An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.
ABSTRACT 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 Neisseria 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, preincubation 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 10 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.
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ABSTRACT: Scabies is a ubiquitous and contagious skin disease caused by the parasitic mite Sarcoptes scabiei. Epidemiological studies have identified scabies as a causative agent for secondary skin infections caused by Staphylococcus aureus and Streptococcus pyogenes. This is an important notion, as such bacterial infections can lead to serious downstream life-threatening complications. Since the complement system is the first line of host defense that confronts invading pathogens, both the mite and bacteria produce a large array of molecules that inhibit the complement cascades. It is hypothesized that scabies mite complement inhibitors may play an important role in providing a favorable micro-environment for the establishment of secondary bacterial infections. This review aims to bring together the current literature on complement inhibition by scabies mites and bacteria associated with scabies and to discuss the proposed molecular link between scabies and bacterial co-infections. This article is protected by copyright. All rights reserved.Parasite Immunology 07/2014; · 1.85 Impact Factor
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ABSTRACT: Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus.PLoS Neglected Tropical Diseases 06/2014; 8(6):e2928. · 4.49 Impact Factor
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ABSTRACT: Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (Pn), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae. In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of Pn (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the Pn treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of Pn treated mice presented with sepsis and recoverable levels of live Meningococci. In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of Pn at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of Pn administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
The Journal of Immunology
An Evaluation of the Role of Properdin in Alternative
Pathway Activation on Neisseria meningitidis and
Sarika Agarwal,*,1Viviana P. Ferreira,†,‡,1Claudio Cortes,†,‡Michael K. Pangburn,‡
Peter A. Rice,* and Sanjay Ram*
Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against in-
vasive Neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological sur-
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 Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated pro-
perdin. C3deposition on Neisseria decreasedmarkedly whenproperdin functionwas blockedusing ananti-properdin mAbor 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, preincubation 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 10 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. The Journal of Immunology, 2010, 185: 507–516.
trimers (P3), and tetramers (P4) formed by head-to-tail association
of monomers (1–3). The AP C3 convertase, C3b,Bb, has a t1/2of
only 1.5 min. Properdin carries out the important function of bind-
ing to and stabilizing the C3b,Bb complex, thereby increasing its
half life 5- to 10-fold (4).
When properdin was first discovered more than 50 y ago, it was
thought to be an initiator of the alternative pathway (AP) (5). This
original theory was later changed in favor of the more widely ac-
cepted role of properdin, that of stabilizing the AP C3 convertase.
Recent studies have shown that properdin can bind directly to
roperdin is the only known positive regulator of the com-
plement system. Each properdin monomer is a 53 kDa
molecule. In plasma, properdin existsas cyclic dimers (P2),
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. (6) reported
that properdin bound to a strain of Neisseria gonorrhoeae and
a “rough” LPS mutant of Escherichia coli K12, and that bacteria-
bound properdin was capable of enhancing C3 deposition on these
bacteria after the addition of properdin-deficient serum. These data
have important implications because properdin deficiency in
humans is associated with an increased incidence of invasive in-
fections with Neisseria meningitidis (8–20). The finding that pro-
perdin 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 commer-
cially 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” proper-
din can promote complement activation and consumption when
added to serum (35). In addition, aggregates are also likely to bind
with higher avidity, or perhaps nonspecifically, 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
*Division of Infectious Diseases and Immunology, University of Massachusetts Med-
ical School, Worcester, MA 01605;†Department of Medical Microbiology and Im-
munology, University of Toledo College of Medicine, Toledo, OH 43614; and
‡Department of Biochemistry, Center for Biomedical Research, University of Texas
Health Science Center, Tyler, TX 75708
1S.A. and V.P.F. contributed equally to this work.
Received for publication November 6, 2009. Accepted for publication April 29, 2010.
This work was supported by National Institutes of Health Grants AI054544 (to S.R.),
AI32725, AI084048 (to P.A.R), and DK-35081 (to M.K.P.), and American Heart
Association National Scientist Development Grant 0735101N (to V.P.F.).
Address correspondence and reprint requests to Dr. Sanjay Ram, Division of Infec-
tious Diseases and Immunology, University of Massachusetts Medical School, Lazare
Research Building, Room 322, 364 Plantation Street, Worcester, MA 01605. E-mail
Abbreviations used in this paper: AP, alternative pathway; CMP-NANA, 59-
cytidinemonophospho-N-acetylneuraminic acid; DGI, disseminated gonococcal in-
fection; Glc, glucose; Hep, heptose; lgt, LOS glycosyl transferase; LOS, lipooligo-
saccharide; MFI, median fluorescence intensity; NHS, normal human serum; P,
contrasted this with unfractionated properdin. These studies pro-
vide important mechanistic insights into the role of properdin in
complement activation on the pathogenic Neisseriae.
Materials and Methods
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 excep-
tions (36, 37), almost all meningococci isolated from the blood or cere-
brospinal 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 isolates must downregulate
capsule production while traversing epithelial barriers (40, 41). To mini-
mize regulation of the AP by sialylation of lipooligosaccharide (LOS)
[reviewed in Ref. 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
NANA), the donor molecule for LOS sialylation, and therefore can sialy-
late 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 mole-
cules (lgtE mutant, glucose [Glc]→heptose [Hep]I; lgtF mutant, HepI
unsubstituted). Sialylation of F62 gonococcal LOS was achieved by adding
CMP-NANA to the growth media to a final concentration of 1 mg/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.
Sera, C3, Abs, and zymosan
Properdin-depleted serum (P-depleted serum) was purchased from Quidel
(Santa Clara, CA) (catalog no. A512). Serum depleted of C3 by immu-
noaffinity chromatography was purchased from Complement Technology
Tyler, TX) (catalog no. A314). C3 was purified from human plasma by
PEG precipitation and DEAE Sephacel chromatography as described pre-
viously (44). Two anti-properdin mAbs were purchased from Quidel (cat-
alog nos. A233 and A235). MAb A233 blocks properdin function;
whereas, mAb A235 binds to properdin but does not block the function
of properdin. C3 deposition on bacteria and zymosan was detected by anti-
encapsulated strains of N. meningitidis (one representative strain from each of the five major serogroups A, B, C, W-135, and Y) and their isogenic
unencapsulated mutants. Bacteria-bound P was detected by flow cytometry using an anti-properdin mAb (Quidel; catalog no. A235) followed by anti-mouse
IgG conjugated to Alexafluor 647. In all graphs, the x-axis represents fluorescence on a log10scale and the y-axis the number of events. Controls, where P
was omitted from the reaction mixture, are shown by the broken line histograms. Numbers adjacent to the histograms represent median fluorescence
intensities (MFIs) of P binding; numbers in the box with the dashed border represents fluorescence of the control bacteria, numbers in the gray shaded
box binding to encapsulated bacteria and the box with a solid black outline binding to unencapsulated meningococci. B, P binding to five strains of
N. gonorrhoeae and (C) properdin binding to zymosan. One experiment of two reproducibly repeated experiments is shown for each of the histograms.
Binding of properdin (P) to Neisseriae after incubation with commercially available unfractionated purified human P (10 mg/ml). A, Five
508 PROPERDIN IN NEISSERIAL INFECTIONS
human C3c conjugated to FITC from Biodesign (now Meridian Life Science,
Saco, ME). 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 permit selective activation of the AP.
Purification and fractionation of properdin
Properdin was purified from normal human plasma as described (45) and
stored at 280˚C until further fractionated. Alternatively, purified human
properdin was obtained either from Complement Technology or from
Quidel. Commercially available properdin or properdin that was purified in
the laboratory and stored at 280˚C is referred to in this study as unfrac-
tionated properdin. Pure, frozen properdin was thawed and the physio-
logical (P2–P4) forms and the aggregated Pnforms were separated by
cation exchange chromatography, followed by size exclusion chromatog-
raphy (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 proper-
din sample, in PBS, was loaded onto a 600 3 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 wk to minimize re-
aggregation of the properdin that can occur with prolonged storage (2).
Properdin binding and C3 deposition by flow cytometry
Briefly, 108bacteria were harvested from a 12–14 h culture on a chocolate
agar plate and suspended in HBSS containing 0.15 mM CaCl2and 1 mM
MgCl2(HBSS++). Zymosan was also suspended in HBSS++at a concen-
tration of 3 3 108particles/ml. Bacteria or zymosan were washed once
with HBSS++and 5 3 107bacteria or zymosan particles were incubated
for 30 min at 37˚C with either: properdin (10 mg/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 ml; 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 catalog
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 in-
strument (Becton Dickinson) and analyzed using the FlowJo analysis soft-
ware program (Version 7.2.4, TreeStar, 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: 1) the function of properdin was blocked with an
anti-properdin mAb, and 2) 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 mg/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 catalog no. A233) to Mg/EGTA-
NHS to a final concentration of 50 mg/ml. Total C3 deposition on bacteria
was detected after incubation of bacteria with Mg/EGTA-NHS (20% v/v)
containing the anti-properdin mAb in a final volume of 100 ml. 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 com-
pared with C3 deposition using properdin-depleted serum that was recon-
stituted either with unfractionated properdin or with properdin fractions
(described previously), each to a concentration of 10 mg/ml.
To determine whether preincubation of bacteria with properdin would
properdin or properdin fractions (10 mg/ml in a final volume of 100 ml) 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 pro-
perdin form to a final concentration of 10 mg/ml; this reconstituted serum
was added to the bacteria to achieve a final serum concentration of 20%
(v/v) in the reaction mixture. Baseline C3 deposition independent of pro-
perdin 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).
Binding of unfractionated purified properdin to Neisseriae
meningococcal strains and their isogenic unencapsulated mutants
previously to bind to purified properdin directly by flow cytometry
(6) and was used as a positive control. Only minimal binding of
subpopulations of serogroups A, C, and W-135 strains was seen
relative to controls; capsule expression further decreased properdin
binding to these serogroups (Fig. 1A). Gonococcal strains bound
varying amounts of purified properdin; the highest levels of pro-
perdin binding were observed with N. gonorrhoeae F62; whereas,
minimal binding was seen with strains WG and 24-1 (Fig. 1B).
No correlation was apparent between the ability of a gonococcal
Table I.Bacterial strains used in this study
A:4:P1.9:ST-5; Germany, 1991; encapsulated; LOS not sialylated
Insertional inactivation of mynB (mynB:: CmR) of A2594; unencapsulated,
LOS not sialylated
Insertional inactivation of lst of H44/76 (B:15:P1.7,16: ST-32; Norway, 1976); (lst::KmR);
encapsulated, LOS not sialylated
Insertional inactivation of siaD of H44/76 lst (siaD::CmR); unencapsulated,
LOS not sialylated
Insertional inactivation of lst of strain C2120 (C:NT:P1.5,2:ST-11; Germany, 1997)
(siaD::CmR); encapsulated, LOS not sialylated
Insertional inactivation of siaD of strain C2120 lst (siaD::CmR); unencapsulated, LOS not sialylated
Insertional inactivation of lst of W171 (W135:NT:P1.10:ST-11) (lst::KmR); encapsulated, LOS not sialylated
Insertional inactivation of siaD of W171 lst (siaD::CmR); unencapsulated, LOS not sialylated
Insertional inactivation of lst of Y2220 (Y:21:P1.15:ST-172) (lst::KmR); encapsulated, LOS not sialylated
Insertional inactivation of lst of Y2220 siaD (lst::KmR); unencapsulated, LOS not sialylated
H44/76 siaD lst
C2120 siaD lst
W171 siaD lst
Y2220 siaD lst
PorB.1B, serum sensitive
PorB.1B, serum sensitive
PorB.1B, serum sensitive
PorB.1B, serum resistant (DGI isolate)
PorB.1A, serum resistant (DGI isolate)
F62 lgtE::kan; Glc→HepI
F62 lgtF::spc; HepI unsubstituted
DGI, disseminated gonococcal infection.
The Journal of Immunology 509
strain to resist complement-dependent killing (Table I) and the
amount of properdin binding (Fig. 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
Properdin plays a critical role in AP-mediated C3 deposition
on meningococci and gonococci
C3 deposition on N. meningitidis and N. gonorrhoeae was assessed
by two methods: 1) use of an anti-properdin mAb to block the
function of properdin present in serum, and 2) use of properdin-
Meningococcal and gonococcal strains described in Fig. 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 mg mAb
A233/ml serum). Shown in Fig. 2A, C3 deposition on both encap-
sulated (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 Fig. 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).
unencapsulated mutants (lower panel) were incubated with NHS-Mg/EGTA (20% [v/v]) either in the absence (gray shaded histograms) or in the presence
(histograms depicted by solid lines) of an anti-properdin mAb that blocks P function (Quidel; catalog no. A233). Total C3 deposited on the bacterial surface
was measured by flow cytometry using sheep polyclonal anti-human C3c conjugated to FITC. B, C3 deposition was assessed on the 10 strains of
N. meningitidis on incubation with P-depleted serum-Mg/EGTA (20% [v/v]) (histograms depicted by solid lines), or with P-depleted serum-Mg/EGTA
reconstituted with purified unfractionated properdin to a concentration of 10 mg/ml (gray shaded histograms). Detection of C3 was performed as indicated
above. Axes are as described in Fig. 1. Controls (no serum added to bacteria) are shown by the broken lines. Controls with heat-inactivated serum show
nearly identical tracings and have been omitted for simplicity. Numbers adjacent to histograms represent the MFIs of C3 binding. One representative
experiment of two separately performed and reproducibly repeated experiments is shown for each of the histograms.
P augments AP-mediated C3 deposition on N. meningitidis. A, Five encapsulated strains of N. meningitidis (upper panel) and their isogenic
510 PROPERDIN IN NEISSERIAL INFECTIONS
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
strain 15253 (Fig. 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 Fig. 4, in-
creasing 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 with sim-
ilar experiments in which 20% serum was used (Fig. 2B).
The presence of physiologic concentrations of properdin in
serum greatly enhanced C3 binding (Figs. 2B, 4). These results
show that properdin is required to boost AP activation on me-
ningococci even in the presence of high complement concen-
trations (as would be encountered by bacteria in the bloodstream).
Preincubation of Neisseriae with unfractionated properdin is
less efficient in depositing C3 than serum reconstituted with
N. gonorrhoeae incubated with unfractionated properdin were
reported to show enhanced C3 deposition after addition of pro-
perdin-deficient serum compared with diminished C3 deposition
EGTA (20% [v/v]) alone (gray shaded histograms) or with added anti-properdin mAb (Quidel mAb A233) that blocks properdin function (histograms
depicted by solid lines). B. C3 deposition on gonococcal strains after incubation with P-depleted serum-Mg/EGTA (20% [v/v]) (histograms depicted by
solid lines) or with P-depleted serum-Mg/EGTA reconstituted with purified unfractionated P (gray shaded histograms). Numbers adjacent to histograms
represent the MFIs of C3 binding. Detection of C3 fragments was performed as in Fig. 1A. One representative experiment of two separately performed and
reproducibly repeated experiments is shown for each of the histograms.
P augments AP-mediated C3 deposition on N. gonorrhoeae. A, C3 deposition on five strains of N. gonorrhoeae incubated with NHS-Mg/
A2594 and Y2220 incubated either with P-depleted serum-Mg/EGTA (final concentration 80% [v/v]; solid black histograms) or P-depleted serum-Mg/
EGTA (final concentration 80% [v/v]) reconstituted with purified unfractionated properdin (10 mg/ml; shaded gray histograms). Numbers adjacent to
histograms represent the MFIs of C3 binding. Detection of C3 was performed as in Fig. 1A. One representative experiment of two separately performed and
reproducibly repeated experiments is shown for each of the histograms.
P mediated deposition of C3 on Neisseriae at 80% serum concentration. Total C3 deposition on the unencapsulated derivatives of strains
The Journal of Immunology 511
on bacteria that were incubated with properdin-deficient serum
in fixing C3 on gonococci, the relative balance between properdin
bound to bacteria directly versus properdin bound indirectly to
bacteria via C3bBb, which is necessary to maximally deposit C3,
was not determined. To address this, we compared C3 deposition
on Neisseriae that were handled as follows: 1) first incubated with
unfractionated purified properdin, followed by the addition of pro-
perdin-depleted serum, or 2) incubated with properdin-depleted se-
rum that had been reconstituted with unfractionated properdin. The
unencapsulated mutant of A2594 and gonococcal strain F62 were
chosen because these strains bound the highest amounts of unfrac-
tionated properdin. Bacteria either were incubated with unfractio-
nated purified properdin (10 mg/ml in a final volume of 100 ml),
washed once to remove unbound properdin, followed by the addi-
tion of properdin-depleted serum (final concentration 20% [v/v]) or
were incubated in 20% properdin-depleted serum with unfractio-
nated properdin added to achieve a final serum properdin concen-
tration of 10 mg/ml. The concentration of properdin in the reaction
mixture where bacteria had been preincubated with properdin
(10 mg/ml) was 5-fold higher than the final concentration of pro-
perdin in the reaction mixture that contained 20% reconstituted
serum ([properdin] 2 mg/ml). Shown in Fig. 5, bacteria preincubated
with unfractionated properdin, followed by the addition of proper-
din-depleted serum bound less C3 than bacteria that were incubated
with properdin-sufficient serum.
Neisseriae do not bind to native properdin
Unfractionated properdin contains high-order oligomers of proper-
din 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 Neis-
seriae, properdin was fractionated by size exclusion chromatogra-
phy 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 with unfractionated properdin, there was barely any
detectable binding of native properdin to any meningococcal or
gonococcal strains tested. Representative examples with N. men-
ingitidis strain A2594 (unencapsulated mutant) and N. gonor-
rhoeae strain F62, the strains that bound the highest amounts of
unfractionated properdin, are shown in Fig. 6A (left and middle
graphs). No binding was seen to P2, P3, or P4fractions; (P3data
only are shown for simplicity). In contrast, P2, P3, and P4all bound
to a similar extent to zymosan (data with P3shown in Fig. 6A,
right graph). High amounts of Pnand unfractionated properdin
bound to zymosan.
It was previously reported that binding of unfractionated pro-
perdin to E. coli K12 or Salmonella typhimurium strains increased
with LPS truncation [loss of the O-Ag, which contain repeating
saccharide structures (6, 47)]. Although unfractionated properdin
did not bind to wild-type enterobacterial strains that expressed the
O-Ag, 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-Ags and all Neis-
serial strains examined in this study, except gonococcal strain
15253, express 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 exten-
sions 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 ob-
served (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 con-
vertases 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 (Fig. 6B, broken red histograms) and simulated
controls with properdin-depleted serum (Fig. 6B, green histo-
grams). 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 in
Fig. 6B, respectively).
Preincubation of Neisseria with native forms of properdin does
not result in C3 deposition; native properdin augments C3
deposition by stabilizing C3 convertases
We assessed a downstream functional consequence of the in-
teraction 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.
C3 deposition. Unencapsulated mutant of meningococcal strain A2594 and
N. gonorrhoeae strain F62 were incubated either with P-depleted serum-
Mg/EGTA (thin solid lines) or preincubated with unfractionated P (10 mg/
ml). Organisms were washed then P-depleted serum-Mg/EGTA (labeled
“Unfractionated P→P-depleted serum-Mg/EGTA”) was added (thick black
lines) or organisms were incubated with P-depleted serum-Mg/EGTA
reconstituted with unfractionated P (shaded gray histograms). Numbers
adjacent to histograms represent the MFIs of C3 binding. Detection of
C3 fragments was performed as in Fig. 1A. One representative experiment
of two separately performed and reproducibly repeated experiments is
shown for each of the histograms.
Preincubation of Neisseriae with unfractionated P enhances
512PROPERDIN IN NEISSERIAL INFECTIONS
Unencapsulated derivatives of strains A2594 and H44/76, encap-
sulated 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 Fig. 7, prein-
cubation of bacteria with P2, P3or P4did not enhance C3 binding
to any of the strains (shaded green histograms) compared with
properdin-deficient serum alone (blue histograms). Preincubation
of meningococcal strains A2594 and H44/76 (both unencapsu-
lated), but not Y2220 (encapsulated) or F62, with Pnresulted in
enhanced C3 binding to bacteria (Fig. 7, shaded green histograms,
right column) compared with incubation with properdin-depleted
serum alone (solid blue histograms). In contrast, reconstitution of
properdin-depleted serum with each of the native fractions resulted
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.
Ab-dependent bactericidal activity is important for protection
against meningococcal infection (49, 50). The AP plays an impor-
tant 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 com-
ponents can lead to C5b-9 insertion into the membrane of Gram-
negative pathogens, resulting in complement-dependent killing.
Deficiencies of the terminal complements (C5–C9) and AP com-
ponents, such as factor D and properdin, predispose individuals to
invasive meningococcal infections (11, 20, 51). Properdin defi-
ciency is rare, but individuals with properdin deficiency are pre-
disposed 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,
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 “conven-
tional” mechanism, namely, 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, respec-
tively) 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
properdin in the void volume of a size-exclusion chromatograph). Binding of each of these fractions to N. meningitidis A2594 (unencapsulated) and
N. gonorrhoeae F62 was measured by flow cytometry. Binding of P2, P3, and P4yielded nearly identical results; results with the P3fraction (only) are
shown for simplicity (solid green line). Binding of Pnis shown by the red line; binding of commercially available unfractionated P by the blue line. B,
Serum P binds to Neisseriae in the presence of the alternative pathway of complement. P binding to unencapsulated N. meningitidis A2594 and N.
gonorrhoeae F62 after incubation with C3-depleted serum-Mg/EGTA (broken red histograms), P-depleted serum-Mg/EGTA (green histograms), C3-
depleted serum-Mg/EGTA reconstituted with purified C3 (solid red histograms), or normal human serum-Mg/EGTA (blue histograms). All sera contained
10 mM Mg2+and 10 mM EGTA and the final concentration of sera in all reaction mixtures was 20%. Axes are as described in Fig. 1. Controls (no added
serum) are shown by the black broken lines. Numbers adjacent to histograms represent the MFIs of P binding. One representative experiment of two
separately performed and reproducibly repeated experiments is shown for each of the histograms.
Binding of fractionated P to Neisseriae. A, P was fractionated into dimers (P2), trimers (P3), tetramers (P4), and higher-order oligomers (Pn;
The Journal of Immunology 513
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 nonspecifically to live cell surfaces where they promote
complement activation (45).
Therewas awidevariation in C3 deposition among strains (Figs.
2, 3), which could reflect differences in the ability of strains to
activate the AP and/or availability of targets for C3 on the bac-
terial 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 Fig.
2A, 2B) as evidenced by less C3 deposition compared with their
isogenic unencapsulated mutants (gray shaded histograms in the
lower panels of Fig. 2A, 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 strain F62
with the unfractionated commercial properdin preparation, fol-
lowed by the addition of properdin-depleted serum (Fig. 5, right
graph, “Unfractionated P → P-depleted serum-Mg/EGTA”)
resulted in increased levels of C3 deposition compared with
preincubation of strain F62 with Pn (void volume eluate of
a molecular sieve column), shown in Fig. 7 (shaded green
histogram, lower right graph). This may be explained by lower
amounts of Pnbinding to F62 relative to unfractionated properdin
(Fig. 6A). High-order oligomers present in commercial properdin
preparations may have also been retained by the molecular sieve
column. Potentially, these retained aggregates in Pnmay 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 Pnprepa-
ration, which enhanced C3 deposition when added either before
serogroup B strain H44/76, encapsulated serogroup Y strain 2220 and gonococcal strain F62 were preincubated either with properdin dimers (P2), trimers
(P3), tetramers (P4) or higher-order oligomers (Pn) each to a final concentration of 10 mg/ml and washed. P-depleted serum-Mg/EGTA was then added to
a final concentration of 20%. C3 deposition on bacteria was measured by flow cytometry (shaded green histograms). Bacteria incubated only with
P-depleted serum-Mg/EGTA (final concentration 20%) are shown by the blue histograms. C3 deposition was also measured on bacteria that were incubated
with P-depleted serum-Mg/EGTA that had been reconstituted with each of the purified properdin (P2, P3, P4, or higher-order oligomer [Pn]) fractions (final
concentration of each P fraction was 10 mg/ml; shown by the red histograms). Axes are as described in Fig. 1. Controls (serum lacking from the reaction
mixture) are indicated by the broken lines. Numbers adjacent to histograms represent the MFIs of P binding. One representative of two separately
performed and reproducibly repeated experiments is shown for each of the histograms.
Preincubation with native P does not enhance C3 deposition on Neisseriae. Unencapsulated derivatives of serogroup A strain 2594 and
514PROPERDIN IN NEISSERIAL INFECTIONS
P-depleted serum-Mg/EGTA (Fig. 7, shaded green histogram in the
two upper graphs in the Pncolumn) or together with P-depleted
serum-Mg/EGTA (Fig. 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 unfractio-
nated properdin (6), although a recent study shows that the native
properdin forms do not (45). Studies that define ligands or func-
tions 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 sur-
faces (52) and may limit the ability of properdin to initiate com-
plement 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 pro-
perdin function, which is to bind to and stabilize AP C3 con-
vertases, 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 (Universita ¨t Wu ¨rzburg, Germany) for providing
meningococcal strains and mutants used in this study, Dr. Daniel Stein for
providing the plasmid to make F62 lgtF, Dr. Asesh Banerjee for the plas-
mid to make F62 lgtE, and Staci Snyder and Connie Elliot for their ex-
cellent technical assistance.
M.K.P. is an officer of and has a financial interest in Complement Tech-
nology, Inc., a supplier of complement reagents.
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516PROPERDIN IN NEISSERIAL INFECTIONS