In addition to its pivotal role in protection against Neisserial infections (67
), complement also plays an important role in innate immune defenses against several other bacterial pathogens. Over 50 years ago, Roantree and Rantz reported that gram-negative bacteria isolated from the bloodstream were almost always resistant to the killing activity of complement (68
). Defects in antibody and/or early components of the classical pathway that impair opsonophagocytosis predispose to recurrent respiratory infections and sepsis caused by bacteria such as Haemophilus influenzae
and Streptococcus pneumoniae
). The link between deficiencies of the alternative and terminal pathways of complement and recurrent, disseminated meningococcal disease is well established (69
Previous studies have shown that the classical pathway of complement is essential to mediate complement-dependent killing of N. gonorrhoeae
). Gonococcal strains that are otherwise sensitive to killing by non-immune normal human serum (all pathways of complement intact) survive when the classical (and lectin) pathway is blocked with Mg/EGTA-NHS (to selectively chelate Ca2+
) or with serum that lacks C1q (35
), while permitting activation of the alternative pathway (34
). However, such strains can be killed when the alternative pathway alone is selectively blocked, as with factor B-depleted serum (35
). N. gonorrhoeae
have evolved several mechanisms to resist killing by human complement. Effective strategies used by N. gonorrhoeae
to escape killing by complement include binding to human complement inhibitors such as factor H and C4BP through their PorB molecule (43
). Sialylation of gonococcal LOS that expresses the lacto-N
-neotetraose structure also enhances factor H binding to bacteria.
The rapid and widespread development of antibiotic resistance by N. gonorrhoeae
) has severely limited therapeutic options and there is an urgent need to develop safe and effective vaccines that prevent gonococcal carriage and disease. Antibody-based vaccines against N. gonorrhoeae
could depend on efficient complement activation to either mediate direct killing through membrane attack complex insertion or promote opsonophagocytosis in order to protect the host. To effectively activate complement on the bacterial surface, vaccine induced antibodies need to overcome the complement inhibitory effects of molecules such as factor H and C4BP that bind to the gonococcal surface. mAb 2C7 kills gonococci that inherently resist killing by complement ( and Ref. (52
)) via any of several mechanisms (31
) and is therefore an attractive vaccine candidate. Antibodies elicited by immunization of mice with a peptide that mimics the 2C7 epitope, bind the 2C7 epitope on gonococcal LOS and kill gonococci whose LOS displays the epitope (31
). Binding of mAb 2C7 to gonococcal LOS requires expression of LgtG, which is encoded by lgtG
that contains a poly-C tract and may therefore be subject to phase variation (49
). Nevertheless, over 90% of minimally passaged clinical isolates of N. gonorrhoeae
bind to 2C7 (72
) suggesting an important, but undefined role for lactose substitution of the HepII chain (a requirement for 2C7 epitope expression (29
)) of gonococcal LOS.
A novel finding in this study was the requirement of the alternative pathway and in particular properdin for specific antibody-mediated complement-dependent killing of N. gonorrhoeae
strains that bind C4BP. Strains that bind C4BP and limit classical pathway activation require recruitment of the alternative pathway to assemble sufficient C3/C5 convertases for subsequent membrane attack complex (C5b-9) and bacterial killing. In the absence of the alternative pathway or when properdin is blocked, mAb 2C7 deposits only a small amount of C3 on strains such as FA1090, 442089 and 15253 because these strains actively regulate the classical pathway through recruitment of C4BP to their surfaces. However, an intact alternative pathway appears to effectively amplify the C3b that ‘breaks through’ the barrier posed by C4BP and effect bacterial killing. The stabilizing effects of properdin on the alternative pathway C3 convertases are crucial to amplify C3 deposition and mediate bacterial killing. On the other hand, strains that resist complement by mechanisms independent of C4BP binding and do not actively cleave C4b or dissociate the classical pathway convertase (C4b,2b according to a newly proposed nomenclature (32
)), do not require properdin for specific antibody-mediated bactericidal activity.
Importantly, the role of properdin in facilitating antibody-dependent killing was not restricted to murine mAb 2C7 but also extended to specific immune human serum that contained antibodies directed against the 2C7-specific LOS epitope. These findings have implications for antibody-based vaccine development because PMNs are a major reservoir and source of properdin (37
). In addition, properdin is synthesized by primary cervical epithelial cells (39
) and would also be exuded into the cervical lumen from serum. The concomitant presence of neutrophils is likely to ensure high levels of properdin that may enhance complement activation on gonococci. Enhanced complement activation in the vicinity of PMNs may also facilitate opsonophagocytic disposition of organisms. As shown by Braconier et al (73
), phagocytic killing was reduced in Streptococcus pneumoniae
serotype 23F due to defective opsonization in the presence of P-deficient sera. Addition of native properdin to the P-deficient sera restored opsonization of S. pneumoniae
serotype 23F by human granulocytes (73
) showing the significance of properdin in phagocytosis. Indeed, a recent report showed that PMNs stimulated by cytokines such as TNF-α activate the alternative pathway of complement and result in C3 fragment deposition on these cells (74
). Properdin secreted by PMNs was detected on cell surfaces and it was proposed that the cell-bound properdin could serve as a focus for additional activation of the alternative pathway. Complement activation in turn resulted in further activation of PMNs, which was accompanied by an increases in CD11b expression and the oxidative burst (74
). The presence of classical pathway activation by specific antibodies, coupled with this “positive feedback loop” involving PMNs and the alternative pathway could prove important in curtailing gonococcal infection by vaccine-elicited antibody.
In conclusion, we have demonstrated that both human and murine antibodies that are directed against an LOS epitope that is currently being investigated as a potential vaccine candidate (31
), as well as specific anti-OMV antibodies more generally, require a functional alternative pathway and in particular properdin, to kill serum-resistant gonococcal strains that bind to the complement inhibitor C4BP. Strains that do not bind to C4BP are efficiently killed by specific antibodies via the classical pathway alone and do not require properdin. These findings provide insights into how complement is activated on N. gonorrhoeae
by specific antibody and also shed light on the requirements for effective complement-dependent bactericidal activity by potential vaccine candidates.