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Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive Neisserial infections. Recently, commercially-available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological ‘native’ (dimeric, trimeric and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of N. meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, pre-incubation of bacteria with unfractionated properdin followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of ten Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the ‘conventional’ mechanism of stabilizing AP C3 convertases.
Properdin is the only known positive regulator of the complement system. Each properdin monomer is a 53 kDa molecule. In plasma, properdin exists as cyclic dimers (P2), trimers (P3), and tetramers (P4) formed by head-to-tail association of monomers (1–3). The AP C3 convertase, C3b, Bb, has a half life of only 1.5 min. Properdin carries out the important function of binding to and stabilizing the C3b, Bb complex, thereby increasing its half-life 5–10 fold (4).
When properdin was first discovered more than 50 years ago, it was thought to be an initiator of the AP (5). This original theory was later changed in favor of the more widely accepted role of properdin, that of stabilizing the AP C3 convertase. Recent studies have showed that properdin can bind directly to AP activator surfaces such as zymosan and rabbit erythrocytes (6) and serve to initiate the AP by forming a platform for assembly of AP C3 convertases (6, 7). Of note, Spitzer et al reported that properdin bound to a strain of N. gonorrhoeae and a ‘rough’ LPS mutant of E. coli K12, and that bacteria-bound properdin was capable of enhancing C3 deposition on these bacteria following the addition of properdin-deficient serum (6). These data have important implications because properdin deficiency in humans is associated with an increased incidence of invasive infections with N. meningitidis (8–20). The finding that properdin binds to N. gonorrhoeae and activates complement has been extrapolated to N. meningitidis (21, 22). This newly proposed (or perhaps more appropriately, rediscovered) mechanism provided an attractive theory to explain why properdin-deficient individuals are exclusively predisposed to meningococcal disease (21, 22).
We questioned the ability of Neisseriae to bind to properdin under physiological conditions for the following reasons. First, both N. meningitidis and N. gonorrhoeae have evolved several intricate mechanisms to evade killing by human complement (23–34); binding of properdin by bacteria would place these pathogens at a distinct disadvantage for survival in their human host. Second, the study of properdin binding to N. gonorrhoeae used commercially available properdin that has undergone freeze-thawing and contains high-order oligomers, or aggregates of properdin (2, 35). These aggregates are also called ‘activated’ properdin or Pn (35). Unlike the physiological forms of properdin, ‘activated’ properdin can promote complement activation and consumption when added to serum (35). In addition, aggregates are also likely to bind with higher avidity, or perhaps non-specifically, to surfaces that native forms of properdin may not.
In this study, we have evaluated the role of native properdin in activating the AP on N. meningitidis and N. gonorrhoeae and have contrasted this with unfractionated properdin. These studies provide important mechanistic insights into the role of properdin in complement activation on the pathogenic Neisseriae.
One representative strain from each of the five major meningococcal serogroups that cause disease worldwide (A, B, C, W-135 and Y) and their isogenic unencapsulated mutants were used in this study. With rare exceptions (36, 37), almost all meningococci isolated from the blood or cerebrospinal fluid are encapsulated (38). Isogenic unencapsulated mutants were also studied because strains carried in the nasopharynx often do not express capsules (39) and further, invasive is olates must down-regulate capsule production while traversing epithelial barriers (40, 41). To minimize regulation of the AP by sialylation of lipooligosaccharide (LOS) (reviewed in (32)), the LOS sialyltransferase gene (lst) of serogroups B, C, W-135 and Y strains was abrogated by insertional inactivation (lst::KanR) as described previously (42). Serogroup A N. meningitidis do not synthesize 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA), the donor molecule for LOS sialylation, and therefore can sialylate its LOS only when CMP-NANA is added to growth media. Five strains of N. gonorrhoeae that differed in their PorB type, sensitivity to the bactericidal action of normal human serum and the clinical syndrome they caused were used in this study. In addition, LOS mutations of one of the strains, F62, was used to examine the role of LOS truncation on interaction of this strain with properdin. Insertional inactivation of LOS glycosyl transferase lgtE and lgtF of F62 using methods described previously (43) yielded isogenic mutant strains that expressed truncated LOS molecules (lgtE mutant, Glc → HepI; lgtF mutant, HepI unsubstituted). Sialylation of F62 gonococcal lipooligosaccharide (LOS) was achieved by adding CMP-NANA to the growth media to a final concentration of 1 μg/ml. All Neisserial strains were routinely cultured on chocolate agar plates at 37°C in the presence of 5% CO2. Strains and their relevant characteristics are listed in Table I.
Properdin-depleted serum (P-depleted serum) was purchased from Quidel (Catalog no. A512). Serum depleted of C3 by immunoaffinity chromatography was purchased from Complement Technology, Inc (Cat. No. A314). C3 was purified from human plasma by PEG precipitation and DEAE Sephacel chromatography as described previously (44). Two anti-properdin mAbs were purchased from Quidel (Cat. Nos. A233 and A235). MAb A233 blocks properdin function while mAb A235 binds to properdin but does not block the function of properdin. C3 deposition on bacteria and zymosan was detected by anti-human C3c conjugated to FITC from Biodesign (now Meridian Life Science, Inc.). Sera obtained fresh from 10 normal adults (normal human serum; NHS) were pooled and stored at −80°C until used. All sera contained 10 mM EGTA and 10 mM Mg2+ to selectively activate the AP.
Properdin was purified from normal human plasma as described (45) and stored at −80°C until further fractionated. Alternatively, purified human properdin was obtained either from Complement Technology, Inc. or from Quidel Corporation. Commercially available properdin or properdin that was purified in the laboratory and stored at −80°C is referred to in this study as unfractionated properdin. Pure, frozen properdin was thawed and the physiological (P2-P4) forms and the aggregated Pn forms were separated by cation exchange chromatography, followed by size exclusion chromatography (2). Briefly, thawed properdin was separated using a 1 mL Mono S cation exchange column and the recovered oligomers further separated by gel filtration on Phenomenex Bio Sep-Sec-S4000 column. The properdin sample, in PBS, was loaded onto the 600 × 7.8 mm molecular sieve column and eluted at a flow rate of 0.5 ml/min. Fractionated properdin was stored at 4°C and was used in experiments within 2 weeks to minimize reaggregation of the properdin that can occur with prolonged storage (2).
Briefly, 108 bacteria were harvested from a 12–14 hour culture on a chocolate agar plate and suspended in Hanks’ balanced salt solution (HBSS) containing 0.15 mM CaCl2 and 1 mM MgCl2 (HBSS++). Zymosan was also suspended in HBSS++ at a concentration of 3 × 108 particles/ml. Bacteria or zymosan were washed once with HBSS++ and 5 × 107 bacteria or zymosan particles were incubated for 30 min at 37°C with either: properdin (10 μg/ml); NHS-Mg/EGTA;C3-depleted serum-Mg/EGTA; properdin-depleted serum-Mg/EGTA or C3-depleted serum-Mg/EGTA reconstituted with purified C3 (1 mg/ml), each used in a final reaction volume of 100 μl; the final concentration of sera in these experiments was 20%. Properdin that bound to bacteria or zymosan was detected using anti-human properdin mAb (Quidel Cat. No. A235) at a dilution of 1:100, followed by anti-mouse IgG conjugated to Alexafluor 647 (1:400). Data were collected either on a LSR II flow cytometer (Becton Dickinson [Franklin Lakes, NJ]) or a FACSCalibur instrument (Becton Dickinson) and analyzed using the FlowJo analysis software program (Version 7.2.4, TreeStar Inc. [Ashland, OR]).
The functional importance of properdin in mediating AP-dependent C3 deposition (includes both the C3b and iC3b fragments) on Neisseria was assessed by two methods: i) the function of properdin was blocked with an anti-properdin mAb and ii) C3 deposited on bacteria by properdin-depleted serum was compared with C3 deposited by properdin-depleted serum reconstituted with purified properdin. The concentration of properdin in reconstituted sera was 10 μg/ml. All reaction mixtures contained 10 mM Mg2+ and 10 mM EGTA to inhibit classical and lectin pathway activation (both dependent on Ca2+) and restrict complement activation to the Mg2+-dependent AP. In the first method, properdin function in serum was blocked by adding anti-properdin mAb (Quidel cat. No. A233) to Mg/EGTA-NHS to a final concentration of 50 μg/ml. Total C3 deposition on bacteria was detected following incubation of bacteria with Mg/EGTA-NHS (20% v/v) containing the anti-properdin mAb in a final volume of 100 μl. Bacteria incubated with Mg/EGTA-NHS (no anti-properdin mAb added) served as a control. Reaction mixtures were incubated for 30 min at 37°C and total C3 (C3b plus iC3b) binding to bacteria was detected by flow cytometry using anti-C3c FITC as described previously (26, 28). In the second method, C3 deposition on bacteria incubated with properdin-depleted serum was compared with C3 deposition using properdin-depleted serum that was reconstituted either with unfractionated properdin or with properdin fractions (described above), each to a concentration of 10 μg/ml.
To determine whether preincubation of bacteria with properdin would enhance C3 deposition, bacteria were incubated with purified unfractionated properdin or properdin fractions (10 μg/ml in a final volume of 100 μl) for 20 min at 37°C, washed once to remove unbound properdin, followed by the addition of P-depleted serum to a final concentration 20% (v/v)). As a comparator, we measured C3 deposition on bacteria that were incubated with properdin-depleted serum reconstituted with the corresponding properdin form to a final concentration of 10 μg/ml; this reconstituted serum was added to the bacteria to achieve a final serum concentration of 20% (v/v) in the reaction mixture. BaselineC3 deposition independent of properdin function was measured by incubating bacteria with properdin-depleted serum. Total C3 (C3b plus iC3b) deposited on bacteria was detected using anti-human C3c conjugated to FITC (1:100 dilution in HBSS++/1% BSA) as described previously (26, 28).
We quantified the binding of unfractionated purified properdin to five meningococcal strains and their isogenic unencapsulated mutants and also to five strains of N. gonorrhoeae. Zymosan has been shown previously to bind to purified properdin directly by flow cytometry (6) and was used as a positive control. Only minimal binding of unfractionated purified properdin to unencapsulated meningococcal subpopulations of serogroups A, C and W-135 strains was seen relative to controls; capsule expression further decreased properdin binding to these serogroups (Figure 1A). Gonococcal strains bound varying amounts of purified properdin; the highest levels of properdin binding were observed with N. gonorrhoeae F62, while minimal binding was seen with strains WG and 24-1 (Figure 1B). No correlation was apparent between the ability of a gonococcal strain to resist complement-dependent killing (Table 1) and the amount of properdin binding (Figure 1B). Furthermore, sialylation of the LOS of N. gonorrhoeae F62 by growth in media containing CMP-NANA did not affect properdin binding (data not shown). As expected, zymosan bound unfractionated properdin well (Figure 1C).
The functional importance of properdin in mediating AP-dependent C3 deposition on N. meningitidis and N. gonorrhoeae was assessed by two methods: i) use of an anti-properdin mAb to block the function of properdin present in serum and ii) use of properdin-depleted serum.
Meningococcal and gonococcal strains described in Figure 1 were used in the following experiments. First, the function of properdin in AP-mediated C3 deposition on Neisseriae was evaluated by using a mAb that blocks properdin function (50 μg mAb A233/ml serum). Shown in Figure 2A, C3 deposition on both encapsulated (upper panel, all except serogroup C) and unencapsulated (lower panel, all) meningococcal strains decreased markedly when properdin function was blocked with the anti-properdin mAb. These data point to a central role for properdin in enhancing AP activation on meningococci. This observation was confirmed by using properdin-depleted Mg/EGTA-treated human serum, shown in Figure 2B, where minimal deposition of C3 occurred as a result of marked diminution of AP activation in properdin-depleted serum; reconstitution of depleted serum with physiologic concentrations of unfractionated properdin increased C3 binding to all strains except encapsulated serogroup C and W-135 isolates. These studies were performed with unfractionated properdin to simulate prior observations made by Spitzer et al (6).
Similarly, the importance of properdin in promoting AP activation on N. gonorrhoeae was demonstrated. A 4- to 10-fold decrease in C3 binding (fluorescence) to all 5 strains occurred when properdin function was blocked with anti-properdin mAb (Figure 3A). Incubation of N. gonorrhoeae in properdin-depleted Mg/EGTA-NHS resulted in small amounts of C3 deposited; addition of properdin to Mg/EGTA-NHS augmented C3 deposition on all strains, minimally to strain15253 (Figure 3B). N. gonorrhoeae strain F62 was chosen for further studies because it bound the greatest amount of C3.
Because activity of the AP is concentration dependent (46), we tested whether higher serum concentrations would permit optimal AP activation in the absence of properdin. Shown in Figure 4, increasing the concentration of properdin-deficient Mg/EGTA-NHS to 80% resulted in no increase in the amount of C3 deposition at 30 min on two meningococcal strains tested, compared to similar experiments where20% serum was used (Figure 2B).
The presence of physiologic concentrations of properdin to serum greatly enhanced C3 binding (Figure 2B and and4).4). These results show that properdin is required to boost AP activation on meningococci even in the presence of high complement concentrations (as would be encountered by bacteria in the bloodstream).
N. gonorrhoeae incubated with unfractionated properdin were reported to show enhanced C3 deposition following addition of properdin-deficient serum compared with diminished C3 deposition on bacteria that were incubated with properdin-deficient serum alone (6). While those data suggested a role for bacteria-bound properdin in fixing C3 on gonococci, the relative balance between properdin bound to bacteria directly versus properdin bound indirectly to bacteria via C3b, Bb, which is necessary to maximally deposit C3, was not determined. To address this, we compared C3 deposition on Neisseriae that were: i) first incubated with unfractionated purified properdin followed by the addition of properdin-depleted serum or ii) incubated with properdin-depleted serum that was reconstituted with unfractionated properdin a priori. The unencapsulated mutant of A2594 and gonococcal strain F62 were chosen because these strains bound the highest amounts of unfractionated properdin. Bacteria either were incubated with unfractionated purified properdin (10 μg/ml in a final volume of 100 μl), washed once to remove unbound properdin, followed by the addition of properdin depleted serum (final concentration 20% (v/v)) or were incubated in 20% properdin depleted serum with unfractionated properdin added to achieve a final serum properdin concentration of 10 μg/ml. The concentration of properdin in the reaction mixture where bacteria were preincubated with properdin (10 μg/ml) was 5-fold higher than the final concentration of properdin in the reaction mixture that contained 20% reconstituted serum ([properdin] 2 μg/ml). Shown in Figure 5, bacteria preincubated with unfractionated properdin followed by the addition of properdin-depleted serum bound less C3 than bacteria that were incubated with properdin-sufficient serum.
Unfractionated properdin contains high order oligomers of properdin formed as a result of freeze-thawing (35). Properdin in serum contains only dimers, trimers and tetramers (native properdin) (2, 35). We hypothesized that an artificial increase in avidity caused by high order oligomerization and aggregation could result in binding of properdin to surfaces that otherwise do not bind the native forms of properdin. To explore this possibility on Neisseriae, properdin was fractionated by size exclusion chromatography and properdin dimers, trimers and tetramers (called P2, P3 and P4, respectively) were tested for direct binding (in the absence of C3 convertases) to Neisseriae by flow cytometry. Properdin that eluted in the void volume (higher order oligomers, or Pn) and commercially available unfractionated pure properdin were used as controls.
Compared to unfractionated properdin there was barely any detectable binding of native properdin to any meningococcal or gonococcal strains tested. Representative examples with N. meningitidis strain A2594 (unencapsulated mutant) and N. gonorrhoeae strain F62, the strains that bound the highest amounts of unfractionated properdin, are shown in Figure 6A (left and middle graphs). No binding was seen to P2, P3 or P4 fractions;(P3 data only are shown for simplicity). In contrast, P2, P3 and P4 all bound to a similar extent to zymosan (data with P3 shown in Figure 6A, right graph). High amounts of Pn and unfractionated properdin bound to zymosan.
It was previously reported that binding of unfractionated properdin to E. coli K12 or Salmonella typhimurium strains increased with LPS truncation (loss of the O-antigen, which contain repeating saccharide structures (6, 47)). While unfractionated properdin did not bind to wild-type enterobacterial strains that expressed the O-antigen, mutants that expressed only the core oligosaccharide, or that lacked part or all of the core oligosaccharide, bound well to unfractionated properdin (6). Neisseria lack O-antigens; all Neisserial strains examined in this study, except gonococcal strain 15253, express at least 4–6 hexose residues extending outward from HepI of the LOS core; the HepI of 15253 is substituted with a lactose residue (48). To rule out the possibility that LOS hexose extensions present in, for example, wild-type strain F62 may have blocked binding of native properdin, we examined the binding of properdin fractions to the LOS truncated lgtE(Glc → HepI) and lgtF(Hep1 unsubstituted) mutants of F62. No binding of any of the native forms of properdin to these mutants was observed (data not shown). As expected, unfractionated properdin bound well to the mutants with truncated LOS (not shown).
To determine whether serum components other than C3 convertases could affect binding (either negatively or positively) of properdin to Neisseriae, we measured properdin binding to bacteria in the presence of C3-depleted serum. Properdin-depleted serum served as a negative control and C3-depleted serum reconstituted with C3 and NHS served as positive controls; all sera contained Mg/EGTA to permit selective activation of the AP. No properdin binding was measured on bacteria that were incubated with C3-depleted serum (Figure 6B, broken red histograms) and simulated controls with properdin-depleted serum (green histograms). Properdin binding was observed in sera that contained both properdin and active C3 (NHS and reconstituted C3-depleted serum, depicted by solid red and blue histograms, respectively).
We assessed a downstream functional consequence of the interaction of native properdin with Neisseria, which distinct from unfractionated properdin, had not resulted in direct binding of properdin to bacteria. We used native oligomers of properdin (2, 35) to confirm its ‘conventional’ role in promoting C3 deposition on bacteria that result from secondary (or indirect) binding of properdin to bacteria via C3b, Bb to stabilize AP C3 convertases. Unencapsulated derivatives of strains A2594 and H44/76, encapsulated serogroup Y strain 2220, and gonococcal strain F62 were preincubated with either P2, P3, P4 or Pn followed by the addition of Mg-EGTA treated properdin-depleted serum. Controls included bacteria plus properdin-depleted serum that was reconstituted with each of the properdin fractions separately. Total C3 deposited on bacteria was measured by flow cytometry. Shown in Figure 7, preincubation of bacteria with P2, P3 or P4 did not enhance C3 binding to any of the strains (shaded green histograms) compared to properdin-deficient serum alone (blue histograms). Pre-incubation of meningococcal strains A2594 and H44/76 (both unencapsulated), but not Y2220 (encapsulated) or F62, with Pn resulted in enhanced C3 binding to bacteria (shaded green histograms, right column) compared to incubation with properdin-depleted serum alone (solid blue histograms). In contrast, reconstitution of properdin-depleted serum with each of the native fractions resulted in an increase in C3 deposition on all strains (solid red lines).
Collectively, these data indicate that the primary mechanism of native properdin-mediated augmentation of C3 deposition on Neisseria involves stabilization of C3 convertases. C3 deposition seen when bacteria are preincubated with unfractionated properdin is likely mediated by the high-order oligomers in the preparations and may not reflect physiological conditions in vivo.
Antibody-dependent bactericidal activity is important for protection against meningococcal infection (49, 50). 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, 20, 51). 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, 20, 51). 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, P3 and P4, respectively) that are present in the ratio of 26:54:20 (2, 35). 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, 35). 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 (Figures 2 and and3),3), 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 Figures 2A and 2B) as evidenced by less C3 deposition compared to their isogenic unencapsulated mutants (grey shaded histograms in the lower panels of Figures 2A and 2B). 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 (Figure 5, 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 Figure 7 (shaded green histogram, lower right graph). This may be explained by lower amounts of Pn binding to F62 relative to unfractionated properdin (Figure 6A). 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 (Figure 7, shaded green histogram in the two upper graphs in the Pn column) or together with P-depleted serum-Mg/EGTA (Figure 7, red histograms in the same graphs).
It is clear that certain complement activator surfaces such as zymosan bind to purified native properdin (Fig. 6A). 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.
We thank Dr. Ulrich Vogel (Universität Würzburg, Germany) for providing meningococcal strains and mutants used in this study. We thank Dr. Daniel Stein for providing the plasmid to make F62 lgtF, Dr. Asesh Banerjee for the plasmid to make F62 lgtE, and Mrs. Staci Snyder and Connie Elliot for their excellent technical assistance.
1This work was supported by National Institutes of Health grants AI054544 (S.R.), AI32725 and AI084048 (P.A.R), and DK-35081 (M.K.P.), and American Heart Association National Scientist Development Grant 0735101N (V.P.F.).
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The authors declare the following disclosure: M.K.P. is an officer of and has a financial interest in Complement Technology, Inc., a supplier of complement reagents.