, University College London, London, United Kingdom
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Introduction |
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MBL, which functionally resembles a hybrid of IgM and Clq (4, 8), contains a collagenous domain (N-terminal) and carbohydrate recognition domains (CRDs; C-terminal) (9, 10). It is the major serum collectin in humans and has been shown to bind to repeating sugar moieties on numerous microorganisms, leading to activation of the lectin complement pathway. It has a specificity defined by the orientation of hydroxyl groups at the 3 and 4 positions around the carbon ring in hexose sugars, with affinity for mannose and N-acetylglucosamine derivatives, and by the spacing and orientation of these sugars on target surfaces (11, 12). On binding to its targets, MBL activates the complement system via MBL-associated serine protease-1 (MASP-1) and MASP-2, of which the latter cleaves C4 and C2 to generate a C3 convertase independently of Ig to promote bacterial lysis (13, 14). MBL is also associated with MASP-3 (15) and a truncated form of MASP-2, MAp19 (16), although the function of these two proteins is not yet known.
There is also evidence that MBL can promote opsonophagocytosis (8). This may be due to complement activation (4) or interactions of MBL with the cellular receptors, cC1qR (17), and CR1/CD35 (18), which are found on cells of the myeloid lineage, vascular endothelium, and platelets (19). Recently, we have shown that MBL is able to promote phagocytosis of meningococci by neutrophils in the absence of other opsonic factors (20).
Adult levels of MBL are present soon after birth and throughout life (8). The presence of MBL during the period in infancy when the lack of bactericidal Abs and the immaturity of the immune system put children at greatest risk for meningococcal disease supports the concept that the MBL pathway plays an important role in host defense. Serum concentrations of MBL are genetically determined via three structural gene mutations and three promoter region polymorphisms (8). Inheritance of structural gene variants of MBL predisposes individuals to meningococcal infection, although this may be more pronounced in the young (21, 22).
We have previously reported a detailed examination of MBL binding to Neisseria meningitidis serogroup B (23) and more recently to serogroup C (24). We showed that MBL was able to bind to and activate complement and to increase killing of these organisms (23, 24). Previous work had indicated that bacterial encapsulation significantly inhibited the binding of MBL to whole organisms (25). However, our studies indicated that the structure and sialylation of lipooligosaccharide (LOS; a phase variable major virulence factor of N. meningitidis), rather than and independently of encapsulation, appear to be the major determinants of MBL binding (23).
The dependence of MBL binding on LOS structure and sialylation suggested that the lectin bound to this outer membrane component when it was in certain conformations. However, based on the known structures of the major meningococcal LOS, the terminal oligosaccharide sugars are not recognized ligands for MBL (26, 27, 28). There is precedence in the literature of LOS sialylation decreasing the binding of mAbs to a neisserial outer membrane, porin protein (29). In addition, the interaction of meningococcal outer membrane proteins opacity protein (Opa) and opc with eukaryotic cells is markedly inhibited by LOS sialylation (30, 31, 32). We have also noted previously that it is only possible to partially and not completely inhibit the binding of MBL to microorganisms using monosaccharide inhibitors of lectin binding (33). This suggested to us that MBL may bind to nonsugar targets on the surface of meningococci.
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Materials and Methods |
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N. meningitidis serogroup C strain 8026 is a case isolate (1, 2, 3) that has previously been reported to bind MBL (24). Group C strains 15501 and 15502 were both isolated from a child with disseminated meningococcal disease and have identical protein profiles, except that 15502 is the Opa-negative variant of 15501, suggesting that the two strains are clonal variants (34). Both express porin protein (PorA) (P1.2) and porin protein B (PorB) (2a) (34). Meningococcal group C strain 15029 is a throat isolate (1, 2, 3). N. meningitidis strain 8032 is a group Y blood isolate (35).
Purification of MBL
MBL was purified from human ethanol-fractionated plasma paste (donated by C. Dash, Blood Products Laboratory, Elstree, United Kingdom) by a modification of the method of Kilpatrick (36), as recently described (20, 37). Briefly, MBL was prepared by a two-step mannan-agarose affinity purification followed by positive removal of trace Ig impurities as described (20).
LOS and outer membrane complex (OMC) purification
LOS were extracted from acetone-dried organisms by the hot phenol-water method (38, 39). OMCs were prepared as previously described (38, 40). The OMC contains LOS and outer membrane proteins (40).
Dot blot analyses
Purified LOS and OMCs were diluted in 50 mM MgCl2-PBS and applied to nitrocellulose. After drying, the membranes were blocked in 5% BSA-TBS and then incubated in 5 µg/ml MBL in 1% BSA-TBS containing 5 mM CaCl2. The blots were then incubated with anti-MBL (clone 131-1; Statens Serum Institut, Copenhagen, Denmark) followed by secondary goat anti-mouse IgG Ab conjugated to alkaline-phosphatase (Sigma-Aldrich, St. Louis, MO). The blots were developed with a solution of 50 mM Tris-HCl (pH 8.0), 0.1% Naphthol AS-MX phosphate disodium salt (Sigma-Aldrich), and 0.2% fast red Texas Red salt (Sigma-Aldrich). All washes were with TBS. A positive control membrane was incubated in the LOS-specific mAb D6A (41, 42). A negative control membrane was incubated as described, but without MBL.
ELISA analyses
MBL binding was investigated using a series of ELISAs. For OMC or LOS ELISA, wells were coated with purified OMC or LOS diluted in PBS with 0.1% deoxycholic acid. Preliminary experiments showed that 0.1% deoxycholic acid-PBS optimized the binding of LOS-specific mAbs to wells coated with purified LOS but had little affect on their binding to OMC, which was already strong. The wells were blocked with 1% BSA-TBS and incubated with 5 µg/ml MBL in 1% BSA-TBS containing 5 mM CaCl2. The wells were then developed as described for the dot blot, except that the substrate was p-nitro-phenyl phosphate (2 mg/ml in 100 mM bicarbonate buffer (pH 9.5) with 10 mM MgCl2; Sigma-Aldrich). The absorbance was read at 405 nm with a MAXline Microplate Reader (Molecular Devices, Sunnyvale, CA). Wells that received all steps except Ag served as a negative control, and values from these wells were subtracted from the experimental wells. Positive control wells coated with LOS or OMC measured the binding of mAb D6A to LOS.
Whole-cell ELISA as previously described (2) was used to show MBL binding to whole organisms. Microtiter wells were coated with 8026 bacteria diluted in PBS and then blocked with 50% SuperBlock (SB) blocking buffer (Pierce, Rockford, IL) diluted in TBS. The replacement of BSA with SB decreased the background binding of MBL on ELISA. The wells were then incubated with serial 2-fold dilutions of MBL in TBS containing 5 mM CaCl2. Primary and secondary Abs were diluted in 10% SB-TBS. All washes except the final two were with TBS-0.05% Tween 20 (Surfact-Amps 20; Pierce). To determine whether calcium was required for MBL binding, 15 microtiter wells coated with whole organisms were incubated with MBL diluted in TBS with 5 mM CaCl2, and 15 microtiter wells coated with whole organisms were incubated with MBL diluted in TBS without 5 mM CaCl2.
For sugar inhibition experiments, MBL (5 µg/ml) was preincubated for 1 h at room temperature in TBS-5 mM CaCl2 with 100, 50, or 25 mM N-acetyl-D-glucosamine (GlcNAc), D-galactose (Gal), or D(+)-mannose (Sigma-Aldrich) before being added to the microtiter wells coated with 8026 whole organisms. The binding of MBL was detected as above. As a control, microtiter wells were coated with mannan (Sigma-Aldrich; 500 µg/ml in 15 mM carbonate (pH 9.6)) overnight at 4°C and then were washed (43). MBL was preincubated with 100, 50, or 25 mM mannose before being added to the wells. The inhibition experiments were repeated with microtiter wells coated with purified OMC from strain 8026. MBL (5 µg/ml) was preincubated with 200 mM Gal or GlcNAc.
SDS-PAGE and immunoblot analysis
The proteins and LOS in purified OMC were separated by SDS-PAGE by a previously described modification of the method of Laemmli (2, 44). The LOS molecules were visualized by silver stain and the proteins were stained with Coomassie R-250 (Sigma-Aldrich).
The separated LOS and proteins of the strains were electroblotted to nitrocellulose, blocked in either 1% BSA-TBS or undiluted SB. The immunoblots were incubated in 3–5 µg/ml MBL diluted in TBS-0.05% Tween 20 containing 5 mM CaCl2. The anti-MBL mAb and alkaline phosphatase-conjugated secondary Abs were diluted in 10% SB-TBS-0.05% Tween 20. The immunoblots were then developed as described for the dot blot. All TBS washes except the final two included 0.05% Tween 20. Positive control immunoblots were incubated in LOS-specific mAb 06B4 (45), and negative control blots included all steps except MBL.
In separate experiments to assess the requirement for calcium, the SDS-PAGE-separated OMC was incubated with MBL diluted in buffer with or without calcium or with MBL that was preincubated with 10 mM EDTA for 10 min before being added to the immunoblots. To evaluate binding characteristics, MBL in calcium-containing buffer was allowed to react with the OMC on immunoblots, and then the blots received three 5-min washes with or without 10 mM EDTA to determine whether the MBL could be eluted from the proteins. In another experiment, MBL was preincubated in buffer with calcium and 500 mM NaCl before being added to the immunoblots.
The SDS-PAGE-separated LOS and proteins in OMC were also incubated with MBL, mAb 4B12 that binds most neisserial Opa proteins (46), or mAb 4D11 that binds all meningococcal class 1, 2, and 3 porin proteins (D. Zhou, unpublished observation). The separated proteins of strain 15501 were also immunoblotted with mAbs specific for PorA (P1.2) and PorB (2a), which were provided by W. D. Zollinger (Walter Reed Army Institute of Research, Washington, DC). Purified PorB, a gift from M. Blake (Baxter Healthcare, Columbia, MD), was analyzed by SDS-PAGE, transferred to nitrocellulose, and incubated with MBL.
N-terminal amino acid sequencing of MBL binding proteins
To confirm the identification of the proteins that bound MBL, the OMC proteins of 8026 were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was cut, and one portion was stained with Coomassie and the other was blotted with MBL. After careful realignment of the two membranes, the two bands that bound MBL were excised from the Coomassie-stained membrane. The N-terminal sequence of the two proteins was determined by the University of California, San Francisco, Biomolecular Resource Center as described (www.ucsf.edu/brc/proseqprotocol.html). They performed a basic local alignment search tool (BLAST) search using the National Center for Biotechnical Information’s database through the University of California, San Francisco, Sequence Analysis Consult Service with the method described (47).
Inhibition of MBL binding to whole organisms by purified PorB
To assess the ability of MBL to specifically bind PorB in the membranes of whole organisms, an inhibition, whole-cell ELISA was done. MBL (5 µg/ml) in TBS containing 5 mM CaCl2 was preincubated for 1 h at room temperature with varying concentrations of purified PorB (0–20 µg/ml) before being added to microtiter wells coated with 8026 organisms. MBL binding was detected as described above. Percent inhibition of MBL binding was calculated as follows: [(absorbance without PorB - absorbance with PorB)/absorbance without PorB] x 100.
Binding of MBL to Escherichia coli expressing gonococcal Opa protein
To examine the binding of MBL to surface-expressed Opa on bacteria, an Opa-producing clone4in E. coli DH5
(gift of G. Gorby, Creighton University School of Medicine, Omaha, Nebraska) and the control strain E. coli DH5 that contained only the vector were used (48). The Opa corresponds to Opa I from gonococcal strain FA1090.
A whole-cell ELISA was done first to confirm the expression of Opa on the E. coli surface. Microtiter wells were coated with the Opa-producing strain or the control strain diluted in PBS to an identical OD, blocked, and reacted with serial 2-fold dilutions of mAb B33, which binds most neisserial Opa proteins (49). This mAb was substituted for mAb 4B12 because of insufficient quantity of that Ab. mAb B33 binding was detected as described for mAb 4B12. The background binding of B33 to the control strain was subtracted from the binding to the Opa-producing strain.
To determine whether the Opa producing E. coli bound MBL, microtiter wells were coated with the two strains as above, blocked, and reacted with 5 µg/ml MBL diluted in TBS without calcium to minimize the lectin binding of MBL to the E. coli LPS. MBL binding was detected as described above. Wells that received all steps except MBL served as a negative control.
Inhibition of factor H binding to PorB
Meningococci have been reported to bind factor H to PorB (50). To investigate the ability of MBL to block this binding, an inhibition ELISA was performed. Microtiter wells were coated overnight at room temperature with 50 µg/ml purified PorB in 0.05 M sodium carbonate buffer (pH 9.6). After washing, the wells were blocked with 1% human serum albumin-TBS (Sigma-Aldrich) for 1 h. Mixtures of 0.5 µg/ml human factor H (Quidel, Santa Clara, CA) and varying concentrations of MBL (0–5 µg/ml) in TBS containing 5 mM CaCl2 were then added to the wells and incubated for 90 min at room temperature. The binding of factor H was detected with goat antisera to human factor H (Quidel), followed by alkaline phosphatase-labeled secondary Ab. Wells that received all steps except factor H served as a negative control. Percent inhibition of factor H binding was calculated as follows: [(absorbance without MBL - absorbance with MBL)/absorbance without MBL] x 100.
Binding of MBL to organisms incubated in serum
A modification of the method described by Gulati et al. (51) was used to determine whether whole organisms incubated in human serum bound MBL. Strain 8032 was grown overnight on Mueller Hinton agar in a candle jar at 37°C. Organisms were harvested off the plate and suspended in veronal buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2 (VBS++) to a concentration of 6 x 108 bacteria/ml. A reaction mixture of 100 µl of bacteria and 100 µl of hypogammaglobulinemic serum (HGS) was incubated at 37°C for 30 min. HGS (52) was used to avoid Ab mediated complement lysis of the organisms and had normal total hemolytic C activity. The MBL concentration of the HGS was determined by ELISA and was 5 µg/ml. The bacteria were centrifuged and washed twice with VBS++, and the pellet was then suspended in 200 µl of VBS++. Aliquots were placed in the wells of a microtiter plate and incubated for 3 h on a horizontal shaker at 37°C. After washing, the anti-MBL mAb or anti-LOS mAb D6A was added to the wells. Monoclonal Ab D6A binds strongly to the LOS of strain 8032 and was used to show attachment of organisms to the wells. As a negative control, aliquots of bacteria not incubated in HGS were incubated in microtiter wells. The wells were developed as described above.
Statistical analysis
Data from groups are expressed as mean ± SD. Differences between groups were analyzed by unpaired sample, two-tailed t test.
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Results |
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Purified LOS and OMC containing LOS and proteins of N. meningitidis strain 8032 were applied to nitrocellulose and incubated with MBL, which bound to OMC but did not bind to the LOS. A control membrane showed strong binding of LOS-specific mAb D6A to both the OMC and LOS dots, confirming the presence of LOS in both samples (data not shown).
ELISA analyses of MBL binding
MBL did not bind to 8032 LOS-coated wells (OD405 = 0.001 ± 0.002). Attachment of the LOS to the wells was confirmed by the strong binding of anti-LOS mAb D6A (OD405 
1.9 for all assays). MBL did bind to 8032 OMC coated wells (OD405 = 0.545; mean of two trials). Whole-cell ELISA showed that strain 8026 bound MBL in a dose-dependent manner (Fig. 1). Calcium in the buffer had little effect on the binding of MBL to 8026 by whole-cell ELISA (binding without calcium, OD405 = 0.763 ± 0.192; binding with calcium; OD405 = 0.651 ± 0.142).
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The results of the dot blot and ELISA analyses indicated that MBL did not bind to 8032 LOS molecules but did bind to other structures in the meningococcal outer membrane. Four meningococcal strains were used to further investigate the binding of MBL to these OMCs. Fig. 3 shows the Coomassie-stained OMC proteins of these strains and confirms that strain 15502 is the Opa-negative variant of strain 15501.
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N-terminal amino acid sequencing of MBL binding sites
The N-terminal amino acid sequences of the two MBL binding sites on strain 8026 were determined as a direct confirmation of the Ab-based identification of Opa and PorB. The OMC proteins of 8026 were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The two protein bands that bound MBL were excised from the Coomassie-stained membrane, and the N-terminal sequence of the two proteins was determined. The sequencing result for the 29-kDa band was ASEDSSRPPYYVQAD, and a BLAST search identified 50 matches (e.g., GenBank accession number AF001185); all were N. meningitidis Opa proteins. Six matched 14 of 15 aa (93%) and the rest matched 13 of 15 (86%). The sequence of the 40-kDa band was DVTLYGVVKAGVETS, which resulted in 45 matches (13 of 15, 86%). All were meningococcal PorB (e.g., GenBank accession number U07188) or N. gonorrhoeae porin (analogous to PorB; GenBank accession number AF200745).
Inhibition of MBL binding to whole organisms by purified PorB
Preincubation of MBL with purified PorB inhibited the binding of MBL to strain 8026 organisms in a dose-dependent manner when analyzed by whole-cell ELISA (Fig. 8). These results supported the conclusion that MBL interacts specifically with this outer membrane protein when expressed on meningococci.
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Because a lectin-like interaction between Neisserial Opa and LOS has been described (55), the binding of MBL to surface-expressed Opa was investigated with an Opa-producing clone in E. coli. The expression of Opa on the surface of E. coli organisms was confirmed by whole-cell ELISA with dose-dependent binding of the Opa-specific mAb (data not shown). As can be seen in Fig. 9, the Opa-producing strain bound significantly more MBL than did the control strain that contained only the vector.
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In an ELISA format, factor H bound well to purified PorB (OD405 = 0.864 ± 0.167). The binding of factor H to PorB was markedly inhibited by MBL in a dose-dependent manner (Fig. 10). The inhibition of the binding of 0.5 µg/ml factor H was nearly complete with 5 µg/ml MBL and was still >50% with 0.1 µg/ml MBL.
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The incubation of meningococcal strain 8032 in 50% human serum resulted in the binding of MBL to the whole bacteria as detected by the anti-MBL mAb on ELISA (OD405 = 0.391). The binding of the anti-LOS mAb D6A was OD405 = 0.611. The binding of MBL to bacteria incubated in buffer was OD405 = 0.104, and the binding of D6A was similar (OD405 = 0.544).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MBL binding to Opa and PorB that we observed did not require calcium and was not inhibitable by mannose or GlcNAc, but it was sensitive to 0.5 M salt, which may suggest a mechanism of the interaction between Opa/PorB and MBL. These proteins are not glycosylated (56), and the homologues in N. gonorrhoeae, Opa, and protein I (PorB equivalent) lack any associated carbohydrates (57, 58), so it appears reasonable that the MBL binding to them was not found to be sugar sensitive or calcium dependent in this study. The N-terminal collagenous region of MBL is an important site of interaction with the immune system and is the calcium-independent binding site of the MASPs (59, 60). The collagenous region is thought to mediate interactions of MBL with cellular receptors based on evidence that MBL receptor binding is not calcium dependent and is favored at low ionic strength (4). Recently, CR1/CD35 (18) was identified as a receptor for MBL. The binding of MBL to this receptor was not inhibited by GlcNAc and did not require calcium but was inhibited in a high salt buffer. The binding characteristics of MBL to Opa and PorB that we have described suggest that the collagenous region of MBL might be involved, although further investigation will be needed to determine the binding site(s) on the MBL molecule for these meningococcal outer membrane proteins. Interestingly, McNeely and Coonrod (61) reported that human C-type lectin surfactant protein A did not bind to Haemophilus influenzae LOS or capsule, but rather bound only to the outer membrane porin protein.
We postulate that MBL binds to surface-exposed conserved regions of Opa because it bound Opa proteins of different sizes on the same and on different meningococcal strains. N. meningitidis typically have up to four or five Opa genes that share conserved regions (62). A single strain can express up to four distinct Opa proteins, and a single organism can simultaneously express up to two (63). MBL also bound to a gonococcal Opa when expressed on the surface of E. coli. PorB is a second binding site for MBL and it is expressed by all meningococci, although variability in this protein is used to serotype N. meningitidis (53, 54). Similarly, MBL must bind to a conserved region of this porin.
Purified PorB was used in an inhibition ELISA to assess the specific binding of MBL to this porin in the membrane of whole meningococci. An inhibition ELISA with purified Opa analogous to that described for PorB was not feasible because a lectin-like interaction between neisserial Opa and LOS has been described (55). This would indicate that purified Opa would bind MBL and would also bind to the LOS on whole organisms, thus acting as a link between MBL and the bacteria. We chose instead to look at MBL binding to E. coli expressing Opa protein from N. gonorrhoeae (48). Neisserial Opa proteins form a family of structurally and functionally related proteins. Antigenic diversity stems from semi- and hypervariable regions within a conserved framework (62, 64, 65), and it was expected that gonococcal Opa would bind MBL.
Purified PorB inhibited the binding of MBL to whole meningococcal organisms, showing that MBL interacted specifically with this major outer membrane protein when expressed on the bacterial surface. Similarly, E. coli that surface-expressed Opa bound significantly more MBL than did the control strain when analyzed by whole-cell ELISA. These data taken together with our previous observation that MBL binds directly to meningococci and increases complement activation and killing of these organisms (23, 24) support the conclusion that Opa and PorB bind MBL to the bacterial surface and are involved in complement activation.
Opa and PorB both interact extensively with the human host. Opa mediates interactions with human cells, including epithelial cells and phagocytes, through interaction with heparan-sulfate proteoglycans and members of the CD66 family, with different Opa variants showing variable specificities for these different ligands (66). We and others have shown that Opa mediates nonopsonic phagocytosis of meningococci (3, 67). PorB modifies apoptosis of host cells and phagocyte function (68, 69, 70, 71) and binds factor H, an important regulator of the alternative complement activation pathway (50). The effect of MBL binding to these molecules remains to be investigated, but we can speculate that it might modulate their interaction with human cells.
The available evidence indicates that complement activation on meningococcal surfaces is regulated predominantly by factor H (50), which acts as a cofactor for factor I-mediated cleavage of C3b to iC3b, limiting the number of functionally active C3 convertases (72, 73). Importantly, meningococci have been reported to bind factor H to PorB but not to sialylated LOS (50). We have recently reported that the binding of MBL to strain 8026 led to accelerated complement activation and increased killing of this organism (24). This is likely due, in part, to an observed increase in C4 cleavage by attendant MBL-associated serine proteases (24), but we were intrigued by the possibility that MBL might also directly influence complement regulation by competing with factor H for binding to PorB.
We confirmed by ELISA the results of Ram et al. (50) that factor H bound to purified PorB. We were also able to show that the simultaneous addition of MBL with factor H was able to inhibit factor H binding to porB in an MBL dose-dependent manner. Preincubation of PorB with MBL was not necessary. Inhibition of factor H binding was >50% at 0.1 µg/ml, similar to the serum concentration of MBL that would be expected in an individual heterozygous for an MBL structural gene mutation (8). Inhibition of factor H binding increased through the physiological range of MBL concentration to 90% at 5 µg/ml, which is at the higher limit of normal MBL serum levels. The changing concentration of MBL in the human host relative to factor H, for example either in serum during an acute phase or locally as a result of inflammation, may profoundly change the outcome of complement activation.
A recent study by Gulati et al. (51) confirmed that the binding of MBL to serum-resistant N. gonorrhoeae resulted in significant complement-mediated kill, but only when the organisms were preincubated with MBL. It was found that if MBL was preincubated with a mixture of C1-inhibitor, which inhibits C4 cleavage by MASP, and 2-macroglobulin, that the MBL-dependent killing was ablated. The regulation of MBL-mediated complement activation in vivo is undoubtedly complex and would vary for different organisms, as evidenced by the different binding characteristics of factor H to meningococci and gonococci (50). We have shown that whole meningococcal organisms bind MBL when incubated with human serum and that MBL inhibits the binding of factor H to purified PorB. The role of MBL bound to meningococci remains to be further elucidated, but likely includes opsonization for phagocytosis and competition with factor H for binding sites on PorB.
In the present study, when SDS-PAGE-separated Opa and PorB were transferred to nitrocellulose and immunoblotted with MBL in 10 mM EDTA, the binding (if anything) appeared to increase. Furthermore, MBL could not be eluted from Opa and PorB with 10 mM EDTA washes once it had bound to these proteins on immunoblot. EDTA in this concentration is used to elute MBL from a mannan-agarose column in the purification process of MBL from serum (37). These findings support the conclusion that the binding of MBL to Opa and PorB is not calcium dependent. We have described elsewhere (24) that at least some of the binding of MBL to strain 8026 as measured by flow cytometry was dependent on the presence of calcium, as demonstrated through calcium chelation by EDTA. Part of this discrepancy might be explained by the potential disruption of the outer membrane of whole organisms by EDTA. The ELISA experiment reported in the present paper was powered to detect a 30% or greater change in MBL binding with and without calcium to whole organisms. Flow cytometry may be more sensitive to small differences in MBL binding to bacteria because of the larger number of individual binding events sampled.
We also previously reported (24) that preincubation of MBL with GlcNAc, a known sugar ligand, partially decreased MBL binding to strain 8026 by flow cytometry, suggesting that LOS was one of the MBL-binding moieties. In the current study, we were unable to demonstrate by ELISA any sugar-sensitive binding to 8026, nor was direct binding to LOS detected by dot blot, ELISA, or SDS-PAGE immunoblot. The apparent lack of binding to LOS was unlikely to be due to sialylation of these molecules, in that strains 8026 and 15029 are known to have little or no LOS sialylation (1, 2, 3). Again, differences in the sensitivity or differences in the configuration of LOS in these solid phase assays relative to flow cytometry are the most likely explanations for these discrepancies.
When taken together, the results of the current study and those of our previous investigation suggest that the major binding of MBL to the meningococcal strains that we tested is to Opa and PorB in a non-lectin-mediated manner, but that minor binding through the CRDs is occurring as well. The most likely candidate for this binding is LOS expressed in small amounts that could not be detected by dot blot, ELISA, SDS-PAGE, or immunoblot. LOS made by N. meningitidis are short glycolipids in the bacterial membranes that are surface expressed. The higher molecular mass (longer) LOS molecules represent the sequential addition of hexoses or hexosamines to a common core region (74). The LOS of meningococcal serogroups B, C, and Y are serotyped L1–L8, and LOS structure has recently been reviewed (75). The major branch of the LOS molecule of all of these serotypes except L6 terminates in galactose or sialylated galactose, which would not bind MBL. L6, however, terminates in GlcNAc, a known MBL ligand, but is a very minor LOS serotype, in that it is a precursor for longer molecules (76). Each meningococcal strain makes from one to eight or more LOS molecules of different structure, and single organisms can express more than one type of LOS molecule in variable quantity (77, 78). When analyzed by SDS-PAGE and silver stain, strain 8026 expresses a large amount of L7 LOS that terminates in galactose but also expresses a very minor, smaller LOS molecule whose size could be consistent with L6 (3, 76). Further study will be necessary to determine whether L6 LOS plays a role in the interaction of meningococci with MBL.
We conclude that MBL can bind to meningococci by a novel target recognition of two outer membrane proteins, Opa and PorB. The binding does not appear to be lectin mediated and occurs when the bacteria are incubated in human serum. These outer membrane proteins mediate a number of interactions of the pathogen with the host, including complement activation and serum killing, binding and internalization/invasion, and the responses of host cells central to the pathogenesis of meningococcal disease. We have shown that MBL inhibits the binding of factor H to purified PorB. The interaction of MBL-bound meningococci with the human immune system will require further characterization. An understanding of the interaction between meningococcal protein ligands and MBL would serve as a model for investigations into the binding of MBL to other proteins and would extend our knowledge of the biology of this important molecule in the pathogenesis of other infectious diseases and autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michele M. Estabrook, Veterans Affairs Medical Center, 111W1, 4150 Clement Street, San Francisco, CA 94121. E-mail address: mme{at}itsa.ucsf.edu
3 Abbreviations used in this paper: MBL, mannose-binding lectin; CRD, carbohydrate recognition domain; MASP, MBL-associated serine protease; LOS, lipooligosaccharide; Opa, opacity protein; PorA, porin protein A; PorB, porin protein B; OMC, outer membrane complex; SB, SuperBlock; GlcNAc, N-acetyl-D-glucosamine; Gal, D-galactose; HGS, hypogammaglobulinemic serum.
4 S. J. Lund, D. J. Carlson, and G. L. Gorby. Gonococcal opacity-associated proteins promote variable degrees of bacterial invasion of human fallopian tube organ culture. Submitted for publication.
Received for publication December 30, 2002. Accepted for publication January 12, 2004.
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References |
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2-macroglobulin. J. Immunol. 168:4178.
2-glycoprotein I, and the Ba fragment of B2. J. Immunol. 136:3407.[Abstract]
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 S Lear, E Eren, J Findlow, R Borrow, D Webster, and S Jolles Meningococcal meningitis in two patients with primary antibody deficiency treated with replacement intravenous immunoglobulin. J. Clin. Pathol., November 1, 2006; 59(11): 1191 - 1193. [Abstract] [Full Text] [PDF]
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G. Madico, J. A. Welsch, L. A. Lewis, A. McNaughton, D. H. Perlman, C. E. Costello, J. Ngampasutadol, U. Vogel, D. M. Granoff, and S. Ram The Meningococcal Vaccine Candidate GNA1870 Binds the Complement Regulatory Protein Factor H and Enhances Serum Resistance J. Immunol., July 1, 2006; 177(1): 501 - 510. [Abstract] [Full Text] [PDF]
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M. C. Schneider, R. M. Exley, H. Chan, I. Feavers, Y.-H. Kang, R. B. Sim, and C. M. Tang Functional Significance of Factor H Binding to Neisseria meningitidis. J. Immunol., June 15, 2006; 176(12): 7566 - 7575. [Abstract] [Full Text] [PDF]
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H. Jarva, S. Ram, U. Vogel, A. M. Blom, and S. Meri Binding of the Complement Inhibitor C4bp to Serogroup B Neisseria meningitidis J. Immunol., May 15, 2005; 174(10): 6299 - 6307. [Abstract] [Full Text] [PDF]
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T. Sprong, A.-S. W. Moller, A. Bjerre, E. Wedege, P. Kierulf, J. W. M. van der Meer, P. Brandtzaeg, M. van Deuren, and T. E. Mollnes Complement Activation and Complement-Dependent Inflammation by Neisseria meningitidis Are Independent of Lipopolysaccharide Infect. Immun., June 1, 2004; 72(6): 3344 - 3349. [Abstract] [Full Text] [PDF]
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), GlcNAc (•), or Gal (
). As a positive control for monosaccharide inhibition of MBL binding, separate microtiter wells were coated with mannan (crosses) and incubated with MBL that had been preincubated with mannose. Values are the percent binding of MBL that was preincubated with sugar relative to that of MBL preincubated with buffer. Error bars denote SD.
29 kDa) on 15029. Porin: MBL bound to one of the two bands bound by 4D11 on 15501. MBL bound the same band on 8026 as mAb 4D11 and bound to one of the two bands bound by 4D11 on 15029.
