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The Virulence Function of Streptococcus
pneumoniae Surface Protein A Involves
Inhibition of Complement Activation and
Impairment of Complement
Receptor-Mediated Protection
Bing Ren, Mark A. McCrory, Christina Pass, Daniel C.
Bullard, Christie M. Ballantyne, Yuanyuan Xu, David E.
Briles and Alexander J. Szalai
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:7506-7512; ;
doi: 10.4049/jimmunol.173.12.7506
http://www.jimmunol.org/content/173/12/7506
The Journal of Immunology
The Virulence Function of Streptococcus pneumoniae Surface
Protein A Involves Inhibition of Complement Activation and
Impairment of Complement Receptor-Mediated Protection1
Bing Ren,* Mark A. McCrory,† Christina Pass,† Daniel C. Bullard,‡ Christie M. Ballantyne,§
Yuanyuan Xu,† David E. Briles,* and Alexander J. Szalai2†
S
treptococcus pneumoniae is a major cause of bacterial
pneumonia, otitis media, meningitis, and septicemia, especially in infants, the elderly, and immunocompromised individuals. The thick and rigid cell wall of the Gram-positive pneumococcus is protective against lysis by the complement membrane
attack complex, so the main mechanism of complement-mediated
elimination of pneumococci from infected hosts is by phagocytosis. Phagocytosis relies on the interaction of Ab and/or complement, deposited on the pathogen’s surface, with phagocyte receptors for the Fc portion of Igs or various complement proteins.
Pneumococcal surface protein A (PspA)3 is an important virulence factor for S. pneumoniae (1–3). Accordingly, in a murine
model of bacteremia, PspA-deficient (PspA⫺) pneumococci have
significantly reduced virulence compared with pneumococci that
express PspA (PspAⴙ) (2, 3). Earlier studies from our laboratory
and others’ have shown that PspA inhibits complement activation,
thereby limiting opsonization of pathogens by complement protein
Departments of *Microbiology, †Medicine, and ‡Genetics, University of Alabama,
Birmingham, AL 35294; and §Department of Medicine, Baylor College of Medicine,
Houston, TX 77030
Received for publication March 30, 2004. Accepted for publication October 15, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI21548 and
AI42183 (to A.J.S.) and the Carsten Cole Buckley Pediatric Meningitis Research
Fund (to D.E.B.).
2
Address correspondence and reprint requests to Dr. Alexander J. Szalai, Division of
Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, 437B Tinsley Harrison Tower, 1530 Third Avenue South, Birmingham, AL
35294-0006. E-mail address: [email protected]
3
Abbreviations used in this paper: PspA, pneumococcal surface protein A; CR, complement receptor; FB, factor B; FD, factor D; MBL, mannose-binding lectin; WT,
wild type.
Copyright © 2004 by The American Association of Immunologists, Inc.
3 (C3) (4 –7). Ipso facto, PspA⫺ pneumococci that are avirulent in
normal mice become virulent in C3-deficient (C3⫺/⫺) and factor
B-deficient (FB⫺/⫺) mice (5).
It is currently accepted that three different pathways can lead to
complement activation: the classical pathway, the mannose-binding lectin (MBL) pathway, and the alternative pathway (8). All
three pathways converge upon activation of C3, which is cleaved
into C3a and C3b. This process exposes a thioester group on the
␣-chain of C3b that allows C3b to covalently attach to any activating surface in close proximity (9). With the assistance of various cofactors, surface-bound C3b is then sequentially processed,
via the enzymatic activity of factor I, into smaller fragments (iC3b
and C3d/C3dg). Importantly, C3b and its downstream cleavage
products remain covalently bound to the target surface and act as
ligands for different complement receptors (CRs) on various host
phagocytes (10). Consequently, microbial pathogens sufficiently
coated with C3 fragments are recognized by phagocytes.
The phagocyte CRs include CR1 (also called CD35), CR3 (also
called ␣M␤2, Mac-1, or CD11b/CD18), and CR4 (also called
␣X␤2, p150,95, or CD11c/CD18) (11). CR1 reportedly has high
affinity for C3b and lower affinity for iC3b, and also binds C4b,
C1q, and MBL (12–14). Likewise, murine CR1 has binding sites
for both C3b and C4b (15, 16). Because CR1 has cofactor activity,
it also participates directly in complement activation by facilitating
the factor I-mediated conversion of C3b to iC3b. This action of
CR1 can down-regulate further complement activation (17). A
closely related, but nonphagocytic, receptor, CR2 (CD21), binds
C3dg/C3d and thereby influences B cell activation and development (16, 18). In the mouse, CR1 and CR2 are alternatively spliced
products of a single Cr2 gene (19). In contrast to CR1, CR3 and
CR4 have high affinity for iC3b and lower affinity for C3b. CR3
and CR4 are both members of the ␤2 integrin family, which also
0022-1767/04/$02.00
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Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of
complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A
(PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated
host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of
PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2ⴚ/ⴚ), CR3
(CR3ⴚ/ⴚ), or CR4 (CR4ⴚ/ⴚ) were challenged with WU2, a PspAⴙ capsular serotype 3 pneumococcus, and its PspAⴚ mutant
JY1119. Pneumococci also were used to challenge factor D-deficient (FDⴚ/ⴚ), LFA-1-deficient (LFA-1ⴚ/ⴚ), and CD18-deficient
(CD18ⴚ/ⴚ) mice. We found that FDⴚ/ⴚ, CR3ⴚ/ⴚ, and CR4ⴚ/ⴚ mice had significantly decreased longevity and survival rate upon
infection with WU2. In comparison, PspAⴚ pneumococci were virulent only in FDⴚ/ⴚ and CR1/2ⴚ/ⴚ mice. Normal mouse serum
supported more C3 deposition on pneumococci than FDⴚ/ⴚ serum, and more iC3b was deposited onto the PspAⴚ than the PspAⴙ
strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable
for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway.
Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against
pneumococcal infection. The Journal of Immunology, 2004, 173: 7506 –7512.
The Journal of Immunology
7507
includes LFA-1 (also called ␣L/␤2 or CD11a/CD18) and ␣D/␤2
(CD11d/CD18) (20). CR3 and LFA-1 are important adhesion molecules involved in the migration of polymorphonuclear neutrophils
and monocytes from the periphery to sites of inflammation (20).
In the present study the impact of deletion of various CRs on
pneumococcal virulence and on PspA’s virulence function was
assessed by comparing the outcome of infections with PspA⫹ and
PspA⫺ pneumococci in wild-type and various CR-deficient mice.
Our results suggest that in the mouse, CR1, CR3, and CR4 each
contribute to host defense again pneumococcal infection.
diluted in lactated Ringer’s buffer to achieve the desired working concentration. The number of bacteria injected into mice was confirmed by plating
residual inoculums on blood agar. To assess the net clearance/growth of
pneumococci in the circulation, mice were challenged i.v. with 2 ⫻ 105
CFU/mouse, and 50 ␮l of blood was collected from the retro-orbital plexus
at 5 min, 1 h, 2 h, 4 h, and 6 h postinoculation. Blood was serially diluted
and plated on blood agar plates to determine the number of viable S. pneumoniae recovered. The limit of detection was 102 CFU of bacteria/ml
blood; thus, when no pneumococci grew on culture plates, a level of bacteremia of 102 CFU/ml was reported. The mortality of mice was monitored
for 10 days after challenge.
Materials and Methods
Complement deposition assays
Animals
Pneumococci were grown in Todd-Hewitt broth supplemented with 0.5%
yeast extract until the OD600 nm was 0.45 (⬃108 CFU/ml), and 150 ␮l of
bacterial culture was decanted. Bacteria were washed twice with PBS and
resuspended in 50 ␮l of 10% normal mouse serum (isolated and pooled
from five naive WT C57BL/6J mice) diluted in gelatin veronal buffer with
calcium (0.15 mM) and magnesium (0.5 mM; GVB2⫹; Sigma-Aldrich, St.
Louis, MO). The bacteria were thus opsonized at 37°C for 5, 10, or 30 min,
after which ice-cold PBS buffer containing 10 mM EDTA was added to
stop the reaction. Opsonized bacteria were washed, and the bacteria were
resuspended in 50 ␮l of reducing SDS sample buffer, boiled for 10 min,
and loaded onto SDS-polyacrylamide gels for electrophoresis. Subsequent
immunoblots were prepared and developed using goat anti-mouse C3 polysera (Bethyl Laboratories, Montgomery, TX) in concert with biotin-conjugated rabbit anti-goat IgG (H and L chains) and alkaline phosphataseconjugated streptavidin. Immunoreactive bands of protein were quantitated
by densitometry using the Bio-Rad gel documentation system (Bio-Rad,
Hercules, CA). To validate the findings and determine their clinical relevance, the experiment was repeated using normal human serum (lot
OM1013; Quidel, San Diego, CA). In this case, purified human C3, C3b,
and iC3b proteins were used as controls, and the immunoblot was developed using goat anti-human C3 polysera (both from Quidel).
Wild-type (WT) C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Factor D-deficient (FD⫺/⫺), Cr2-deficient (CR1/
2⫺/⫺), CD11b-deficient (CR3⫺/⫺), CD11c-deficient (CR4⫺/⫺), CD11a-deficient (LFA-1⫺/⫺), and CD18-deficient (CD18⫺/⫺; mice that lack
functional CR3, CR4, and LFA-1) mice were from our own colonies. The
construction and phenotype of each targeted mutant have been fully described previously (19, 21–23). All these mice are either C57BL/6J congenic or were backcrossed to C57BL/6J for at least six generations before
use. All animals were housed in separate cages, and none exhibited any
evidence of infection before pneumococcal challenge at 10 –12 wk of age.
Bacteria and infections
A capsular type 3 pneumococcal strain (WU2, PspA⫹) (24, 25) and its
isogenic mutant (JY1119, PspA⫺) (4, 7, 26) were used. Bacterial stocks
were frozen at ⫺80°C in Todd-Hewitt broth supplemented with 0.5% yeast
extract containing 10% glycerol. To maintain the mutant PspA⫺ genotype,
which carries an erythromycin resistance cassette, the culture medium for
JY1119 always contained erythromycin (0.3 ␮g/ml). Frozen stocks of each
strain containing a known concentration of viable bacteria were thawed and
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FIGURE 1. Blood clearance/growth of PspA⫹
WU2 and PspA⫺ JY1119 pneumococci in normal
vs mutant mice. WT mice (solid lines) and mutant mice (dashed lines; deficient protein indicated) were infected i.v. with ⬃2 ⫻ 105 CFU of
WU2 (F) or JY1119 (E). Bacteremia was determined at different time points. Data are presented as the mean ⫾ SEM CFU per milliliter of
blood for six to 12 mice (indicated) in each
group. The asterisks indicate that bacteremia in
deficient mice is significantly different (p ⬍
0.05) from that in WT mice infected with the
same strain of pneumococcus.
7508
PspA INHIBITS COMPLEMENT AND COMPLEMENT RECEPTORS
Flow cytometry was used to further quantitate the amount of C3 deposited onto bacteria. Pneumococci were incubated with 30% FD⫺/⫺ or WT
serum in GVB2⫹ at 37°C for 30 min. Bacteria incubated with only GVB2⫹
served as a negative control. After washing, bacteria were stained with
FITC-conjugated goat IgG to mouse C3, and the fluorescence intensity of
each sample was determined using a FACScan instrument (BD Biosciences, Mountain View, CA)
Surface expression of CRs on peripheral blood cells
The expression of CR1/2 and CR3 on peripheral blood cells was determined by staining freshly isolated cells with fluorochrome-conjugated goat
anti-mouse CD21/35 or anti-mouse CD11b, respectively. Briefly, heparinized blood from WT, CR1/2⫺/⫺, and CD18⫺/⫺ mice was washed with
HBSS containing 0.1% BSA and stained with FITC-conjugated rat antimouse mAb 7G6 (anti-CD21/35) or R-PE-conjugated mAb M1/70 (antiCD11b), both obtained from BD Pharmingen (San Diego, CA). After one
wash, each blood sample was incubated with FACS lysing solution (BD
Biosciences) to deplete erythrocytes. By flow cytometry, total leukocytes
were gated based on forward and side scatter properties, and the fluorescence intensity profiles of the gated cell populations were determined.
Measurement of serum Abs
Statistical analysis
To compare levels of bacteremia, Student’s t tests were applied to logtransformed data. To compare survival times (longevity) of WT and mutant
Results
PspA⫺ pneumococci persist in the blood and cause fatal
bacteremia in FD⫺/⫺ mice
We reported previously that when PspA⫹ vs PspA⫺ pneumococci
were used to challenge FB⫺/⫺ mice, the PspA⫺ bacteria were as
virulent as PspA⫹ bacteria (5). However, because the mouse factor
B-encoding gene, Bf, resides within the H-2 locus, we could not
formally rule out the possibility that disruption of H-2, rather than
absence of FB, was responsible for the increased susceptibility of
FB⫺/⫺ mice to PspA⫺ pneumococci. FB and FD are both essential
for formation of the alternative pathway C3 convertase. C3b, the
activated form of C3, binds FB and presents it for cleavage by FD,
yielding C3bBb, the alternative pathway C3 convertase. C3bBb
sequentially activates more C3 and triggers the alternative pathway
amplification loop. Consequently, like FB⫺/⫺ mice, FD⫺/⫺ mice
have no operable alternative pathway and have impaired classical
pathway activity due to the absence of an amplification loop. Importantly, FD⫺/⫺ mice are not likely to have H-2 impairment.
We determined the ability of FD⫺/⫺ to resist infection with the
PspA⫹ and PspA⫺ strains. The clearance/growth of pneumococci
in WT and FD⫺/⫺ mice was not different for the first 4 h after
infection with PspA⫹ WU2, but pneumococcal burden was significantly higher (⬃5-fold higher) in FD⫺/⫺ than WT by 6 h (Fig.
1A, F). As a consequence, the longevity and survival rate of
FD⫺/⫺ mice challenged with WU2 were significantly lower than
those in WT mice ( p ⫽ 0.013; Fig. 2, left panel). In contrast, the
PspA⫺ JY1119 strain was completely cleared from the blood of
FIGURE 2. Survival times of WT and mutant mice infected i.v. with ⬃2 ⫻ 105 CFU of WU2 (left panel) or JY1119 (right panel). Each dot or cross
represents the death of a single mouse, and the mutant gene carried by mice is indicated on the abscissa. For animals infected with each kind of bacteria,
the median time to death (horizontal lines) is shown. The longevity of mutants was compared with that of WT mice, and mutants whose survival time was
significantly less than that of WT mice infected with the same bacteria (p ⬍ 0.05) are indicated with an asterisk.
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Sera from CD1/2⫺/⫺, CD18⫺/⫺, and WT mice were collected, and the
levels of Abs directed against heat-killed pneumococci and phosphocholine
Ag therein were determined by ELISAs. Microtiter plates were coated with
heat-killed WU2 (108 CFU/well in PBS) or phosphocholine-conjugated
BSA (5 ␮g/well) overnight at 4°C. Mouse serum was serially diluted and
added to the plates. ELISA plates were washed, sequentially developed
with biotin-conjugated, goat anti-mouse Ig and streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL), and
scanned at 405 nm.
mice infected with PspA⫹ and PspA⫺ S. pneumoniae, Mann-Whitney U
tests were used.
The Journal of Immunology
WT in ⬍4 h, but persisted in FD⫺/⫺ (Fig. 1A, E). WT mice challenged with JY1119 all survived at least 10 days, whereas FD⫺/⫺
mice challenged with JY1119 had significantly shortened longevity (3.5 days; p ⫽ 0.004 compared with WT) and only 14% survived (Fig. 2, right panel). In vitro, WT serum supported more C3
deposition onto pneumococci than did FD⫺/⫺ serum regardless of
whether WU2 or JY1119 was being opsonized, and the amount of
C3 deposited onto JY1119 was consistently greater than that deposited onto WU2 (Fig. 3). Together, these results reinforce those
we reported previously using FB⫺/⫺ mice, confirming the importance of an intact alternative pathway for the efficient clearance/
killing of pneumococci in the mouse. They also support our hypothesis that PspA circumvents the role of this pathway in
protection.
Clearance/killing of blood-borne PspA⫺ pneumococci is slower
in CR1/2⫺/⫺ mice, leading to fatal infection
FIGURE 3. C3 deposition on pneumococci in the presence of WT and
FD⫺/⫺ mouse sera. Pneumococci were incubated in 30% pooled WT or
FD⫺/⫺ serum at 37°C for 30 min, and C3 deposition was detected by flow
cytometry using FITC-conjugated goat anti-mouse C3 IgG. For WU2 incubated with WT serum, FD⫺/⫺ serum, and buffer (no serum), the mean
fluorescence intensities achieved were 71.2, 36.8, and 4.1 U, respectively.
For JY1119 incubated with WT serum, FD⫺/⫺ serum, or buffer, the mean
fluorescence intensities were 168.9, 64.5, and 3.6, respectively.
tion with JY1119. These data indicate that CR3 and CR4 also are
required for full protection from pneumococci.
Clearance/killing of blood-borne pneumococci is marginally
improved in LFA-1⫺/⫺ and CD18⫺/⫺ mice
Both PspA⫹ and PspA⫺ bacteria disappeared from the blood of
LFA-1⫺/⫺ marginally faster than from WT mice (Fig. 1E, dashed
lines), and the longevity of WU2-infected LFA-1⫺/⫺ mice was
somewhat prolonged (Fig. 2, left panel). Similarly, and despite
their inherited absence of functional CR3 and CR4, WU2 was
removed more quickly from the blood of CD18⫺/⫺ than from WT
mice (Fig. 1F, dashed lines), and no difference was observed in the
survival time of CD18⫺/⫺ and WT mice infected with either pneumococcus (Fig. 2).
Low expression of CR1/2 and high anti-pneumococcal Ab titers
in CD18⫺/⫺ mice
CD18 is the common ␤-chain shared by CR3, CR4, and LFA-1;
thus, deficiency of CD18 results in a lack of all three functional
molecules; yet CD18⫺/⫺ mice resisted infection. A compensatory
increase in the expression of CR1/2 in CD18⫺/⫺ mice could explain this seeming paradox. However, CD18⫺/⫺ mice had ⬎2-fold
less expression of CR1/2 on their PBL than WT mice (Fig. 4A,
compare bottom left and bottom right panels). Alternatively, a
compensatory heightened level of naturally occurring Abs in
CD18⫺/⫺ could also explain our observation. Indeed, we observed
that CD18⫺/⫺ did have much higher levels of Igs reactive with
heat-killed pneumococci (Fig. 4B, left panel). It is therefore likely
that bacteremia is lessened, and survival rate is unaffected in
CD18⫺/⫺ mice compared with WT mice, because they make
higher than normal levels of protective anti-pneumococcal Abs.
Likewise, although CR1/2⫺/⫺ mice had normal expression of CR3
(Fig. 4A) and WT-like levels of anti-pneumococcal Abs (Fig. 4B,
bottom left panel), their lower levels of anti-phosphocholine Abs
(Fig. 4B, lower right panel) might account for their extreme
susceptibility.
Degradation of C3 on the pneumococcal surface
Because CR1/2, CR3, and CR4 have variable affinities for similar
C3-derived ligands, we sought to determine which C3-derived
fragments are present on the surface of serum-opsonized pneumococci. To this end, WU2 and JY1119 were incubated in 10% normal serum to allow opsonization to proceed. The incubation was
terminated at different time points by the addition of cold PBS
buffer containing EDTA. C3 retained on the surface of the opsonized bacteria was then revealed by immunoblot analysis (Fig.
5A). Based on comparison with purified human C3, C3b, and iC3b
controls and the presence of the ␣2 fragment (Fig. 5B), the major
form of C3 deposited on WU2 and JY1119 appears to be iC3b,
although C3b was also present in some samples. As incubation
time was increased, iC3b accumulated on both types of bacteria
(Fig. 5A). Importantly, however, at each time point comparatively
more iC3b was present on JY1119 than on WU2. With the BioRad gel documentation system, we measured the densities of ␣2
and ␤ subunits in each sample. At 5, 30, and 60 min, the ␤-chain
signal was 1.44-, 1.47-, and 1.32-fold greater, respectively, for
JY1119 than for WU2. This indicates that deposition of C3 per se
was consistently greater for the PspA⫺ strain. Contemporaneously,
the ␣2 fragment was 2.03-, 1.91-, and 1.09-fold more intense, respectively, for JY1119 than for WU2. This indicates that processing of deposited C3 is more rapid on JY1119 than on WU2. Based
on the ␣2/␤ ratio, processing of C3b to iC3b was estimated to be
1.4- and 1.3-fold greater for JY1119 than WU2 at 5 and 30 min,
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For up to 6 h postinfection, no significant difference in blood clearance/killing of WU2 was observed in CR1/2⫺/⫺ mice compared
with WT mice (Fig. 1B, F); however, the clearance of JY1119 was
significantly delayed in CR1/2⫺/⫺ compared with WT mice (Fig.
1B, E). Thus, at 4 h postinfection, 10-fold more PspA⫺ pneumococci were recovered from the blood of CR1/2⫺/⫺ mice than from
contemporaneous samples from WT mice. In WU2-infected CR1/
2⫺/⫺ mice, the 10-day survival rate (12%; one of eight) was lower
than that in WU2-infected WT (42%; five of 12), although median
longevity was not significantly different (Fig. 2, left panel). All
WT mice challenged with JY1119 survived infection, but only
20% (one of five) of CR1/2⫺/⫺ mice did ( p ⫽ 0.018; Fig. 2, right
panel). These data reveal that, like FB and FD, CR1/2 is required
for full protection from pneumococci.
Clearance/killing of blood-borne PspA⫺ pneumococci in CR3⫺/⫺
or CR4⫺/⫺ mice is slower, but does not lead to fatal infection
The residence time of WU2 in the bloodstream of CR3⫺/⫺ and
CR4⫺/⫺ mice was not substantially different from that observed in
WT mice; bacteremia was fairly constant throughout the experiment in all cases (Fig. 1, C and D, F). Despite this, both kinds of
receptor-deficient mice showed increased death rates; 88% of
CR3⫺/⫺ and 75% of CR4⫺/⫺ died within 72 h compared with 58%
of WT mice (Fig. 2, left panel). The median longevity of CR3⫺/⫺
and CR4⫺/⫺ mice was significantly reduced compared with that of
WT mice ( p ⫽ 0.003 for CR3⫺/⫺; p ⫽ 0.007 for CR4⫺/⫺). In
contrast to WU2, clearance/killing of JY1119 was significantly
slower in both CR3⫺/⫺ and CR4⫺/⫺ compared with WT mice;
thus, at 4 h postinfection, 100-fold more PspA⫺ pneumococci
were recovered from CR3⫺/⫺ than WT mice (Fig. 1C), and 10-fold
more was recovered from CR3⫺/⫺ (Fig. 1D). However, unlike
CR1/2⫺/⫺ mice, all CR3⫺/⫺ and all CR4⫺/⫺ mice survived infec-
7509
7510
PspA INHIBITS COMPLEMENT AND COMPLEMENT RECEPTORS
respectively, but by 60 min, the advantage was negated. The difference in the amount of iC3b deposited onto the two strains of
pneumococci was more obvious when mouse, rather than human,
serum was used as the complement source, but the reaction was
slower. These findings are consistent with the proposal that more
C3 is deposited on JY1119 than WU2, the majority of C3 deposited is iC3b, and the rate of conversion of C3b to iC3b is increased
on JY1119. The fact that similar observations were made using
mouse vs human serum suggests that this has clinical relevance.
Discussion
In this study and previous ones (4 –7), we showed that substantially
less C3 is deposited onto the PspA⫹ S. pneumoniae strain WU2
than onto its isogenic PspA⫺ counterpart JY1119 regardless of the
serum source, and that WT mouse serum supports more deposition
of C3 onto the pneumococcal surface than FB⫺/⫺ (7) or FD⫺/⫺
serum (Fig. 3). This decrease in deposition of C3 in vitro correlates
with impaired host resistance to infection. Thus, in FD⫺/⫺ mice
(Figs. 1 and 2), JY1119 becomes as virulent and as lethal as WU2
is in normal mice. Together with observations made previously by
others (27), our initial findings (5), and the new data we report in
this study clearly indicate that in mice an intact alternative pathway is vital for proper host defense against S. pneumoniae infection, and that PspA impairs this pathway of protection. Recently,
Brown et al. (28) argued that the classical pathway is the dominant
pathway for host innate immunity against pneumococcal infection
in mice. Our current findings are not at odds with this proposition.
Indeed, the classical and alternative pathways are probably both
required for full protection against pneumococcal infection, be-
cause the latter pathway amplifies C3 deposition initiated by the
former.
Immunoblots provided indirect evidence that one of the major
forms of C3 on opsonized WU2 and JY1119 is iC3b, a fragment
known to serve as an efficient ligand for CR3, CR4, and perhaps
also CR1/2. For mice infected with WU2, the strain on which C3
deposition and blood clearance/killing is limited by the presence of
PspA, deficiency of any one of these CRs is thus sufficient to
increase the mortality rates of mice. Notably, the negative impact
on survival rate and longevity achieves statistical significance in
CR3⫺/⫺ and CR4⫺/⫺ mice, an outcome consistent with the reported high affinity binding of iC3b to these receptors. In contrast,
in mice challenged with JY1119, to which more C3 binds and
blood clearance/killing is more efficient because PspA is absent,
deficiency of CR3 or CR4 did not impact survival, but deficiency
of CR1/2 significantly decreased it. Furthermore, CR1/2⫺/⫺,
CR3⫺/⫺, and CR4⫺/⫺ mice all showed retarded removal of
JY1119 from the bloodstream.
CR1/2 also plays an important role in the humoral immune response. Haas et al. (19) reported that serum IgM, IgG1, IgG2b, and
IgG3 are all significantly reduced in CR1/2⫺/⫺ mice, whereas
IgG2a and IgA are normal. In our hands, the titer of antipneumococcal Abs in CR1/2⫺/⫺ did not differ from WT mice,
whereas the titer of anti-phosphocholine Abs was indeed substantially lower. Anti-phosphocholine Abs are known to be protective
against pneumococcal infection (29, 30); thus, CR1/2⫺/⫺ mice are
probably highly susceptible to the normally avirulent PspA⫺ pneumococci, because, on the one hand, they lack CR1/2 and, on the
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FIGURE 4. A, Surface expression of CR3 (top
row) and CR1/2 (bottom row) on peripheral blood
cells of WT, CR1/2⫺/⫺, and CD18⫺/⫺ mice. PBL
were stained with anti-CD11b (anti-CR3) mAb or
anti-CD21/35 (anti-CR1/2) mAb (shaded areas) or
were left unstained (solid line). The data shown are
representative of results obtained from groups of
three to five mice. B, Serum Abs to heat-killed pneumococci and phosphocholine Ag. Serum was collected from WT, CR1/2⫺/⫺, and CD18⫺/⫺ mice
(three to five mice per group), and Ab titers (IgG
plus IgM) determined by ELISA. The results are
presented as the mean ⫾ SE for each titration.
The Journal of Immunology
7511
other, they have reduced titers of this (and perhaps other) protective Abs. CR1 probably also interacts with C1q and MBL (12–14),
and CR1 and Fc receptors exert synergetic effects to enhance
phagocytosis (31), so the host defense role of CR1/2 in mice infected with JY1119 probably has additional complexity and importance. The relative importance of factor H-mediated clearance
of opsonized bacteria by platelets, which presumably direct uptake
of the organism by the liver and spleen (32), also remains to be
determined.
Previous studies showed that CD18-deficient animals have increased spontaneous infections and earlier mortality compared
with WT mice (33). Others reported that LFA-1⫺/⫺ mice have
increased mortality and a higher incidence of otitis media. These
mice are more likely to develop meningitis after being challenged
with S. pneumoniae i.p., and the LFA-1-ligand interaction is the
stimulatory signal for neutrophils to express full activation. In our
hands, there was no significant difference in mortality between
CR3⫺/⫺, CR4⫺/⫺, and CD18⫺/⫺ mice after infection with either
PspA⫹ or PspA⫺ pneumococci. The fact that CR3⫺/⫺ and
CR4⫺/⫺ mice express normal amounts of CR1/2 (data not shown)
partially explains why these animals are still resistant to JY1119.
LFA1⫺/⫺ and CD18⫺/⫺ mice cleared WU2 somewhat more rapidly than WT, and the survival time of WU2- and JY1119-infected
animals was not adversely affected by deficiency of LFA-1. This
difference between our observations and those of others might be
due to the different outcome parameters and routes of infection
used; i.e., we focused on blood clearance/killing after i.v. infection, whereas others used an i.p. infection model to emphasize the
migration ability of phagocytes into the sites of infection. Moreover, in an earlier i.p. experiment a serotype 6 pneumococcal strain
was used (34), whereas we used a serotype 3 strain. Notably, LFA1⫺/⫺ animals reportedly have a higher number of neutrophils in
the peripheral blood than WT (33, 34), which could also account
for their improved resistance to i.v. infection.
The seeming paradox that CD18⫺/⫺ mice remain fully resistant
can be explained. CD18⫺/⫺ mice lack CR3 and CR4 and have
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FIGURE 5. A, Western blot analysis of C3 deposition on PspA⫹ (WU2) and PspA⫺ JY1119 (JY) pneumococci. A, Pneumococci were opsonized with
human serum (left panel) or mouse serum (right panel) for the times indicated, washed with PBS, boiled in reducing buffer, and electrophoresed, then
transferred to nitrocellulose. Membranes were developed with anti-human or anti-mouse C3 Abs as required. Purified human (Hu) C3, C3b, and iC3b were
included as controls. The identity of each chain or degraded fragment of C3 is indicated. B, Schematic view of C3 processing during complement activation.
After covalent attachment of C3b to pneumococci via the ␣-chain thioester bond (ⴱ), cleavage of the ␣-chain of C3 by the cofactor-dependent action of
factor I (FI) generates iC3b, which remains bound to the opsonized bacterium.
7512
PspA INHIBITS COMPLEMENT AND COMPLEMENT RECEPTORS
Acknowledgments
We thank Drs. Gunnar Lindahl and Susan K. Hollingshead for reviewing
our work and for their many helpful suggestions.
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lower expression of CR1/2, which tends to make them more susceptible. In contrast, these mice are predicted to have neutrophilia
associated with their lack of LFA-1 (33, 34), and we showed that
they express elevated levels of anti-pneumococcal Abs. We propose that in CD18⫺/⫺ mice, the combined benefits of neutrophilia
and high protective Ab titers overcomes the detrimental effects of
low CR1/2 expression and the absence of CR3 and CR4.
As in this study, others have infected mutant mice with S. pneumoniae to study the contributions to protection made by CR1/2,
CR3, CD18, and LFA-1 (19, 33, 34). These earlier studies focused
on the impact of CR1/2 deficiency on the development of protective adaptive immunity or on the effects of CD18, LFA-1, and CR3
on long term survival after i.p. inoculation of pneumococci. Notably, our study is the first comprehensive investigation of the contribution of these receptors to protection against PspA⫹ and
PspA⫺ pneumococcal strains and to date is the only one to study
the impact of CR4 deletion on host defense. In summation, we
conclude that PspA confers virulence onto S. pneumoniae mainly
by interfering with the protective role of the alternative pathway of
complement activation, thus minimizing C3b deposition and slowing iC3b generation. The pneumococcus is thereby shielded from
host CR-mediated pathways of host defense.