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* Centre for Molecular Microbiology and Infection, Imperial College London, London, United Kingdom;
National Institute for Biological Standards and Control, Potters Bar, United Kingdom; and
MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A variety of microbes avoid complement-mediated killing by recruiting fH to their surfaces including parasitic and viral pathogens such as Echinococcus granulosus, Onchocerca volvulus, and HIV (6, 7, 8). fH is also used by a number of bacteria to promote their survival in the host in the face of an immune response. fH binds to OspE, an outer membrane lipoprotein expressed by the spirochete Borrelia burgdorferi, the causative agent of Lyme disease (9, 10). Furthermore the M protein of Group A Streptococcus and YadA of Yersinia enterocolitica both recruit fH leading to reduced opsonophagoctosis but not complement-mediated lysis (11, 12). Neisseria gonorrhoeae, an important cause of sexually transmitted disease, also recruits fH to its cell surface. Isolates of N. gonorrhoeae can be classified as serum sensitive or serum resistant (SR) on the basis of their resistance against complement-mediated killing (13). fH appears to bind to two distinct targets on N. gonorrhoeae (14). The bacterium incorporates exogenous 5'-cytidine monophospho-N-acetyl neuraminic acid (CMP-NANA) into its LPS as sialic acid (15, 16), which can recruit fH to the cell surface (17). However, even in the absence of CMP-NANA some gonococcal strains are SR. This is mediated by binding of fH to a subtype of Por1, the most abundant outer membrane protein. N. gonorrhoeae can express one of two alleles of porin (Por) 1, Por1A or Por1B. Strains expressing Por1A are SR through binding fH, whereas serum-sensitive strains express Por1B instead. Examination of strains expressing hybrids of Por1A and Por1B indicates that fH binds to surface exposed loop 5 of Por1A (18).
An effective complement system is critical for resistance against infections caused by the closely related pathogen Neisseria meningitidis. This is evident from observations of individuals lacking components of the MAC who have between 1,000- and 10,000-fold increased lifetime risk of developing disseminated meningococcal infection compared with the general population (19). The role of the AP in resistance against meningococcal infections is highlighted by the increased susceptibility to neisserial infections in patients lacking properdin, which stabilizes the AP C3 convertase (19). Furthermore individuals with polymorphisms in the promoter of the gene encoding fH have lower serum fH levels and are at increased risk of N. meningitidis infection (20).
N. meningitidis has several mechanisms to avoid lysis by the complement system, including sialylation of its LPS and expression of a polysaccharide capsule (21). Strains which express truncated LPS isoforms or lack a capsule are highly susceptible to complement-mediated lysis, and are markedly attenuated in animal models of bacteraemia (22, 23, 24, 25). The serogroup classification of N. meningitidis is based on the chemical composition of its polysaccharide capsule. Most infections in industrialized countries are caused by serogroup B and C strains (26), which both express capsules composed of polysialic acid, while epidemic disease in subSaharan Africa is usually caused by serogroup A strains which produce a capsule that consists of a polymer of acetylated mannosamine phosphate (27).
Although the binding of fH to N. gonorrhoeae has been extensively characterized, the role of fH in the biology of meningococcal infection has not been investigated. Here, we characterize the basis of fH binding to the meningococcus and define the consequences of this interaction, particularly in relation to resistance against complement-mediated lysis, the most critical aspect of innate immunity against this important human pathogen.
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Materials and Methods |
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N. meningitidis was grown on brain heart infusion medium supplemented with 5% Levanthal’s base at 37°C in the presence of 5% CO2. Antibiotics were added as required at the following concentrations: erythromycin, 2 µg/ml; tetracycline, 2.5 µg/ml; kanamycin (kan), 75 µg/ml. MC58, H44/76 (multilocus sequence typing, ST-32; electrophoretic type, ET-5 (
http://pubmlst.org
) and 528 (ST-18) are serogroup B isolates of N. meningitidis, Z2491 (ST-4, subgroup IV) and F6124 (ST-5, subgroup III) are serogroup A strains, and the serogroup C isolates studied were FAM20 (ET-37) and 90/18311 (ST-11, ET-37) (28). The Streptococcus pneumoniae isolate, D39, and the corresponding mutant lacking PspC were provided by Dr. J. Yuste (University College, London, U.K.) and were grown on blood agar plates.
The construction of MC58
siaD, MC58lst, and MC58siaC has been described previously (29, 30). siaD encodes the enzyme polysialyl transferase that is required for the polymerization of sialic acid during the biosynthesis of serogroup B capsule (31), while Lst is the LPS-specific sialyl transferase (32). The siaC gene is necessary for sialic acid biosynthesis (31); mutants lacking siaC are unable to express a capsule or sialylate their LPS without exogenous CMP-NANA. The MC58porA and MC58porB mutants were derived by insertional inactivation of the genes with a cassette encoding kan resistance. Primers (porA forward, 5'-ATGCGAAAAAAACTTACCGCCCTC-3'; porA reverse, 5'-AATGAAGGCAAGCCGTCAAAAACA-3'; porB forward, 5'-GCGAAGCTTCCCTGATTGCCCTGACTTTG-3'; porB reverse primer, 5'-GCGAAGCTTTGTGGCGCAGACCGAC-3') were designed based on the genome sequence of N. meningitidis strain MC58 to amplify porA and porB from N. meningitidis. The PCR products were ligated into pCR2.1 TOPO (Invitrogen Life Technologies), and the constructs were verified by restriction endonuclease digestion. A kan-rpsL cassette was amplified using primers with KpnI recognition site at the 5' ends, and the product was inserted into the porA gene at a unique KpnI site in the open reading frame. Another pair of primers with BbvCI sites at the 5' ends was used to amplify the kan resistance cassette from Tn5, which was ligated into a unique BbvCI site in porB. The porAkan/rpsL and porBkan constructs were amplified by PCR, and the products were used to transform N. meningitidis by standard methods. Recombinants were selected on medium containing kan, and allelic replacement was confirmed by PCR and Southern blot analysis in back-crossed strains (data not shown).
Preparation of fH-depleted human plasma
Pooled citrated human plasma (30 ml; HD Supplies) was dialyzed at 4°C against 10 mM HEPES, 140 mM NaCl, 0.5 mM EDTA (pH 7.4) (running buffer), and the salt concentration was raised to 1 M to prevent C1q depletion. A 25-ml MRC OX23-Sepharose column (33) was equilibrated in running buffer, and the plasma run through at a rate of 10 ml/h. The unbound protein was collected in 1-ml fractions, and the protein concentration in each fraction was measured by reading the OD at 280 nm. Forty fractions (1 ml each) were pooled and dialysed against the running buffer, and aliquots were frozen rapidly in liquid nitrogen then stored at –80°C. The plasma was diluted from 30 to 40 ml during this procedure. fH was then eluted from the column with 50 ml of 3 M MgCl2, adjusted to pH 6.8–7.0 by the addition of Tris base.
The extent of fH depletion from the plasma was >99.5% as assessed by ELISA as follows. Microtiter plates (MaxiSorp; Nunc) were coated with monoclonal anti-human fH MRC OX23 (50 µl/well) and purified IgG in 0.1 M Na2CO3 (pH 9.6) 2.5 mg/well (34). Plates were left for 1 h at 20°C, washed four times with PBS, and any remaining nonspecific binding sites were blocked with PBS-0.1% Tween20 (PBS-T) for 2 h at room temperature. Samples of plasma were assayed in doubling dilutions from 1/100 to 1/25,600 in PBS-T. Serially diluted samples (50 µl) were added to each coated well and incubated for 1 h at 20°C. fH of a known concentration (diluted 1/2 to 1/512, with a starting concentration of 10 µg/ml) was included as a standard and treated exactly as the plasma samples. The wells were washed four times in PBS-T, and 50 µl of rabbit anti-human fH (MRC Immunochemistry Unit) at a dilution of 1/32,000 in PBS-T was added to each well and incubated for 1 h at 20°C. The wells were washed again with PBS-T, then 100 µl of goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma-Aldrich) at 1/5000 dilution was added to each well. The plate was incubated as before, washed again in PBS-T, and developed with the substrate p-nitrophenyl phosphate (Sigma-Aldrich). The plate was read at 405 nm in a microtiter plate reader (Multiscan Ascent; Labsystems) after optimum color development had occurred.
Flow cytometry
FACS analysis was performed to quantify the binding of fH to N. meningitidis. Bacteria were grown overnight on solid medium and then harvested into PBS. The number of bacteria was estimated by measuring the OD at 260 nm of an aliquot of the bacterial suspension lysed in 200 mM NaOH/1% SDS (v/w). Bacteria (108 CFU) were fixed in 3% paraformaldehyde for 2 h at room temperature, washed three times in PBS, and stored at –80°C in PBS/15% glycerol. To estimate fH binding, 108 cells were resuspended in 10 µl of normal human serum (NHS) or alternative sources of fH for 30 min at 37°C. After two washes in PBS-T, binding was detected following incubation with a murine anti-human fH mAb (MRC OX23 or MRC OX24, 0.1 µg/ml, (34), or a polyclonal goat anti-human fH (1/200 dilution in PBS; Calbiochem) for 30 min at 37°C in 50 µl of PBS. An anti-human Talin IgG1 mAb (Sigma-Aldrich) and polyclonal goat anti-murine LAMP sera (Jackson ImmunoResearch Laboratories) were used as control primary Abs. The cells were washed twice in PBS-T, then resuspended in 50 µl of PBS with a polyclonal donkey anti-mouse IgG-FITC conjugate (1/200 dilution in PBS; Jackson ImmunoResearch Laboratories) or a polyclonal donkey anti-goat IgG FITC conjugate (1/200 dilution; Jackson ImmunoResearch Laboratories) and incubated for 30 min on ice. The cells were examined in a flow cytometer (Calibur FACScan; BD Biosciences), and at least 4 x 104 events were recorded. fH binding to the cell surface was expressed as the percentage of fluorescent bacteria normalized using results from negative controls. Binding of fH to live bacteria was performed as follows: Bacteria (2 x 109 CFU) were incubated with 20 µl of purified fH (870 µg/ml in 10 mM K2PO4, 100 mM KCl, 0.5 mM EDTA (pH 7.4)) or alternative source of fH (serum from a C7-deficient individual or heat-inactivated serum) for 30 min at 37°C. After two washes, the number of bacteria was estimated by measuring the OD at 260 nm, and the bacteria were fixed in 3% paraformaldehyde for 15 min, washed, and analyzed by FACS.
Far Western blot analysis
Whole cell lysates of N. meningitidis were prepared by growing bacteria overnight on solid medium, harvesting around 109 CFU in 400 µl PBS, and centrifugation at 15,700 x g for 2 min. The pellet was resuspended in 100 µl of SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 100 µM 2-ME, 10% glycerol, 0.1% bromophenol blue). Outer membrane vesicles (OMVs) were prepared as described previously (35). Proteins were separated under reducing conditions by SDS-12% PAGE, then the proteins transferred to polyvinylidene difluoride membranes (Millipore). After nonspecific binding had been blocked with PBS containing 5% milk, the blots were incubated for 2 h with purified fH (final concentration of 3.36 µg/ml in PBS-T/0.5% milk) or NHS as fH source (final dilution of 1/25 in PBS-T/0.5% milk), then incubated with MRC OX24 (86 ng/ml) in PBS-T/0.5% milk. Binding of MRC OX24 was detected using a donkey anti-mouse-IgG HRP-conjugated secondary Ab (Invitrogen Life Technologies).
Cofactor activity assay of fH bound to N. meningitidis and serum sensitivity assays
The cofactor activity of fH was assessed by analyzing fI-mediated conversion of C3b to iC3b. Briefly, fixed MC58 (109 CFU) were preincubated with fH (870 µg/ml) or with buffer alone (10 mM K2PO4, 100 mM KCl, 0.5 mM EDTA (pH 7.4)) for 30 min at 37°C. After three washes, the bacteria were incubated with C3b (1 µg) and fI (0.4 µg) in 20 mM phosphate buffer (pH 6) in a final volume of 30 µl for 30 min at 37°C. fH was added directly to samples as a positive control for the assay, and reactions also were performed in the absence of fI as a negative control. The samples were separated under reducing conditions by SDS-PAGE, transferred to membranes, and subjected to Western blot analysis using a polyclonal rabbit anti-C3c Ab (1/1000 v/v; Dako) as the primary Ab, which was detected with a HRP-conjugated donkey anti-rabbit IgG (1/10,000 v/v; Dako).
For serum sensitivity assays, bacteria were grown overnight on solid medium, harvested into 400 µl of PBS, and centrifuged at 450 x g for 2 min to remove clumps. The supernatant was collected and spun at 15,700 x g for 2 min, the pellet was resuspended in 400 µl of PBS, and the number of bacteria was quantified as above. The bacteria (2 x 109 CFU) were centrifuged, resuspended in 20 µl of purified fH (870 µg/ml), then incubated for 30 min at 37°C, or with the same buffer without fH as a negative control. After two washes in PBS-T, bacteria were quantified and added to DMEM with glutaMAX (Invitrogen Life Technologies) at a concentration of 2 x 105 CFU/ml. The bacterial suspension was added to an equal volume of fH-depleted plasma already diluted 1/3 in DMEM (final dilution 1/6), then incubated at 37°C. Surviving bacteria were recovered by plating to brain heart infusion agar.
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Results |
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We used three experimental conditions to detect fH binding to the serogroup B N. meningitidis isolate MC58 by FACS. First, NHS was used as the source of fH, and cells were incubated with PBS as a negative control. Second, MC58 was incubated with fH-depleted plasma supplemented with purified fH added at physiological concentrations (500 µg/ml; normal range, 100–600 µg/ml), and the results were compared with those obtained with bacteria incubated with depleted plasma without added fH. Finally, we incubated bacteria with purified fH alone. Fig. 1 shows fH binding to MC58 after incubation with NHS, evaluated by FACS analysis with a polyclonal anti-fH Ab (Fig. 1A) or with the mAbs MRC OX23 (Fig. 1B) or MRC OX24 (Fig. 1C). Similar levels of binding were detected regardless of the Ab used; no binding was detected using either an irrelevant polyclonal sera (data not shown) or an isotype matched mAb (Fig. 1D). Binding was observed using fH-depleted plasma only when supplemented with additional fH (Fig. 2A), and when fH was provided as a purified protein (Fig. 2B). Thus, fH binds to N. meningitidis independent of other components of the complement cascade such as C3b. To exclude the possibility that the binding, we detected was the result of an artifact from fixing bacteria, we examined whether live N. meningitidis was able to bind fH. Live bacteria recruited fH regardless of whether purified fH, heat-inactivated serum or serum from a C7-deficient individual (which lacks bacteriolytic activity) was used as the source of fH (Fig. 2C).
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The LPS of N. gonorrhoeae can be sialylated in the presence of exogenous CMP-NANA if the strain expresses LPS of the appropriate immunotype (37) and the sialylated LPS modulates fH binding (17, 38). For serogroup B N. meningitidis, the situation is different as the bacterium synthesizes sialic acid, which is incorporated into the capsule as well as LPS. Therefore, we investigated the contribution of sialic acid to fH binding to MC58 as this strain expresses a polysialic acid capsule and LPS immunotype (L3, L7, L9) that can be sialylated (39). The extent of fH binding was examined in strains lacking a capsule (MC58siaD) or unable to synthesize sialic acid (MC58siaC). Following incubation in NHS, both MC58siaD and MC58siaC (Fig. 5A) exhibited similar levels of fH binding as the parental strain, MC58, regardless of which anti-fH mAb was used for detection. To exclude the possibility that fH binds to sialic acid on LPS, we assessed the binding to MC58lst, which lacks LPS sialylation (30). The lst mutant bound fH at wild-type levels (Fig. 5B), demonstrating that fH recruitment by the meningococcus is entirely independent of the presence of surface sialic acid. Binding of fH to live bacteria showed similar results (data not shown).
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To further characterize the basis of fH binding to N. meningitidis, Far Western blot analysis was performed. Whole bacterial cell lysates were separated by SDS-PAGE, transferred to membranes that were then incubated with purified fH. fH binding was assayed with MRC OX23 or MRC OX24, followed by an HRP-conjugated goat anti-mouse Ab for chemiluminescent detection. In lysates from N. meningitidis strains Z2491 and MC58, fH bound principally to a protein with an approximate molecular mass of 33 kDa (Fig. 5C); binding to a protein of the same molecular mass in serogroup C N. meningitidis was detected but was less marked despite equal loading of lysates (Fig. 5D); minor differences between strains were seen in the migration of the band reacting with fH. The specificity of the Far Western blot analysis was confirmed by incubating the membranes without fH or with an isotype-matched (anti-Talin IgG1) mAb instead of anti-fH mAbs (data not shown); the 33-kDa band was not detected under these circumstances. Furthermore, fH binding to S. pneumoniae in a PspC-dependent manner was detected with MRC OX23 (data not shown); PspC is the major fH receptor on the pneumococcus (40). To exclude the possibility that fH was binding to the most abundant protein, whole cell lysates were examined by Coomassie brilliant blue staining. As shown in Fig. 5D, no predominant band of 33 kDa was evident. Binding also was detected by Far Western blot analysis of OMVs from N. meningitidis (Fig. 5C), and was abolished if the lysates were exposed to proteinase K, demonstrating that the 33-kDa band is proteinaceous in nature (data not shown).
In N. gonorrhoeae, fH binds to a region of external loop 5 of an outer-membrane porin, Por1A (18). N. meningitidis can express two outer-membrane porins, PorA and PorB. PorA is a 42-kDa protein (41) (www.tigr.org, NMB1429) with 51% amino acid identity to Por1A, whereas PorB has a molecular mass of 35.6 kDa (42, 43) (NMB2039) and shares 81% amino acid identity with Por1A (Fig. 6A). Therefore, we hypothesized that N. meningitidis PorA or PorB is necessary for recruitment of fH to the bacterial surface. Insertional mutants of porA and porB were constructed in MC58 and examined for fH binding by FACS and Far Western blot analysis. The absence of expression of the PorA and PorB proteins was confirmed by SDS-PAGE of OMV preparations from each mutant (data not shown). No significant difference was observed in the amount of fH binding detected by FACS to MC58, MC58porA, and MC58porB (Fig. 6B). Furthermore, the 33-kDa protein band was still detected by Far Western blot analysis in the insertion mutants (Fig. 6C). On prolonged exposure, additional minor bands of the predicted molecular mass of PorA and PorB were not detected in lysates from the corresponding mutants, suggesting that fH binds to PorA and PorB, but to a lesser extent than the 33-kDa protein.
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Because fH is recruited by N. meningitidis, we next examined the role of fH in protection against complement-mediated killing. First, we determined whether fH bound to the bacterium retains its activity as a cofactor for fI-mediated cleavage of C3b into inactive fragments including iC3b. Cleavage of C3b results in the loss of the 109-kDa band (corresponding to C3b), and the appearance of the 40-kDa component of iC3b and was dependent on the presence of fI (Fig. 7A). Even in the absence of fH, fI was able to cleave C3b, although this was clearly enhanced when fH was added directly to assays. Increased production of iC3b also was detected when N. meningitidis was preincubated with fH but not with buffer alone, demonstrating that the binding of the regulator to the bacterial surface did not affect its ability to act as a cofactor for fI (Fig. 7A).
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100%,compared with bacteria preincubated with buffer alone or with an irrelevant protein, BSA. Therefore, fH recruitment to the bacterial surface contributes to the avoidance of complement-mediated lysis by N. meningitidis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We used two independent methods, FACS and Far Western blot analysis, to demonstrate that N. meningitidis recruits fH. To confirm the specificity of this interaction, binding was detected using any of three separate Abs, but not with irrelevant polyclonal sera or an isotype matched mAb. We confirmed the specificity of the Far Western blots by detecting fH binding to S. pneumoniae in a PspC-dependent fashion (40). Furthermore, we detected fH binding when it was provided either as serum or as a purified protein to live or fixed bacteria. Highest levels of binding were found when live bacteria were incubated in NHS, an environment that is relevant to understanding the pathogenesis of disseminated meningococcal infection. The finding that fH binding is saturable at plasma fH concentrations further supports the physiological significance of our findings.
We found that isolates of N. meningitidis bound fH regardless of their multilocus sequence type or serogroup. As the serogroup A capsule has a fundamentally different chemical composition to the polysialic acid capsule of serogroup B and C strains, it was unlikely that the capsule itself was responsible for the recruitment of fH to the bacterium. In support of this, we found that fH recruitment was not affected by deletion of siaD, which is required for capsule biogenesis. However, siaD mutants can still synthesize sialic acid, and this could have mediated fH recruitment. We excluded this possibility by demonstrating that a siaC mutant, which is defective for the production of sialic acid, also recruits fH at levels comparable to that of the wild-type strain. Thus, the capsule is neither required for nor inhibits the recruitment of fH to N. meningitidis.
The binding of fH to the related pathogen, N. gonorrhoeae, has been extensively characterized (17, 18, 38). We found that, unlike the gonococcus, binding of fH by N. meningitidis was independent of the presence of LPS sialylation; the lst mutant of N. meningitidis bound fH at comparable levels to the wild-type bacterium. A recent report (38) indicates that sialic acid on N. gonorrhoeae LPS is not the actual target of fH binding but instead acts to enhance the interaction with Por1A. Therefore, our findings are entirely consistent with those described with N. gonorrhoeae, because the meningococcus does not express Por1A.
We investigated whether the meningococcal porins, PorA or PorB, are the target of fH binding given the precedent of Por1A mediating binding to N. gonorrhoeae (18). Because the level of fH binding was not reduced in the porA or porB mutant, neither of these outer membrane porins appears to act as the sole receptor for fH on N. meningitidis. However, it is still possible that PorA and PorB recruit fH independently to the bacterial surface but that their functions are redundant. Far Western blot analysis did indicate that fH does bind to PorB (Fig. 6C), in keeping with previous findings with recombinant PorB (53). However, PorB does not appear to play a significant role in fH binding, because loss of porB did not alter overall fH recruitment to the bacterial surface as detected by FACS. Even though there is a degree of identity between meningococcal PorA and PorB with gonococcal Por1A over the sequence that mediates fH binding (18, 43), the regions in the meningococcal proteins contain additional amino acids (Fig. 6A) that might have a critical effect on the accessibility or the conformation of any potential fH binding site.
Far Western blot analysis indicated that a protein with an apparent molecular mass of 33 kDa binds to fH. Inspection of the sizes of bands detected in different isolates is consistent with there being allelic variants of the fH binding partner among circulating meningococcal strains (Fig. 5D). This is not surprising, because many structures expressed on the surface of N. meningitidis, especially those under immune selection, exhibit allelic variation (54). Interestingly, fH is recruited by immunogenic proteins in other bacteria such as M protein, PspC, and OspE of S. pyogenes, S. pneumoniae, and B. burgdorferi, respectively (9, 11, 40). N. gonorrhoeae Por1A also is the target of bactericidal Abs (14). Additionally, it has been shown recently that C4bp, another negative regulator of the complement system, binds to PorA (51), a major immunogenic protein on the surface of the meningococcus (55). Therefore, recruitment of negative regulators by Ags that elicit host immune responses might provide a mechanism to prevent deposition of complement components in a highly localized fashion; identification of the binding partner of fH on N. meningitidis might be informative about novel Ag(s) for inclusion in vaccines to prevent meningococcal disease.
To further understand the biological significance of fH recruitment by N. meningitidis, we examined the cofactor activity of fH bound to the bacterium. Although there have been several reports of cofactor activity when incubated with recombinant proteins (56, 57, 58, 59), there has been only one other example in which the presence of fH on the surface of a bacterium has been demonstrated to retain its ability to enhance fI-mediated cleavage of C3b (60). Furthermore, fH serum sensitivity assays were performed. We examined the protective effect of fH bound to the bacterium against complement-mediated bacteriolysis. fH has a dual role in serum sensitivity assays, because depletion of fH in serum can lead to a reduction in effective C3 levels through unrestricted activation of the AP; individuals who lack fH become functionally C3 deficient and develop membranous glomerulonephritis and hemolytic uremic syndrome through inappropriate complement deposition (61). Despite this, in every assay with fH-depleted plasma, there was sufficient active complement to kill between 80 and 100% of wild-type bacteria. Although we preincubated bacteria with concentrations of fH that were above physiological levels (870 µg/ml, normal range 100–600 µg/ml), the concentration of bacteria (5 x 109 CFU/ml) was much higher than found in disease in meningococcal bacteraemia (105–106 CFU/ml) (62, 63)). We found that preincubation of bacteria with fH significantly protected N. meningitidis from complement killing, with 100% increase in bacterial survival if they were preincubated with fH. N. meningitidis avoids complement-mediated lysis by expression of a polysaccharide capsule and LPS (23). However, during rounds of bacterial replication in vivo in the systemic circulation or at the mucosal surface, binding of fH also will provide the bacterium with an advantage for survival and represents an additional mechanism by which N. meningitidis avoids complement-mediated killing.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the Wellcome Trust and Meningitis Research Foundation. 
2 Address correspondence and reprint requests to Dr. Christoph M. Tang, Centre for Molecular Microbiology and Infection, Flowers Building, Armstrong Road, Imperial College London, London, SW7 2AZ, U.K. E-mail address: c.tang{at}imperial.ac.uk
3 Abbreviations used in this paper: CP, classical pathway; AP, alternative pathway; MAC, membrane attack complex; fH, factor H; fI, factor I; SR, serum resistant; CMP-NANA, 5'-cytidine monophospho-N-acetyl neuraminic acid; Por, porin; kan, kanamycin; OMV, outer membrane vesicle.
Received for publication October 28, 2005. Accepted for publication March 28, 2006.
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2
8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect. Immun. 66: 5939-5947. -2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae. J. Biol. Chem. 271: 28271-28276. 
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 T. Hallstrom, P. F. Zipfel, A. M. Blom, N. Lauer, A. Forsgren, and K. Riesbeck Haemophilus influenzae Interacts with the Human Complement Inhibitor Factor H J. Immunol., July 1, 2008; 181(1): 537 - 545. [Abstract] [Full Text] [PDF]
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P. T. Beernink and D. M. Granoff Bactericidal Antibody Responses Induced by Meningococcal Recombinant Chimeric Factor H-Binding Protein Vaccines Infect. Immun., June 1, 2008; 76(6): 2568 - 2575. [Abstract] [Full Text] [PDF]
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 A. K. Zaiss, M. J. Cotter, L. R. White, S. A. Clark, N. C. W. Wong, V. M. Holers, J. S. Bartlett, and D. A. Muruve Complement Is an Essential Component of the Immune Response to Adeno-Associated Virus Vectors J. Virol., March 15, 2008; 82(6): 2727 - 2740. [Abstract] [Full Text] [PDF]
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R. Casey, J. Newcombe, J. McFadden, and K. B. Bodman-Smith The Acute-Phase Reactant C-Reactive Protein Binds to Phosphorylcholine-Expressing Neisseria meningitidis and Increases Uptake by Human Phagocytes Infect. Immun., March 1, 2008; 76(3): 1298 - 1304. [Abstract] [Full Text] [PDF]
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J. A. Welsch and D. Granoff Immunity to Neisseria meningitidis Group B in Adults despite Lack of Serum Bactericidal Antibody Clin. Vaccine Immunol., December 1, 2007; 14(12): 1596 - 1602. [Abstract] [Full Text] [PDF]
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H. Smith, C. M. Tang, and R. M. Exley Effect of Host Lactate on Gonococci and Meningococci: New Concepts on the Role of Metabolites in Pathogenicity Infect. Immun., September 1, 2007; 75(9): 4190 - 4198. [Full Text] [PDF]
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J. Findlow, A. Holland, D. Martin, P. Oster, P. Balmer, and R. Borrow Inadequacy of Colominic Acid as an Absorbent Intended To Facilitate Use of Complement-Preserved Baby Rabbit Serum in the Neisseria meningitidis Serogroup B Serum Bactericidal Antibody Assay Clin. Vaccine Immunol., May 1, 2007; 14(5): 556 - 561. [Abstract] [Full Text] [PDF]
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G. Madico, J. Ngampasutadol, S. Gulati, U. Vogel, P. A. Rice, and S. Ram Factor H Binding and Function in Sialylated Pathogenic Neisseriae is Influenced by Gonococcal, but Not Meningococcal, Porin J. Immunol., April 1, 2007; 178(7): 4489 - 4497. [Abstract] [Full Text] [PDF]
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