Human L-ficolin, a recognition molecule of the lectin activation pathway of complement, activates complement by binding to pneumolysin, the major toxin of Streptococcus pneumoniae

Abstract

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Figure 1. C1q binding to recombinant PLY exclusively occurs when either a PLY specific antibody or IgG3 or IgM binds to PLYin a non-specific fashion.

Microtiter ELISA plates were coated either with PLY or immune complexes (positive control) formed by incubation of HSA and a polyclonal goat anti-HSA antibody to capture C1q from NMS (A) or NHS (B). Purified human C1q was serially diluted in BBS and incubated for 1 hr at RT in ELISA plates pre-coated with PLY. Purified C1q showed strong binding to PLY in the presence of a specific anti-PLY antibody, but not in the absence of immunoglobulins (C). Non-specific polyclonal antibodies of either all isotypes or isotype-specific preparations of IgM or IgG subclasses were serially diluted and incubated in PLY-coated ELISA plates. PLY binds non-immune IgM and IgG3, but does not bind IgG1 or IgG2 (D). Results are means (±SEM) of 3 independent experiments.

Figure 2. Deposition of C3 activation products, C3b and iC3b, on PLY.

Serial dilutions of NMS or NHS in BBS were incubated in microtiter ELISA plates pre-coated with either PLY or mannan (positive control). ELISA plates were incubated for 1 hr at 37°C. The degree of C3b or iC3b deposition was monitored using a rabbit anti-human C3 antibody (as described in Materials and Methods). In both mouse (A) and human (B) sera, a significant degree of C3 activation occurred on PLY. This was seen even at high serum dilutions where complement activation can only occur by either the LP or the CP. At high serum concentration, using Mg⁺²-EGTA buffer (which inhibits C3 deposition via the calcium-dependent LP & CP), AP-mediated deposition of C3 activation products on PLY was also detected in both NMS (C) and NHS (D). Results are means (±SEM) of 3 independent experiments.

Figure 3. Western blot analysis to monitor C3 cleavage in response to the addition of increasing concentrations of PLY to NHS using a fluid phase assay.

1.25% NHS was incubated with serial dilutions of PLY in BBS buffer (starting concentration 100 µg/ml) for 1 hr at 37°C. Samples of this reaction were collected at different time points and subjected to a Western blot analysis using rabbit anti-human C3 to identify the various C3 activation products as described in Materials and Methods. A dose-dependent increase in the abundance of C3 activation products was observed to correlate with increasing PLY concentrations added to the serum.

Figure 4. In C1q-depleted human serum, C3 can still be activated on PLY in a LP-dependent fashion.

ELISA plates were either coated with PLY, or mannan, or immune complexes (as either positive or negative controls). (A) The deposition of C3 activation products on PLY was measured in serially diluted WT, C1q^−/− and MASP-2^−/− mouse sera. MASP-2 deficiency does not abolish C3 activation on PLY, while in the absence of C1q, no C3 activation can be detected in mouse sera. In human serum, however, absence of C1q has only a minor effect on the level of C3 deposition on PLY (B). In figure C, C1q-depleted human serum was incubated with different concentrations of anti-hMASP-2 mAb OMS721 followed by incubation with ELISA plates coated with PLY or mannan (as positive control) for 1 hr at 37°C. C3 deposition was detected using anti-human C3 as described in Materials and Methods. Inhibition of MASP-2 functional activity using the anti-hMASP-2 mAb OMS721 abolished C3 deposition on PLY in human C1q-depleted serum. Results are means (±SEM) of 3 independent experiments.

Figure 5. Human L-ficolin is the only LP recognition molecule that binds to PLY.

Serial dilutions of NMS or NHS in BBS were incubated in ELISA plates pre-coated with PLY or specific ligands for LP recognition molecules. The binding of specific LP recognition molecules to PLY was subsequently detected using antibodies directed against MBL, ficolins and CL-11. The binding of murine MBL-A and MBL-C (A), murine ficolin-A (C), murine CL-11 (E), human MBL (B), human FCN2 & 3 (D) and human CL-11 (F) were assessed by ELISA. In contrast to mouse ficolin-A (which has no binding affinity to PLY) human L-ficolin (FCN2) (the human orthologoue of murine ficolin-A) showed strong binding to PLY. Results are means (±SEM) of three independent experiments.