Antibody-dependent bactericidal activity is important for protection against meningococcal infection (49
). The AP plays an important role in amplifying C3 deposition on the bacterial surface. C3 activation represents the convergence of the classical, lectin and APs. The subsequent activation of the terminal complement components can lead to C5b-9 insertion into the membrane of gram-negative pathogens, resulting in complement-dependent killing. Deficiencies of the terminal complements (C5 through C9) and AP components such as factor D and properdin predispose individuals to invasive meningococcal infections (11
). Properdin deficiency is rare, but individuals with properdin deficiency are predisposed to severe invasive meningococcal infections, often with a higher mortality than normal individuals (11
). Both N. meningitidis
and N. gonorrhoeae
have evolved several intricate mechanisms to evade complement. The previously reported ability of N. gonorrhoeae
to bind to properdin and activate complement (6
) would provide a distinct disadvantage to the bacteria in vivo
Two important observations have emerged from this study. First, properdin is critical for optimal AP-dependent C3 deposition on the pathogenic Neisseriae and second, native properdin does not bind directly to any of the strains of N. meningitidis
or N. gonorrhoeae
tested and does not initiate AP activation when preincubated with Neisseriae. Together, these results strongly suggest that properdin acts to enhance AP activation on Neisseriae through the “conventional” mechanism – i.e., by stabilizing AP C3 convertases. These studies emphasize the importance of using native properdin for functional assays. The only forms of properdin reported in serum are dimers, trimers and tetramers (P2
, respectively) that are present in the ratio of 26:54:20 (2
). Higher order oligomers (aggregates of properdin) that form when properdin is freeze-thawed (as seen in commercial preparations), or with prolonged storage of native properdin, can promote fluid phase complement activation and consumption when added to serum (2
). Furthermore, a recent study shows that higher order oligomers can bind non-specifically to live cell surfaces where they promote complement activation (45
There was a wide variation among strains in their ability to bind to C3 ( and ), which could reflect differences in the ability of strains to activate the AP and/or availability of targets for C3 on the bacterial surface. An important observation was that expression of groups A, B, C and W-135, but not group Y, capsules all resulted in less AP activation (shaded graphs in the upper panels of ) as evidenced by less C3 deposition compared to their isogenic unencapsulated mutants (grey shaded histograms in the lower panels of ). The mechanism of AP suppression by select meningococcal capsular polysaccharides is currently the subject of a separate investigation.
The importance of properdin in promoting AP activation on Neisseriae was shown using a mAb against properdin that blocked its function and resulted in a marked decrease in C3 deposition on all meningococci and gonococci tested. These findings were confirmed by an independent method where C3 deposition on Neisseriae was enhanced when properdin-depleted serum was reconstituted either with native or unfractionated properdin.
It is noteworthy that preincubating N. gonorrhoeae stra in F62 with the unfractionated commercial properdin preparation followed by the addition of properdin-depleted serum (, right graph, “Unfractionated P → P-depleted serum-Mg/EGTA”) resulted in increased levels of C3 deposition compared to preincubation of strain F62 with Pn (void volume eluate of a molecular sieve column), shown in (shaded green histogram, lower right graph). This may be explained by lower amounts of Pn binding to F62 relative to unfractionated properdin (). High order oligomers present in commercial properdin preparations may have also been retained by the molecular sieve column. Potentially, these retained aggregates in Pn may have influenced binding to strain F62 and consequent C3 deposition that simulated C3 binding brought on by unfractionated commercial properdin. This may have also influenced higher C3 binding by unencapsulated Group A and B N. meningitidis by the Pn preparation, which enhanced C3 deposition when added either before P-depleted serum-Mg/EGTA (, shaded green histogram in the two upper graphs in the Pn column) or together with P-depleted serum-Mg/EGTA (, red histograms in the same graphs).
It is clear that certain complement activator surfaces such as zymosan bind to purified native properdin (). Other complement activator surfaces such as rabbit erythrocytes have also been reported to bind to commercially available unfractionated properdin (6
), although a recent study shows that the native properdin forms do not (45
). Studies that define ligands or functions of properdin using unfractionated properdin that may contain aggregates need to be interpreted with caution. In addition, other molecules such as serum amyloid P component have been reported to interfere with the ability of properdin to bind to surfaces (52
) and may limit the ability of properdin to initiate complement activation in the context of serum.
In conclusion, our results emphasize the importance of using native forms of properdin to analyze the biological and functional roles of this molecule. The ‘conventional’ mechanism of properdin function, which is to bind to and stabilize AP C3 convertases, remains the principal mechanism of function on the surface of Neisseria. The lack of this essential mechanism may explain why properdin-deficient individuals are more susceptible to meningococcal infections.