Species-Specific Interaction of Streptococcus pneumoniae with
Human Complement Factor H1
Ling Lu,* Zhuo Ma,* T. Sakari Jokiranta,†Adeline R. Whitney,‡Frank R. DeLeo,‡
and Jing-Ren Zhang2*
Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in
remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and
disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial
immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway
inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a
major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse
and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in
obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-
specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal
fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional
evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-
mediated host defense in humans. The Journal of Immunology, 2008, 181: 7138–7146.
otitis medium, and sinusitis (1). The nasopharynx of humans is the
only natural reservoir for the pneumococci, although other animal
species can be experimentally infected with the bacterium (2). The
bacterial and host determinants for the strict host tropism of S.
pneumoniae have not been defined. S. pneumoniae can be fre-
quently carried as a commensal organism in healthy adults, but
causes severe infections in individuals without a fully functional
immune system (1). Clinical surveys and experimental evidence in
animal models have indicated the complement system is an essen-
tial element of host defense against the pneumococci (3–8). This
is exemplified by the observations that patients deficient in com-
plement proteins C2 and C3 have increased susceptibility to re-
current pneumococcal infections (9, 10).
Previous studies have also implicated several strategies used by
S. pneumoniae to avoid complement attack. Pneumococcal surface
protein A, a major surface protein, is able to interfere with acti-
vation of the alternative complement pathway by blocking the dep-
he Streptococcus pneumoniae (the pneumococcus) is a
Gram-positive bacterium that causes a wide spectrum of
infections, such as pneumonia, bacteremia, meningitis,
osition of C3 on the pneumococcal surface (11–14). Pneumolysin,
the only well-characterized pneumococcal toxin, is able to deplete
complement by promoting activation of the classical complement
pathway (15, 16). Pneumococcal surface protein A- and pneumo-
lysin-deficient strains of S. pneumoniae are significantly attenuated
in terms of their virulence levels in mice (17, 18). A third com-
plement evasion mechanism has been implicated in S. pneu-
moniae, which involves the recruitment of complement factor H
(FH)3by choline-binding protein A (CbpA) (19–25).
CbpA, also known as PspC (26), SpsA (27), Hic (19), or C3-bind-
ing protein (28), is a major surface-exposed protein of S. pneumoniae
(29). The cbpA locus exists in all virulent strains tested to date (30,
31). CbpA is considered a virulence factor because CbpA-deficient
pneumococcal strains have attenuated capacity to colonize the naso-
pharynx and cause infections in the lungs and bloodstream in animal
mococcal survival in vivo and pathogenesis are not completely un-
on in vitro investigations with epithelial cultures (29, 35, 36). In these
studies, CbpA was shown to interact with sialic acid (29), human
polymeric IgR (pIgR) (35, 37), and complement C3 protein (36). In
addition, CbpA has been shown to bind to free host factors, including
FH (19, 20), C3 (28), secretory component (SC) (35, 37), and secre-
tory IgA (SIgA) (27, 38). The findings from our previous studies (35,
38) and others (39) have demonstrated that CbpA only interacts with
pIgR, SC, and SIgA of humans, but not the counterparts from com-
mon model animals, including mouse, rat, and rabbit, suggesting
CbpA as a bacterial determinant for the host tropism of S. pneu-
moniae. Finally, CbpA confers protective immunity against lethal
challenge of virulent pneumococci in animal models (29, 30, 32, 40).
CbpA is among a few pneumococcal proteins that can stimulate Ab
production in humans (41, 42).
*Center for Immunology and Microbial Disease, Albany Medical College, Albany,
NY 12208;†Department of Bacteriology and Immunology, Haartman Institute and
HUSLAB, University of Helsinki, Helsinki, Finland; and‡Laboratory of Human Bac-
terial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
Received for publication December 4, 2007. Accepted for publication September
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.
1This work was supported in part by the Intramural Research Program of the National
Institutes of Health, National Institute of Allergy and Infectious Diseases, and by a
research grant from the National Institutes of Health/National Institutes of Deafness
and Other Communication Disorders (DC006917).
2Address correspondence and reprint requests to Dr. Jing-Ren Zhang, Center for
Immunology and Microbial Disease, Albany Medical College, M/C 151, Room
MS453, 47 New Scotland Avenue, Albany, NY 12208. E-mail address: zhangj@
3Abbreviations used in this paper: FH, factor H; CbpA, choline-binding protein A;
pIgR, polymeric IgR; PMN, polymorphonuclear leukocyte; PVDF, polyvinylidene
difluoride; RPMI/H, RPMI 1640 medium buffered with 10 mM HEPES; SC, secretory
component; SIgA, secretory IgA.
The Journal of Immunology
Based on extensive sequence variations in the CbpA locus, Ian-
nelli et al. (31) have divided the CbpA allelic variants into 11 PspC
types. The typical CbpA alleles (types 1–6) in the majority of
pneumococcal isolates consist of three N-terminal ?-helical do-
mains and are anchored to the cell wall choline via the C-terminal
choline binding domain (31, 43). In contrast, the CbpA alleles in
PspC types 7–11 (referred to as nontypical CbpA hereafter), in-
cluding the Hic protein, possess nontypical N-terminal domains
and are expressed at the cell surface via an LPXTG anchoring
motif (31, 44). Consistently, the sequence homology levels be-
tween the typical and nontypical CbpA alleles are much lower
(?30% sequence identity) than that among the CbpA alleles
within the two CbpA groups. Our recent study has mapped the
FH-binding activity to the N-terminal domain of CbpA in a type 2
strain D39 (PspC type 3) (25). Hic (PspC type 11) was the first
CbpA allele shown to bind human FH, but our recent study showed
CbpA of strain D39 has 23-fold or higher affinity to human FH as
compared with Hic of a type 3 strain A66 (19, 25). The biological
implications of these differences among the CbpA alleles on pneu-
mococcal carriage and virulence remain unclear.
FH protects host cells from random deposition of C3b and non-
specific complement activation by inhibiting the activation of the
alternative complement pathway (45). Many pathogenic bacteria,
including S. pneumoniae, have been shown to evade complement-
mediated host defense by recruiting FH to the bacterial surfaces
(46–48). The FH proteins from the characterized animal species
are all composed of 20 short consensus repeats and share a similar
molecular size ?155 kDa (45). CbpA and its allelic variants bind
to short consensus repeats 6–10 (23), 8–11 (24, 49), 13–15 (21),
and 19–20 (49) of human FH. In addition to its antiphagocytic
property (24, 50), recent studies also showed CbpA-FH interaction
enhances pneumococcal adhesion to and invasion of host cells (49,
51). The FH proteins from different mammalian species have
extensive sequence variations, which is exemplified by only
61% sequence identity between human (1231 aa) and mouse FH
(1234 aa). Previous studies demonstrate that the OspE and
BBA68 proteins of Borrelia burgdorferi selectively bind to hu-
man, but not mouse FH (52, 53). In this study, we showed that
CbpA binds only to human FH, but not the FH variants from
mouse and other animal species tested to date. This species-
specific interaction of S. pneumoniae with human FH is con-
sistent with our observation that, in the mouse sepsis model, the
FH-binding negative pneumococci had no significant reduction
in virulence or survival defect in the bloodstream. Because hu-
mans are the only natural host for S. pneumoniae, our data
suggest that the CbpA-mediated recruitment of complement FH
may contribute to host tropism of this pathogen.
Materials and Methods
Bacterial strains and growing conditions
The capsular serotype 2 strain D39 and its isogenic derivatives ST588 and
ST650 were previously described (25). Additional pneumococcal isolates
from blood and cerebrospinal fluid were provided by the Active Bacterial
Core Surveillance Program at the Centers for Disease Control and Preven-
tion. The basic characteristics of these strains are provided in Table I. The
bacteria were routinely grown in Todd-Hewitt broth containing 0.5% yeast
extract or on tryptic soy agar plates containing 3% (v/v) sheep blood.
Western blot was performed essentially as described previously (25).
Briefly, rCbpA2 protein with an N-terminal His tag (1 ?g/lane) or bacterial
cell lysates (5 ?g of total protein/lane) were boiled for 5 min in standard
SDS-PAGE gel-loading buffer (54), and subjected to electrophoresis in
10–20% Tris-HCl SDS-PAGE gels (Bio-Rad). Protein concentrations of
the fractions were determined by the Bio-Rad protein assay reagent.
CbpA2 represents the 254 aa of CbpA in strain D39, including the FH
binding domain (35). Serum samples were treated in a similar manner,
except that the final volumes of the serum-loading buffer mixtures were
adjusted to equal volumes with deionized water before boiling. The pro-
teins were blotted to polyvinylidene difluoride (PVDF) membranes (Mil-
lipore) with a semidry electrotransfer apparatus (Bio-Rad), according to the
manufacturer’s instructions. The blots were blocked with 5% milk (w/v)
and washed three times in PBS before the detection step.
The FH proteins in human and mouse serum samples were detected with
a goat anti-human FH Ab (1/2500; Calbiochem) and a rat anti-mouse FH
Ab (1/2000) (55), respectively. The binding of serum FH to CbpA was
assessed by incubating the protein blots of rCbpA2 or pneumococcal ly-
sates with normal human (1/1000 dilution) or mouse serum (1/100 dilution)
overnight at 4°C at dilutions. The reactivity of pneumococcal lysates to
purified human FH (Sigma-Aldrich; 0.4 ?g/ml) and purified human SIgA
(Sigma-Aldrich; 0.4 ?g/ml) was detected, as described previously (25).
The levels of reactivity were visualized with appropriate secondary Ab-
peroxidase conjugates by the ECL Western blot kit (Pierce), according to
the supplier’s instructions. A peroxidase-conjugated rabbit anti-goat IgG
Ab (1/5000; Bio-Rad) was used as secondary Ab to detect CbpA binding
to human FH at a final dilution of 1/5000. CbpA binding to mouse FH was
visualized with a peroxidase-conjugated rabbit anti-rat IgG Ab (Invitrogen)
at a final dilution of 1/2000.
Table I. S. pneumoniae strains used in this study
Reactivity with FHe
Reactivity with SIgAe
Reference or SourceAccession No.f
aThe first line represents the designation of the isolates in our strain collection. The strain identifications of the original source are provided on the second line.
bCapsule types are based on the information from the original provider or publication.
cTissue site information of strains is based on the original provider or publication.
dPspC types of pneumococcal strains are assigned according to the sequence homology of their CbpA alleles to the previously described sequences of known PspC types
eThe positive (?) and negative (?) reactivities of each strain to human FH and SIgA are assigned based on the presence or absence of Western blot signal. The data do not
reflect potential quantitative differences among strains in terms of CbpA expression or binding affinity.
fThe GenBank accession numbers for all cbpA alleles except for that of strain D39 (31) represent new sequence entries as a result of this study. NA ? not applicable.
gCDC, Centers for Disease Control and Prevention; CSF, cerebrospinal fluid.
7139The Journal of Immunology
For dot blot, the methanol-activated PVDF membranes were coated with
purified proteins (1 ?g/spot) or undiluted serum samples (2 ?l/spot) from
various species. After the membranes were air dried, they were rinsed with
water and blocked by 5% milk (w/v). Following three washes with PBS,
the membranes were incubated with biotin-labeled CbpA2 at a final con-
centration of 0.3 ?g/ml for 1 h at room temperature. Biotin labeling of
CbpA was performed using the EZ-link biotinylation kit from Pierce, ac-
cording to the supplier’s instructions. The bound CbpA was detected using
a streptavidin-peroxidase conjugate (1/2000; Pierce) and visualized by the
ECL Western blot kit.
ELISA was performed essentially as described previously (38). Direct
ELISA was performed by coating the wells of 96-well plates (Nalge Nunc
International) with CbpA (0.5 ?g/well) by incubating overnight at 4°C.
Serial dilutions of normal human and mouse sera were added as a source
of FH. The bound FH proteins were reacted with a goat anti-human FH Ab
(1/1000; Calbiochem) and a rat anti-mouse FH Ab (1/500) (55), respec-
tively. The levels of reactivity were detected with peroxidase-conjugated
rabbit anti-goat and anti-rat IgG Abs (Bio-Rad; 1/5000). Absorbance was
read on a microtiter plate reader at a wavelength of 490 nm (Bio-Rad).
Competitive ELISA was performed in a similar fashion with certain mod-
ifications. Purified human FH (0.5 ?g/well) was used to coat to 96-well
plates. After washing and blocking steps, the wells were treated with bi-
otin-labeled CbpA at a final concentration of 0.3 ?g/ml in the presence or
absence of various concentrations of human or mouse serum samples. After
1-h incubation, the wells were washed and further incubated with strepta-
vidin-peroxidase conjugate (1/1000 dilution). The ELISA results for direct
and competitive ELISA are presented as the means of the absorbance units
from triplicate wells after subtraction of background readings. The back-
ground levels were determined by measuring the absorbance of wells with-
out serum (direct ELISA) or biotin-labeled CbpA (competitive ELISA).
Two-way ANOVA was used to perform statistic analysis using the Graph-
Pad Prism 5.0 software for Mac OS X. A p value greater than 0.05 was
Amplification of cbpA allelic variants and DNA sequencing
The cbpA allelic variants were amplified by PCR using primers Pr588
(5?-AATGAGAAACGAATCCTTAGCAATG-3?) and Pr589 (5?-AAGA
TGAAGATCGCCTACGAACAC-3?) representing the highly conserved
flanking sequences of the cbpA locus, as described previously (31). The
high-fidelity DyNAzyme EXT DNA polymerase (New England Bio-
labs) was used to minimize the amplification errors. DNA sequences
were determined by automated sequencing of the cbpA-containing PCR
fragments. Sequence analyses were conducted using the DNASTAR
Lasergene version 7.1. The nucleotide sequences of the cbpA alleles in
the pneumococcal isolates are contained in GenBank accession numbers
as listed in Table I.
Isolation of human and murine polymorphonuclear leukocytes
Human PMNs were isolated from venous blood of healthy individuals, as
described previously (56). Studies were performed in accordance with a
protocol approved by the Institutional Review Board for Human Subjects,
National Institute of Allergy and Infectious Diseases. Purified PMNs were
suspended in RPMI 1640 medium buffered with 10 mM HEPES (pH 7.2)
(RPMI/H) and kept at ambient temperature until used. Purity of PMN
preparations and viability were assessed by flow cytometry (FACSCalibur;
BD Biosciences). Cell preparations contained 98–99% granulocytes, of
which typically 94% were neutrophils and 5% were eosinophils. Murine
PMNs were isolated from femurs and tibias of adult mice, as described
(57). Cells were suspended in RPMI/H and kept at ambient temperature
until used. Cell purity was assessed by microscopy, and ?60% of the cells
were mature neutrophils.
Phagocytosis assays were performed using a previously described method
(58), but with modifications. S. pneumoniae strains were cultured to mid-
exponential phase of growth (OD of 0.35–0.45 at 600 nm), as described
above, washed, and resuspended in Dulbecco’s PBS at 1.25 ? 108CFU/
ml. Bacteria were labeled with 5.0 ?g/ml FITC (Sigma-Aldrich) for 20 min
at 37°C, and unbound label was removed by two washes in DPBS. Labeled
S. pneumoniae were resuspended in RPMI/H and chilled on ice until used.
PMNs (106) were combined with 107S. pneumoniae and pooled normal
human or mouse serum (10% final concentration) in wells of a 96-well
microtiter plate on ice. Assays with human or mouse PMNs were per-
formed with bacteria opsonized with serum from the same species (e.g.,
human PMNs were combined with bacteria opsonized with human serum).
Samples were mixed gently with a pipette and rotated at 37°C for 30 or 60
min. At the desired times, samples were placed on ice and analyzed by flow
cytometry. Samples were analyzed to determine the total number of PMNs
with bound/ingested bacteria and then reanalyzed immediately in the pres-
ence of an equal volume of trypan blue (2 mg/ml in 0.15 M NaCl/0.02 M
citrate buffer (pH 4.4)) to measure the number of PMNs with ingested
bacteria (phagocytosis). Ten thousand events were collected per sample,
and a single gate was used to exclude debris and free bacteria (CellQuest
Pro Software; BD Biosciences). Percentage of phagocytosis was deter-
mined by the percentage of FL1-H (FITC)-positive PMNs after quenching
with trypan blue.
Groups of five female BALB/c mice (Taconic Farms) were infected with S.
pneumoniae strains by i.v. inoculation, as described previously (32). The
pneumococci were grown to middle log phase in Todd-Hewitt broth con-
taining 0.5% yeast extract (OD620? 0.4). Bacterial cultures were centri-
fuged, and the pellets were resuspended in PBS to estimated density ac-
cording to the pre-established correlation between optical absorbance and
bacterial density (CFU/ml). The bacterial suspensions were further diluted
in PBS and plated on blood agar plates to confirm the concentrations of
viable bacteria. Mice were i.v. infected with 0.2 ml of pneumococcal sus-
pensions (5 ? 106CFU/ml in PBS) and bled retroorbitally at 0, 12, 24, and
36 h postinfection. The blood samples were diluted in PBS and spread on
blood agar plates to enumerate recovered bacteria. Mortality was moni-
tored daily for 10 days postinfection. The levels of bacterial density in the
bloodstream are presented as CFU/ml blood after the diluting factors are
considered. All animal infection procedures were in compliance with the
guidelines of the Institutional Animal Care and Use Committee.
The FH binding domain of CbpA and pneumococcal survival in
Our previous study revealed a sequence motif at the N terminus of
CbpA in strain D39 that is responsible for the FH-binding activity
(25). In this study, we first determined whether the pneumococcal
recruitment of FH enhances pneumococcal resistance to the com-
plement-mediated bacterial clearance in vivo using a common
mouse bacteremia model. BALB/c mice were i.v. infected with
similar CFUs of strain D39 or its isogenic mutant strains ST588
and ST650. ST588 lacked the entire coding region of cbpA,
whereas ST650 contains a deletion only in the FH binding domain
and retains the rest of CbpA (25). The mice infected with all three
strains displayed similar bacteremia levels at five time points (0,
12, 24, 36, and 48 h) (Fig. 1A). Similarly, no significant difference
in survival rate was observed among the groups of mice infected
with strains D39, ST588, and ST650 (Fig. 1B). This is in agree-
ment with a previous report indicating that D39 and an isogenic
mutant strain lacking CbpA did not show significant differences in
the mouse bacteremia model (32). We thus conclude that the FH-
binding activity of CbpA does not confer a survival advantage for
the pneumococci in the bloodstream of mice.
Lack of binding activity between mouse FH and CbpA
The above mouse infection experiments raised the question as to
whether the pneumococci are able to interact with mouse FH. All
previous studies on the FH-pneumococcus interaction have been
conducted with purified human FH protein or normal human serum
(19, 20, 22–25, 59), with one exception (60). Quin et al. (60) have
recently shown that the pneumococci recovered from the blood of
mice retained mouse FH in a CbpA-dependent manner, as detected
by flow cytometry using an anti-human FH Ab. It should be noted
that virtually all of the FH-related available reagents are based on
human FH. We first attempted to use this Ab to detect binding
interaction between CbpA and mouse FH by Western blot. The Ab
readily detected FH in human serum, but not FH in mouse serum
7140 HUMAN-SPECIFIC INTERACTION WITH PNEUMOCOCCUS
(data not shown). Because the normal plasma contains high con-
centration of FH (300–450 ?g/ml) (45), our data indicated that
this Ab does not significantly cross-react with mouse FH.
During the course of our investigation, a rat anti-mouse FH mAb
became available (55). We initially assessed the reactivity of the
mouse FH Ab in recognizing FH in mouse serum. Different
amounts (0.125–2 ?l) of normal mouse or human serum were sub-
jected to SDS-PAGE. As shown in Fig. 2A (right panel), the Ab
was able to recognize the mouse FH in mouse serum at a 1/2000
dilution, although its potency was lower than the polyclonal Ab
against human FH (left panel). Four-fold more mouse serum was
needed for the mouse FH Ab to yield a similar level of signal
intensity to that generated with human serum and the cognate Ab.
Consistent with the amino acid sequence diversity between mouse
and human FH proteins (61% amino acid sequence identity), nei-
ther Ab showed detectable cross-reactivity to the noncognate FH
proteins under the same conditions (data not shown). This mAb
was subsequently used to determine the binding between CbpA
and mouse FH.
In agreement with our previous study (25), the FH protein in
normal human serum was capable of binding to CbpA2, a rCbpA
containing the intact FH binding domain (Fig. 2B, left panel). In
contrast, a similar procedure failed to detect reactivity with the
normal mouse serum even with the highest amount of CbpA (4 ?g)
used (Fig. 2B, right panel). We confirmed this finding with a more
sensitive and quantitative ELISA method using normal serum sam-
ples as a source of FH. Although human serum FH showed a
dose-dependent binding to CbpA, the same reactions with mouse
serum did not result in any detectable binding activity (Fig. 2C).
Our separate ELISA experiments showed that the anti-mouse FH
Ab was able to detect mouse serum FH with up to 5000-fold di-
luted serum samples (data not shown). These initial findings sug-
gested that CbpA has very weak binding, if any, to mouse FH. This
result is reminiscent of a previous report by McDowell et al. (53)
that the BBA68 protein of B. burgdorferi binds specifically to hu-
man FH, but not the mouse counterpart. However, the Western blot
and direct ELISA data obtained with two different anti-FH Abs
were not absolutely conclusive. We next verified this finding by
competitive ELISA. Human FH was coated onto 96-well plates
and incubated with biotin-labeled CbpA2 in the presence or ab-
sence of human or mouse serum. Consistent with the Western blot
experiment (Fig. 2B), the CbpA-FH binding was blocked by hu-
man serum in a dose-dependent manner (there was ?50% inhibi-
tion by 2% human serum) (Fig. 2D). In sharp contrast, mouse
serum produced a marginal blocking effect on the CbpA-FH bind-
ing even at its highest concentration (100%). Together, the data
obtained with complementary experimental methods demonstrated
that pneumococcal CbpA allele of strain D39 binds to human FH,
but not mouse FH under these experimental conditions.
Lack of binding activity between CbpA and FH of other animal
We further determined whether CbpA binds to the FH proteins of
other animal species by dot blot. Equal volumes (2 ?l) of undiluted
serum samples from human, mouse, rat, rabbit, horse, and cattle
ulence in mice. A, Pneumococcal replication/survival in the blood. Bacte-
rial levels in the blood of BALB/c mice were monitored at 0, 12, 24, 36,
and 48 h after i.v. infection with 1 ? 106CFU of the D39 wild-type strain
(F), the isogenic CbpA-null mutant of strain D39 (ST588) (E), and the
isogenic mutant lacking the FH binding domain of CbpA (ST650) (Œ). The
data are presented as the mean ? SE of CFU/ml blood from three separate
experiments. B, Percentage of survival rate of mice. The same groups of
five BALB/c mice as described in A were monitored daily for death for 10
days after the i.v. infection. This is a representative of three separate
CbpA is dispensable for pneumococcal bacteremia and vir-
A, Detection of mouse and human serum FH by Western blot. Various
volumes (0.125–2 ?l) of normal human serum (left panel) or mouse serum
(right panel) were subjected to SDS-PAGE and blotted with a goat anti-
human FH (left panel) or a rat anti-mouse FH Ab (right panel). Molecular
mass markers are indicated in kilodaltons on the left. B, Detection of
CbpA-FH binding by Western blot. Different amounts of CbpA2 (0.25–4
?g) were subjected to SDS-PAGE. Identical blots were blotted with either
human serum (left panel; 1/1000 dilution) or normal mouse serum (right
panel; 1/100 dilution), followed by incubating with corresponding anti-FH
Ab and peroxidase-conjugated secondary Ab. Molecular mass markers are
indicated in kilodaltons on the left. C, Detection of CbpA-FH binding by
direct ELISA. Immobilized CbpA was mixed with serial dilutions of nor-
mal human or mouse serum for 1 h. CbpA-FH binding was detected by Ab
against either human or mouse FH and peroxidase-conjugated secondary
Abs by ELISA. D, Detection of CbpA-FH binding by competitive ELISA.
Human FH-coated wells of microtiter plates were mixed with biotin-la-
beled CbpA2 in the presence or absence of various concentrations of nor-
mal human or mouse serum for 1 h. CbpA-FH binding was detected with
a streptavidin-peroxidase conjugate by ELISA.
CbpA specifically binds to human FH, but not mouse FH.
7141The Journal of Immunology
bovine were each spotted in duplicate onto a PVDF membrane and
probed with biotinylated CbpA. Multiple proteins were also in-
cluded as positive (human FH and SIgA) and negative (BSA) con-
trols. Although the positive controls from the human origin (FH,
SIgA, and serum) showed strong binding activity to CbpA, no
CbpA binding signal was detected with the serum samples from
mouse, rat, rabbit, horse, and bovine serum along with BSA (neg-
ative control). This reactivity pattern was reproducible in our ad-
ditional experiments (data not shown). These observations have
convinced us that the pneumococci interact with FH in a human-
specific fashion. This finding is consistent with the fact that the
pneumococci are naturally carried only by humans (2).
Human-specific FH binding by multiple other pneumococcal
All virulent strains of S. pneumoniae tested to date contain the
cbpA locus (30, 31). However, there are extensive sequence vari-
ations among CbpA allelic variants from pneumococcal strains
(30, 31). Because our previous experiments regarding CbpA-FH-
binding activity were all conducted with CbpA alleles from strains
D39 and TIGR4 (25) (Figs. 2 and 3), we were interested in whether
this species-specific host-pathogen interaction occurs in additional
S. pneumoniae isolates. We determined the FH-binding activity of
11 pneumococcal isolates from invasive (blood and cerebrospinal
fluid) infections by Western blot. The rationale was that FH-me-
diated evasion of the complement system by S. pneumoniae is
most likely to operate in the invasive infections due to high con-
centrations of complement proteins in the bloodstream. These iso-
lates represent 11 different capsular serotypes (types 3, 6A, 6B, 7F,
10A, 15C, 19A, 19F, 22F, 23F, and 38). None of the strains
showed detectable binding to mouse FH when mouse serum was
used as a source of FH at a 1/100 dilution (data not shown). When
the same blot was stripped and reprobed with human serum at a
1/1000 dilution as described for Fig. 2B, all but one (ST865) of the
isolates reacted with FH (Fig. 4A). These results have further ver-
ified the species-specific binding between CbpA and human FH.
We further attempted to address why these CbpA alleles have
different levels of FH-binding activity and reasoned that sequence
polymorphisms and differential expression of the CbpA alleles are
two major possibilities. Variable sizes of the FH-binding bands
suggested sequence variations in these CbpA alleles (Fig. 4A),
which could in turn affect the FH binding. We first assessed the
sequence variations of the CbpA allelic variants by determining
the full cbpA-coding sequences of 10 strains. Repeated attempts to
amplify the cbpA locus of strain ST873 by PCR were unsuccessful.
Consistent with variable sizes of the FH-binding bands (Fig. 4A),
sequence analysis showed extensive sequence variations among
the CbpA alleles (data not shown). The sequence variations may in
part explain different FH-binding levels of the CbpA alleles. As
reflected from the order of the CbpA alleles in Fig. 4B, the highly
reactive strains (D39, ST866, ST869, ST860, ST863, ST872, and
ST861) shared higher levels of sequence homology in the FH bind-
ing domain of CbpA (25). Consistent with this notion, the FH
binding domains of the weakly reactive strains (ST858 and ST862)
have reduced sequence homology compared with that of the highly
reactive strains. The CbpA allele in ST864 is an exception, which
showed relative strong FH binding, but possesses weak sequence
homology with the FH-binding motif of D39. Finally, the sequence
information also revealed the lack of the FH binding domain in
strain ST865, a type 3 cerebrospinal fluid isolate (Fig. 4B). ST865
possesses a PspC type 8 allele, which is highly similar to the CbpA
allele in another type 3 strain G396. Our previous study could not
detect FH-binding activity in G396 (25). This result thus explains
the absence of the FH-binding activity in ST865 (Fig. 4A).
The variable FH-binding intensities among the pneumococcal
isolates could also reflect different expression levels of the CbpA
Normal serum samples from human, mouse, rabbit, horse, and bovine (2 ?l
each) along with purified protein human SIgA, human FH, and BSA (1 ?g
each) were coated onto a methanol-activated PVDF membrane as duplicate
spots. The membrane was probed with biotinylated CbpA.
CbpA does not bind to FH of additional animal species.
lysates of 11 pneumococcal isolates were subjected to SDS-PAGE. CbpA-FH binding was detected by Western blot as in Fig. 2B. Molecular mass markers
are indicated in kilodaltons on the left. B, Sequence comparison in the FH binding domain of the CbpA alleles. The isolates are listed below strain D39
according to the sequence homology level between each CbpA allelic variant and CbpA of D39 in the FH binding domain. Sequence homology was
determined by the DNASTAR MegAlign program. Identical amino acids are shaded. Gaps were introduced for optimal alignment, as indicated by dashed
lines. The previously identified FH-binding motif is marked with a line (25). C, Detection of CbpA allelic variants with purified human SIgA. CbpA variants
of the pneumococcal isolates were detected by Western blot using purified human SIgA (0.4 ?g/ml), as described previously (25).
Human FH-specific binding operates in other pneumococcal isolates. A, Binding of human serum FH to CbpA allelic variants. Cellular
7142 HUMAN-SPECIFIC INTERACTION WITH PNEUMOCOCCUS
variants, although an approximately equivalent amount of total
proteins (5 ?g) was used for each strain (Fig. 4A). We further
assess the CbpA variants with human SIgA because the SIgA bind-
ing site is highly conserved among many pneumococcal isolates
(31, 39). All isolates except for ST862 and ST865 showed positive
binding to purified human SIgA. The levels of the reactivity were
also somewhat variable, which was most likely due to uneven ex-
pression levels of different CbpA alleles and/or sequence varia-
tions in the SIgA-binding motif (Fig. 4C). The lack of SIgA bind-
ing in strain ST865 is consistent with the lack of the SIgA binding
site in this strain as reported for all CbpA variants from type 3
isolates tested to date (31). Multiple reactive bands detected with
human FH and SIgA in certain strains were likely to represent
degradation products of CbpA because this phenomenon appeared
to occur only when the reactivity was strong. Taken together, our
data demonstrate that the species-specific binding of pneumococ-
cal CbpA to human FH occurs in the majority of clinical isolates,
but levels of the binding interaction may vary from strain to strain.
Impact of the FH binding domain on phagocytosis of
The human-specific pneumococcal recruitment of FH suggested
that this interaction may enhance bacterial adaptation in the human
host, thus contributing to the tropism of this pathogen to humans.
Because our previous experiments indicated that mouse models are
not appropriate to address this possibility, we approached this issue
by assessing the impact of the pneumococcus-FH interaction on
phagocytosis in vitro. Strains D39 and ST650 (isogenic mutant
lacking the FH binding domain of CbpA) were tested in both hu-
man and mouse phagocytosis systems (human serum plus human
PMNs vs mouse serum plus mouse PMNs). Serum was used as a
source of FH and other proteins of the complement system. There
was in general equivalent association of D39 with human and
mouse PMNs, indicating that PMN binding in these assays is me-
diated primarily by molecules not impacted by CbpA FH-binding
activity (Fig. 5A). Although on average ST650 were associated
(surface bound and ingested combined) with more PMNs than D39
in either phagocytosis system, this difference was greater in the
human system and significant only with the human system (Fig.
5A). There was also comparable, albeit relatively limited, ingestion
of D39 in the human and mouse phagocytosis systems, suggesting
that phagocytosis of S. pneumoniae occurs in part in the presence
of CbpA (Fig. 5B). By comparison, phagocytosis of S. pneumoniae
was more pronounced in the absence of CbpA FH-binding activity,
because there were significantly more human PMNs containing
ingested ST650 at the two time points tested (p ? 0.01) (Fig. 5B).
Thus, absence of the CbpA FH binding domain enhanced up-
take of S. pneumoniae by PMNs in the human phagocytosis
system, whereas levels of D39 and ST650 phagocytosis were
not significantly different in the mouse system at 30 min
(43.3 ? 6.5% vs 33.5 ? 8.2%, respectively) (Fig. 5B). There
were significantly more mouse PMNs with ingested ST650 than
D39 by 60 min, but the difference between these two strains was
greater (15.5%) in assays with human PMNs and serum (Fig.
5B). Taken together, the lack of CbpA FH-binding activity led
to a more profound impact in the human phagocytosis system
compared with the mouse system.
Previous in vitro studies have implicated the CbpA-mediated
pneumococcal interaction with FH as an important strategy for
bacterial evasion of complement-mediated host defense (24, 50,
60). However, it is unclear whether the pneumococcal recruitment
of FH affects bacterial survival and virulence during pneumococcal
infections. By using the pneumococcal strains with deletion only in
the FH binding domain (25), we have shown that the FH binding
domain is critical for pneumococcal resistance to complement-me-
diated phagocytosis in a well-defined phagocytosis cell culture
model (61, 62). However, the deletion of the FH binding domain
in CbpA or the entire CbpA did not significantly affect pneumo-
coccal bacteremia or virulence in mice. This finding fully agrees
with the lack of detectable binding interaction between S. pneu-
moniae and mouse FH in our molecular analyses. Together with
the species-specific interaction between CbpA and human pIgR/
SC/SIgA (35, 38, 39), our data further suggest that CbpA is a
bacterial determinant for natural host tropism of S. pneumoniae to
The results of the phagocytosis experiments using a mouse system
are intriguing, because the lack of the CbpA FH binding domain had
an impact on pneumococcal phagocytosis (but not binding) with
mouse PMNs in the presence of mouse serum (complement system).
The CbpA FH binding domain may possess another uncharacterized
activity that is involved in pneumococcal interaction with mouse
phagocytes. This agrees with the previous observations indicating that
CbpA-deficient pneumococci are attenuated in nasal colonization and
lung infection in mouse infection models (29, 32, 33). It is also pos-
sible that this domain directly or indirectly influences pneumococcal
interaction with serum factors including complement C3 and other
members of the FH family (or FH-like proteins). This notion is con-
sistent with the previous reports that CbpA binds to C3 (28), which
A, Percentage of PMNs with associated (bound and ingested) pneumo-
cocci. Association of strains D39 and ST650 (isogenic mutant lacking the
FH binding domain of CbpA) with human or mouse PMNs was compared
by flow cytometry, as described in Materials and Methods. Results are the
mean ? SEM for (n) human blood donors or (n) experiments with murine
PMNs, as indicated. Statistics were performed using a one-way ANOVA
and Bonferroni’s posttest for multiple comparisons. B, Percentage of
PMNs with ingested pneumococci. Samples in A were mixed with trypan
blue to quench fluorescence of extracellular (surface-bound) bacteria, as
described in Materials and Methods.
CbpA enhances pneumococcal resistance to phagocytosis.
7143The Journal of Immunology
promotes pneumococcal adhesion to host cells (36). Many pathogenic
bacteria have been shown to bind to FH-like proteins (45, 46). Lastly,
we cannot completely exclude the possibility of a low-affinity binding
between CbpA and mouse FH due to the detection limit of Western
blot and ELISA, although the mouse infection data argue against this
possibility. Such a low-affinity binding of CbpA to mouse FH would
inhibit complement-mediated phagocytosis by mouse phagocytes.
This would resolve the discrepancy between our results and observa-
tions reported by Quin et al. (60). The low-affinity binding between
ing analysis, but can be detected when both TCR and MHC are fo-
cally enriched in cellular context (63).
Nasopharyngeal colonization and subsequent dissemination to
other tissue sites including the bloodstream are the key aspects of
pneumococcal pathogenesis. CbpA has been shown to contribute
to nasopharyngeal colonization of S. pneumoniae in rat and mouse
infection models (29, 32, 64). However, the contribution of CbpA
to pneumococcal sepsis is not entirely clear. Rosenow et al. (29)
first showed that CbpA is not required for pneumococcal sepsis in
an infant rat model. Other investigators have reported marginal
effects of CbpA mutations on bacterial survival in the bloodstream
(32) and virulence in mice when the animals were i.v. infected (32,
65). Conversely, other studies have recently reported that CbpA
significantly contributes to pneumococcal sepsis in mice (34, 64).
Our mouse infection experiments have confirmed the previous ob-
servations that CbpA is not essential for pneumococcal infection in
the bloodstream of mice (29, 32). The pneumococcal mutant strain
lacking the FH binding domain or the entire CbpA protein pro-
duced a phenotype comparable to the wild-type strain. The mice
infected with CbpA mutant or wild-type strains had similar levels
of bacteremia at various time points post-i.v. infection. Consistent
with that observation, survival rates were similar for the mice i.v.
infected with the wild-type and isogenic CbpA mutant strains. The
discrepancy among different studies regarding the contribution of
CbpA to pneumococcal sepsis in mice may be explained in part by
technical variations, including the use of different bacterial strains,
infection doses, and mouse strains.
It is well documented that S. pneumoniae is able to bind to
human FH (19–25). Our molecular analysis has shown that pneu-
mococcal CbpA specifically interacts with human FH, but the
same approach did not detect obvious binding between CbpA and
mouse FH. This human-specific interaction was initially identified
with purified CbpA of strain D39 by Western blot analyses and
ELISA. The species-specific binding between CbpA and human
FH is not limited to a single pneumococcal strain because the
native CbpA variants of multiple pneumococcal isolates were able
to bind to human FH, but not the mouse counterpart. Similarly, no
detectable binding was observed between CbpA and the FH pro-
teins of rat, rabbit, horse, and bovine. The lack of detectable bind-
ing interaction between pneumococcal CbpA and mouse FH is
fully consistent with the mouse infection data of this study and
previous studies (29, 32). Deleting the entire CbpA protein in
strain D39 did not have any significant effect on pneumococcal
survival in the bloodstream or virulence post-i.v. infection, which
was also observed previously by other investigators (29, 32). The
molecular basis for this human-specific binding is not clear. It
appears that multiple domains of the FH protein are involved in
CbpA binding (21, 23, 49). It is likely that amino acid sequence
difference(s) in these regions between the human and mouse FH
proteins determines the binding specificity to CbpA.
Our observations in this study are not in full agreement with a
recent report by Quin et al. (60), despite that both studies used the
same D39 strain. These investigators reported that the pneumo-
cocci recovered from the blood after i.v. or i.p. infection had sig-
nificant binding to mouse FH, as detected by flow cytometry with
an anti-human FH Ab (60). In this study, this Ab readily recog-
nized human FH, but could not detect mouse FH in serum samples
by Western blot (data not shown). A plausible explanation is that
the flow cytometry method used by Quin et al. (60) may be able to
detect a potentially weak interaction between CbpA and mouse FH
due to its higher detection sensitivity. This possibility is consistent
with our observation that high concentrations of mouse serum had
a marginal blocking effect on CbpA binding to human FH in our
ELISA experiments, although the inhibition was not statistically
The noted mouse-human difference in the context of interaction
with S. pneumoniae is probably a reflection of human-specific in-
teraction between CbpA and other mucosal factors in humans. The
biochemical analyses from our previous studies and others have
revealed that CbpA also binds to human pIgR/SC/SIgA, but not to
the counterparts of common model animals, including mouse, rat,
and rabbit (35, 38, 39). Because SIgA and SC represent the extra-
cellular portion of pIgR, CbpA apparently binds to these host fac-
tors by the same mechanism (37, 38). pIgR is predominantly ex-
pressed by the mucosal epithelial cell to transport IgA to mucosal
surfaces membrane (66). We (35) and others (37) have shown that
CbpA-pIgR interaction promotes pneumococcal adhesion to and
invasion of respiratory epithelial cells, thus suggesting this human-
specific pneumococcal-host interaction enhances pneumococcal
adhesion and thereby colonization in humans, although their actual
biological impact has not been defined due to the lack of appro-
priate animal models. Likewise, it is reasonable to postulate that
the species-specific pneumococcal interaction with human FH pro-
motes bacterial evasion of complement-mediated host defense in
This study and others (35, 38, 39) have suggested that CbpA
may be one of the bacterial factors that define pneumococcal host
specificity. Humans are the only natural host for S. pneumoniae,
but the underlying mechanisms for this strict host tropism remain
to be elucidated. Identifying human-specific binding interactions
of S. pneumoniae with FH and pIgR/SC/SIgA has provided the
first line of molecular evidence that the bacterium prefers humans
to other animal species. A challenging, but important issue in fu-
ture studies is to develop appropriate model systems to assess the
in vivo impact of these human-specific pneumococcal interactions
in human host. It should be noted that the CbpA-negative pneu-
mococcal mutants are deficient in the nasopharyngeal colonization
in mice and rats (29, 32, 64). It is thus possible that CbpA interacts
with other unknown host factor(s), which promotes pneumococcal
colonization and/or infection in mice. However, the information
from this study and others (35, 38, 39) has highlighted the clear
differences between humans and mice in terms of interacting with
the pneumococci at the molecular level. Accordingly, cautions are
well justified when future experimental findings from mice are
extrapolated to human infections of S. pneumoniae.
We thank Jun Yang, Daimin Zhao, and Yueyun Ma for technical assis-
tance, and James R. Drake for valuable advice on the phagocytosis exper-
iments. We are also grateful for the pneumococcal strains provided by the
Active Bacterial Core Surveillance/Emerging Infections Programs Net-
work at the Centers for Disease Control and Prevention.
The authors have no financial conflict of interest.
7144 HUMAN-SPECIFIC INTERACTION WITH PNEUMOCOCCUS
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