must survive complement-mediated killing to cause invasive disease. The classical pathway is required to initiate bactericidal activity on Neisseriae
). The importance of Abs in protection against invasive disease has been detailed in elegant studies by Goldschneider et al. (33
). Once the classical pathway is set into motion, the positive feedback loop of the alternative pathway augments C3b deposition on bacteria. Persons deficient in alternative pathway activators such as properdin and factor D (1
) are predisposed to invasive meningococcal disease.
Down-regulation of the alternative pathway may be beneficial for meningococci to survive in the human host. Almost all isolates recovered from the bloodstream or cerebrospinal fluid are encapsulated and capsular polysaccharide is required for high-level serum resistance (37
). However, isolates recovered from the nasopharynx are often shown to be unencapsulated (39
). Encapsulation may actually hinder bacterial invasion of epithelial cells (43
), and it has been suggested that meningococci may need to down-regulate capsule expression to traverse the epithelial barrier (44
). Meningococci, therefore, use redundant mechanisms to evade complement at various stages of disease pathogenesis. Binding of the alternative pathway down-regulatory molecule, fH to microbial surfaces is used by several bacterial species to regulate the alternative pathway of complement (reviewed in Ref. 46
). The major classical pathway regulator, C4b-binding protein (C4bp) may also be important and we have recently demonstrated binding of C4bp to meningococcal PorA under hypotonic conditions, which may constitute a separate mechanism of complement evasion by N. meningitidis
). The other pathogenic Neisseria
species, N. gonorrhoeae
, uses both C4bp and fH to its advantage to regulate complement activation on its surface. Sialylation of the lacto-N
-neotetraose LOS species enhances fH binding to N. gonorrhoeae
). In addition, several gonococcal strains that express the porin 1A (Por1A) molecule bind fH, which confers serum resistance in the absence of LOS sialylation (25
). In this study, we report binding of fH to the meningococcal lipoprotein GNA1870. All meningococcal isolates that have been tested express GNA1870 (6
fH binds to selected polyanions, such as heparin, dextran sulfate, chondroitin sulfate A, and carrageenan (types III and IV), but not others such as chondroitin sulfate C, keratan sulfate, hyaluronic acid, colominic acid (bacterial polysialic acid), or polyaspartic acid (48
). An important observation we made was that fH did not bind to the polyanionic capsular polysaccharides expressed by any of the five major meningococcal serogroups, based on the finding that fH bound equally well to wild-type strains and their unencapsulated (siaD lst
) mutants (). Serogroup B capsular polysaccharide regulates the alternative complement pathway (50
), but our observations suggest that this effect is not attributable to enhanced fH binding.
We previously reported an association between expression of meningococcal PorB3 and the ability to bind to fH (51
). We speculated that PorB3 might be the acceptor for fH, based on sequence similarities between meningococcal PorB3 and gonococcal Por1A (52
), the latter having been shown to bind fH (25
). In this study, we have identified a ligand for fH on N. meningitidis
. Of the five diverse strains of N. meningitidis
that we examined for fH binding, strains C2120 and W171 do not express PorB3 (they express PorB2), yet they still bind fH, suggesting that fH binds to a target(s) other than PorB3. Using Por deletion mutants, as well as allelic replacement (), we confirmed that fH did not bind to either Por molecule on intact N. meningitidis
strain H44/76. Although binding of fH to PorB3 purified from strain H44/76 has been reported previously using ELISA (53
), our results indicate that a fH-PorB3 interaction may not occur on live bacteria.
The ~29 kDa molecule in outer membrane preparations of H44/76 that bound fH in Western blots was identified as GNA1870 by MALDI-TOF MS and peptide mass fingerprinting analysis. Attempts at N-terminal Edman sequencing of this protein failed, consistent with the protein N terminus being modified or blocked, in this instance by palmitoylation (9
). A limitation in the definitive identification of ligands using Western blotting is that surface molecules that are out of context of the membrane and/or denatured could bind fH via regions that may otherwise be cryptic on intact bacteria, thereby yielding false positive results. In addition, binding sites that require a conformational or three-dimensional structure will not be identified by this method. Therefore, we used two independent approaches () to confirm that GNA1870 was the receptor for fH on intact meningococci.
We observed fH binding to all three GNA1870 variant classes, despite sequence diversity among these molecules (). This observation suggests that retaining the ability to bind fH may be beneficial to bacteria in vivo. The lower OD410 nm
reading seen with the variant 3 protein may reflect differences in recognition of the proteins by the polyclonal variant 1–3 anti-GNA1870 Ab (8
), and does not necessarily imply decreased binding of this protein to fH (). The N-terminal ~100 aa of all sequenced GNA1870 molecules bears maximum sequence homology, raising the possibility that the fH binding motif may reside in this region.
Differences in fH binding were attributable to differences in expression levels of GNA1870, and not to differences in amino acid sequences. A comparison of GNA1870 expression and fH binding among strains H44/76, W171, and Y2220 showed a correlation between levels of GNA1870 expression and fH binding (). The polyclonal anti-GNA1870 antiserum used in this assay binds better to variant 2 GNA1870 than variant 1 proteins (8
), thereby making it unlikely that there was preferential detection of H44/76 GNA1870 (variant 1). Furthermore, W171 and Y2220 were chosen for this experiment because their gna1870
genes are identical, allowing for a symmetric and unbiased comparison of GNA1870 expression. Y2220 expresses low amounts of GNA1870, and may bind fH but in amounts below the threshold of detection by flow cytometry. Low levels of GNA1870 expression and concomitant low or no fH binding are not unique to serogroup Y strains; we identified two other serogroup B strains of N. meningitidis
(2996 and NMB) that express low levels of GNA1870 (6
) and did not bind fH. We constructed a GNA1870 deletion mutant in strain 2996 and observed an increase in serum sensitivity (data not shown). This result suggests that even low levels of factor H binding (below the threshold of detection by flow cytometry) may diminish serum killing. This finding may have implications for the use of GNA1870 as a vaccine candidate because curtailing fH binding by specific immune Ab () will enhance killing, even by normal serum.
It is noteworthy that the DNA sequence spanning the region between the gna1870
ORF and the gene 3′ to gna1870
in Y2220 and W171 are identical, which suggests that differential protein expression is not because of differences in promoter sequences identified previously (9
). Y2220 is capable of high-level GNA1870 expression, as evidenced by the observation that Y2220 siaD lst H44/76GNA1870+
expressed similar amounts of GNA1870 as H44/76, the latter having been shown to express high levels of GNA1870 (7
). The factor(s) responsible for variable GNA1870 expression among meningococcal strains is not clear.
Both capsulated and unencapsulated bacteria showed heightened serum resistance when they expressed GNA1870 and bound fH. Capsular polysaccharide is a key determinant of high-level serum resistance in meningococci (38
). The ability to bind fH provides an additional level of protection to encapsulated bacteria against complement-mediated killing. The boosted level of serum resistance seen with encapsulated strains that bind fH could be critical for bacterial survival in the bloodstream, where high levels of complement are present. Although bacteria at mucosal surfaces encounter lower levels of complement (54
), colonizing meningococci (which are often unencapsulated and potentially more serum sensitive) need to evade complement to successfully inhabit the nasopharynx.
The ability of bacterial surface-bound fH to regulate the C3-amplification loop of the alternative pathway of complement was illustrated by increased C3 binding to an isogenic mutant of strain H44/76 that lacked GNA1870 (). It is noteworthy that the GNA1870-deletion mutant bound slightly less IgG and C4 than its parent, suggesting that normal human serum may contain Abs directed against this protein. Despite less C4 binding to the mutant strain (and consequently diminished activation of C3 via the classical pathway of complement), we observed more C3 activation on this strain, which is consistent with uninhibited activation of C3 by the alternative pathway in the absence of fH binding.
In conclusion, we have demonstrated an important function for GNA1870, an immunogenic protein under investigation as a broadly effective meningococcal vaccine candidate. In addition to activating the classical pathway of complement in their own right, Abs directed against GNA1870 could promote bacterial killing and opsonophagocytosis by diverting fH away from the bacterial surface. This potential dual mechanism of bacterial killing makes GNA1870 an attractive vaccine candidate. One reason for vaccine failures is the selection of bacteria that lack the target Ag. Escape mutants that do not express GNA1870 because of selection by Ab pressure would be at a significant disadvantage because they would be more susceptible to complement-dependent killing. Our findings provide a strong rationale for the use of GNA1870 as one component of a meningococcal vaccine. We suggest referring to GNA1870 as fHBP to reflect the important function of this protein.