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The Journal of Immunology
Immune Evasion of Moraxella catarrhalis Involves Ubiquitous
Surface Protein A-Dependent C3d Binding
Teresia Hallstro ¨m,*,†There ´se Nordstro ¨m,* Thuan Tong Tan,* Taras Manolov,*
John D. Lambris,‡David E. Isenman,xPeter F. Zipfel,†Anna M. Blom,{
and Kristian Riesbeck*
The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b de-
position (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is
a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary
disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface
protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to
bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical
isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a
generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to
C3d and that the major bacterial C3d-binding domains were within UspA1299–452and UspA2165–318. The interaction with C3 was
not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative
pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the
activation of complement and to inhibit C3a-mediated inflammation.
pneumococci and Haemophilus influenzae, M. catarrhalis is the
third most common pathogen isolated from middle ear fluid cul-
tures from children with acute otitis media (3). Fifteen to 20%
of all otitis media cases are caused by M. catarrhalis (4). M.
catarrhalis is also an important cause of lower respiratory tract
infections in patients with chronic obstructive pulmonary disease
(5). The carrier rates of M. catarrhalis among chronic obstructive
pulmonary disease patients were 5–32% when sputum samples
were analyzed from patients that did not have any clinical signs of
infection (Ref. 6 and references therein). Additionally, M. catar-
The Journal of Immunology, 2011, 186: 000–000.
oraxella catarrhalis is a Gram-negative diplococcus
that during the last two decades has increasingly gained
recognition as a respiratory pathogen (1, 2). After
rhalis is one of the most common inhabitants of the pharynx of
healthy preschool children, and within this group it becomes
pathogenic upon simultaneous viral infection. During the first year
of life, the carrier rate of M. catarrhalis is ∼67% of all children
and the species colonizes oropharynx and nasopharynx (7). This
implies that M. catarrhalis as a commensal has numerous strate-
gies to survive in the host (1). In recent years, focus has been on
both its outer membrane protein composition and interactions
with the human host (8, 9). Several virulence determinants of
M. catarrhalis have been identified, including M. catarrhalis IgD-
binding protein/hemagglutinin, protein CD, M. catarrhalis ad-
herence protein, and the ubiquitous surface proteins (Usp) (10–
The UspA family consists of UspA1 (88 kDa), UspA2 (62 kDa),
and the hybrid protein UspA2H (92 kDa) (16, 17). All strains
express UspA1 in combination with either UspA2 or UspA2H.
UspA1 and A2 are closely related and share significant sequence
similarity in a region comprising 140 aa (16). UspA1 and UspA2
are multifunctional proteins, have highly conserved epitopes, and
thus are of considerable interest as potential vaccine candidates
(9, 18). The hybrid UspA2H consists of UspA1 and UspA2 with
its N-terminal (head region) and C-terminal (near-end stalk and
membrane-anchored region) having shared sequence similarity
with UspA1 and UspA2, respectively (17, 19, 20). The stalk re-
gion is highly conserved between UspA1, UspA2, and UspA2H of
different strains (17, 19, 21). The UspAs bind the extracellular
matrix proteins laminin and fibronectin and are essential for at-
tachment of M. catarrhalis to epithelial cells (17, 22–24), and they
play important roles in M. catarrhalis serum resistance by inter-
acting with C3, C4b-binding protein (C4BP), and vitronectin (25–
28). Moreover, naturally acquired Abs to UspA1 and UspA2 are
bactericidal (29, 30).
The complement system is a major part of the innate immune
defense against pathogenic microorganisms, and activation of this
system leads to a cascade of protein deposition on the bacterial
*Medical Microbiology, Department of Laboratory Medicine Malmo ¨, Lund Uni-
versity, Ska ˚ne University Hospital, Malmo ¨, Sweden;†Leibniz Institute for Natural
Product Research and Infection Biology, Hans Kno ¨ll Institute, Friedrich Schiller
University, Jena, Germany;
University of Pennsylvania School of Medicine, Philadelphia, PA 19104;xDepart-
ment of Biochemistry, University of Toronto, Toronto, Ontario, Canada; and{Med-
ical Protein Chemistry, Department of Laboratory Medicine Malmo ¨, Lund Uni-
versity, Ska ˚ne University Hospital, Malmo ¨, Sweden
‡Department of Pathology and Laboratory Medicine,
Received for publication August 2, 2010. Accepted for publication December 15,
This work was supported by grants from the Alfred O¨sterlund Foundation, the Anna
and Edwin Berger Foundation, the Janne Elgqvist Foundation, the Johan Kock Foun-
dation, the Krapperup Foundation, the Marianne and Marcus Wallenberg Foundation,
the Swedish Medical Research Council, the Swedish Society of Medicine, and the
Cancer Foundation at the University Hospital in Malmo ¨.
Address correspondence and reprint requests to Dr. Kristian Riesbeck, Medical Micro-
biology, Department of Laboratory Medicine Malmo ¨, Lund University, Ska ˚ne Univer-
sity Hospital, SE-205 02 Malmo ¨, Sweden. E-mail address: email@example.com
Abbreviations used in this article: BHI, brain heart infusion; C4BP, C4b-binding
protein; C3met, methylaminetreated C3; DGVB, dextrose gelatin veronal buffer;
Efb, extracellular fibrinogen-binding protein; MAC, membrane attack complex;
NHS, normal human serum; pAb, polyclonal Ab; Sbi, staphylococcal binder of
IgG; Usp, ubiquitous surface protein.
Published January 26, 2011, doi:10.4049/jimmunol.1002621
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surface resulting in opsonization of pathogens, which facilitates
phagocytosis, release of chemoattractants, as well as formation
of the membrane attack complex (MAC). The classical pathway of
the complement system is activated by target-bound Abs and C-
reactive protein (31), the lectin pathway by binding of mannose-
binding lectin, whereas the alternative pathway is spontaneously
activated through direct contact with foreign particles or cells
(32). All pathways lead to the formation of the C3 convertases,
with subsequent cleavage of C3 to C3a and C3b. The complement
protein C3 is one of the most abundant proteins in serum (1–2 mg/
ml) and one of the most important components in the complement
system. It is a large molecule composed of two chains, an a-chain
(110 kDa) and a b-chain (75 kDa), which are held together with
disulfide bonds (33). Upon activation, C3 undergoes marked
conformational changes and proteolytic cleavages, which results
in amplification of complement, anaphylatoxin (C3a) release, cell
lysis, opsonization, and phagocytosis (34). The presence of an
internal thioester bond is required for C3 deposition on molecular
surfaces. C3 forms a link between all three pathways of comple-
ment activation and in addition it is also involved in the interaction
between the innate and acquired immune systems (35). Patients
with C3 deficiency have shown increased susceptibility to infec-
tions of a variety of bacterial pathogens (36). The C3 convertases
cleave C3 to C3b and C3a and these fragments have important
functions (37). C3a is an anaphylatoxin that induces proin-
flammatory activities. C3b binds factor B, which allows factor D
to cleave factor B, resulting in a surface-bound C3 convertase,
C3bBb. This alternative pathway C3 convertase will cleave more
C3 in an amplification loop. The classical and lectin pathways
form a C3 convertase from activation of C4 and C2, resulting in
C4b2b (37). The alternative pathway functions as an amplification
loop for the classical and lectin pathways and can also be spon-
taneously activated by covalent attachment of C3 to the surface
of a microbe in the absence of complement inhibitors. Thereafter,
C3b can bind covalently to the complement-activating surface,
C4bC2b or C3bBb, forming the C5 convertases of the classical/
lectin (C4bC2bC3b) and alternative (C3bBbC3b) pathways (37).
The C5 convertases bind and cleave C5 to the proinflammatory
analphylatoxin C5a and C5b. C5b initiates the assembly of the late
complement components (C5b, C6, C7, C8, and C9), forming
pores in the membrane. These channels result in the MAC (C5b–
9), which reduces the osmotic pressure and causes lysis of the
target cell. Lysis of the microorganism is one way for the com-
plement system to clear the pathogen.
Human pathogens have evolved mechanisms to escape the in-
nate immunity and complement. One frequent strategy used by
microbes is tousethe inhibitorsofcomplement, including factor H,
factor H-like protein 1, factor H-related protein 1, C4BP, and
vitronectin for protection against complement-mediated killing
(38–41). Importantly, the complement inhibitors are functionally
active when bound to the surface of the pathogen and thus inhibit
the complement-mediated attack. In fact, a number of micro-
organisms, including H. influenzae, Streptococcus pneumoniae,
Streptococcus pyogenes, and Neisseria meningitidis have all been
reported to bind factor H and C4BP (42–49). Another strategy
used by some pathogens is to directly inactivate C3 by produc-
ing C3-binding proteins. Staphylococcus aureus, Porphyromonas
gingivalis, Candida albicans, and Pseudomonas aeruginosa are
pathogens able to bind and/or degrade C3 to impair the immune
response (39, 50–54). Staphylococcal complement inhibitor, ex-
tracellular fibrinogen-binding protein (Efb), Efb homologous pro-
tein, and staphylococcal binder of IgG (Sbi) all expressed by S.
aureus and pH-regulated Ag 1 expressed by C. albicans are mi-
crobial proteins that bind C3 and its fragments and thereby inhibit
the C3 convertases and further activation of complement (52, 55–
59). Additionally, interpain A expressed by Prevotella intermedia
efficiently degrades the a-chain of C3 and thereby inhibits all
complement pathways (60).
M. catarrhalis has been shown to interact with C4BP, the in-
hibitor of the classical pathway, via UspA1 and UspA2, which
bind to the a-chain of C4BP via complement control protein
domains 2, 5, and 7 (26). C4BP bound to the M. catarrhalis cell
surface retains its cofactor activity as determined by analysis of
C4b degradation. A more intriguing strategy to combat comple-
ment is that Moraxella binds and neutralizes C3 (27). M. catar-
rhalis readily absorbed C3 from human serum in which com-
plement was inactivated and noncovalently bound purified methy-
lamine-treated C3 (C3met) via the UspAs. An interesting finding
was that preincubation of serum with recombinant UspA150–770
and UspA230–539results in absorption of C3 and increases the
survival of a M. catarrhalis UspA1/A2H-deficient mutant. In
contrast to M. catarrhalis, several other species including Mor-
axella subspecies lack the capacity to bind C3 or C3met (27).
In the present study we found that Moraxella-dependent C3
binding correlated with UspA expression and that the ability of
M. catarrhalis to bind C3 contributed to serum resistance. Both
UspA1 and UspA2 were able to inhibit activation of the alternative
and classical pathways and C3a generation in activated human
serum and thereby they most likely contribute to survival of M.
catarrhalis in the human host. Interestingly, the UspA1 and A2
bound the C3d fragment of C3, and the C3d binding domains were
located within UspA1299–452and UspA2165–318. M. catarrhalis
also bound murine C3, indicating that the interaction with C3 is
not species specific. Taken together, UspA-mediated C3d binding
of M. catarrhalis inhibits all complement pathways and thus
contributes to the survival of the complement-mediated attack.
Materials and Methods
Bacterial strains and culture conditions
The clinical M. catarrhalis isolates (n = 20) were described earlier (61). M.
catarrhalis were routinely cultured in brain heart infusion (BHI) liquid
broth or on BHI agar plates at 37˚C. M. catarrhalis RH4 mutants were
made previously (26). The UspA1-deficient mutant was cultured in BHI
supplemented with 1.5 mg/ml chloramphenicol (Sigma-Aldrich, St. Louis,
MO), and the UspA2H-deficient mutant was incubated with 7 mg/ml
zeocin (Invitrogen, Carlsbad, CA). The UspA2H of M. catarrhalis RH4
was formerly known as UspA2 (26, 28, 62). Both chloramphenicol and
zeocin were used for growth of the UspA1/A2H double mutants.
Proteins and Abs
C3b-like molecules (C3met) were prepared by incubation of purified C3
with 100 mM methylamine (pH 7.6) for 1 h at 37˚C and subsequent dialysis
against 100 mM Tris-HCl, 150 mM NaCl (pH 7.5). C3, C3b, C3c, and C3d
were purchased from Complement Technology (Tyler, TX). Recombinant
C3d was expressed and purified as described previously (63). S. aureus
Efb was cloned in the expression vector PET200D and expressed as an
N-terminal His-tagged protein in Escherichia coli. Human factor H
and C4BP were from Complement Technology. The manufacture of
recombinant UspA150–770and UspA230–539, which are devoid of their
hydrophobic C termini and recombinant proteins spanning the entire
UspA150–770and UspA230–539, were described previously (24, 26).
The anti-UspA1/A2/A2H (anti-UspA) polyclonal Ab (pAb) was pro-
duced and purified as described (26). The rabbit anti-human C3d pAb, the
mouse anti-human C5b–9 mAb, the FITC-conjugated swine anti-rabbit
pAb, the FITC-conjugated rabbit anti-mouse pAb, the HRP-conjugated
swine anti-rabbit pAb, and the HRP-conjugated rabbit anti-mouse pAb
were purchased from Dakopatts (Glostrup, Denmark), the C3b mAb was
from AbD Serotec (Du ¨sseldorf, Germany), and the goat anti-human C3
and the rabbit anti-C3a pAb were from Complement Technology. The
HRP-conjugated donkey anti-goat pAb was obtained from Serotec
(Oxford, U.K.).
2 UspAs INTERACT WITH C3d
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Flow cytometry analysis
The capacity for different M. catarrhalis strains to bind C3met was ana-
lyzed by flow cytometry. Bacteria were grown on solid medium overnight
and washed once in PBS containing 2% BSA (PBS-BSA) (Saveen Werner,
Malmo ¨, Sweden). Bacteria (108) were incubated with C3met (40 mg/ml) in
PBS-BSA for 1 h at 37˚C. After two washings, the bacteria were incubated
with FITC-conjugated goat anti-rabbit C3 pAb (Dakopatts). After two
additional washes, bacteria were analyzed by flow cytometry (Epics XL-
MCL; Coulter, Hialeah, FL). All incubations were kept in a final volume
of 100 ml PBS-BSA and washings were done with the same buffer. The
FITC-conjugated anti-C3 pAb was used as a negative control for each
strain analyzed. To analyze UspA1/A2/A2H expression, bacteria were
incubated with the anti-UspA pAb and washed as described above. FITC-
conjugated goat anti-rabbit pAb was used for detection. To determine the
efficiency of M. catarrhalis binding to C3, the wild-type strain and the
UspA1/A2H-deficient double mutant were incubated with increasing
concentrations of C3met (range, 1–750 mg/ml) or normal human serum
(NHS) with 10 mM EDTA (NHS-EDTA) (0–60%). After washes the
bacteria were incubated with a FITC-conjugated rabbit anti-human C3
pAb. After two final washes, bacteria were analyzed by flow cytometry.
To analyze the M. catarrhalis interaction with mouse C3, the wild type
and the UspA1/A2H-deficient mutants were incubated with increasing
concentrations of EDTA-treated mouse serum (0–10%) (Innovative Re-
search, Novi, MI), followed by incubation with FITC-conjugated goat anti-
mouse C3 pAb (ICN Biomedicals, Irvine, CA).
Serum bactericidal assay
M. catarrhalis strains were diluted in a buffer allowing activation of all
complement pathways; that is, dextrose gelatin veronal buffer (DGVB2+)
(2.5 mM veronal buffer [pH 7.3], containing 70 mM NaCl, 140 mM
glucose 0.1% [w/v] gelatin, 1 mM MgCl2, and 0.15 mM CaCl2). Bacteria
(104CFU) were incubated in 10% of pooled NHS diluted in DGVB2+in
a final volume of 100 ml. After 30 min, 10-ml aliquots were removed and
spread onto chocolate agar plates. After 18 h incubation at 37˚C, CFU
were determined. NHS was obtained from healthy blood donors with in-
formed consent.
Hemolytic assay
Sheep erythrocytes were washed with DGVB2+and rabbit erythrocytes
with MgEGTA buffer (2.5 mM veronal buffer [pH 7.3] containing 70 mM
NaCl, 140 mM glucose, 0.1% [w/v] gelatin, 7 mM MgCl2, and 10 mM
EGTA). The latter buffer allows study of exclusively the alternative
pathway due to lack of calcium. After washings, the erythrocytes were
resuspended at a concentration of 109cells/ml. Sheep erythrocytes were
preincubated with amboceptor (Dade-Behring, Marburg, Germany) (64)
for 20 min at 37˚C, and thereafter washed twice with DGVB2+. After
washings, the IgG-sensitized sheep erythrocytes (1.25 3 107) were in-
cubated with 40 ml NHS diluted in DGVB2+(1:80), and rabbit erythrocytes
were incubated with 5% mouse serum diluted in MgEGTA buffer, in
a volume of 150 and 100 ml, respectively. After 1 or 3 h at 37˚C, eryth-
rocytes were centrifuged and the amount of lysed erythrocytes was de-
termined by spectrophotometric measurement of released hemoglobin at
414 nm. For inhibition with UspA1 and UspA2, 40 ml NHS (1:80) was
preincubated with 2.5–10 mg/ml recombinant UspA150–770or UspA230–539
for 15 min at 37˚C, and thereafter added to the erythrocytes. For inhibition
of the mouse complement system, 5% mouse serum was preincubated
with 10–100 mg/ml recombinant UspA150–770or UspA230–539for 30 min
at 37˚C.
Complement activation ELISA
The inhibitory effect of UspA1 and UspA2 on the alternative and classical
alternative or classical pathways with specific activators and detection of
C3b or MAC (C5b–9) deposition as previously described (65). Microtiter
plates (F96 Maxisorb; Nunc-Immuno Module, Roskilde, Denmark) were
coated with LPS (10 mg/ml) (activating the alternative pathway) or IgM
(2 mg/ml) (activating the classical pathway) overnight at 4˚C, washed four
times with PBS with 0.1% Tween 20 (PBS-Tween), and blocked for 1 h at
37˚C with PBS containing 2% BSA. NHS was diluted in MgEGTA buffer
(20 mM HEPES, 144 mM NaCl, 7 mM MgCl2, and 10 mM EGTA [pH
7.4]) or gelatin veronal buffer (Complement Technology) and incubated in
plates for 1 h at 37˚C. After washings, complement activation was mea-
sured using mouse anti-human C3b mAb or mouse anti-human C5b–9
mAb and HRP-conjugated rabbit anti-mouse pAbs. The reaction was de-
veloped with 1,2-phenylenediamine dihydrochloride (Dako). Complement
activity was quantified by measuring the absorbance at 492 nm. The
complement activation of NHS alone was set to 100%. For inhibition,
UspA150–770(5–50 mg/ml), UspA230–539(5–50 mg/ml), factor H (5–50 mg/
ml) (alternative pathway), C4BP (5–50 mg/ml) (classical pathway), or BSA
(5–50 mg/ml) were preincubated with 20% NHS (alternative pathway) or
2% NHS (classical pathway) for 15 min at 37˚C and thereafter added to the
Trypsin sensitivity assay
C3 (6 mg) was incubated with or without 15 mg recombinant UspA150–770,
UspA230–539, or Efb in PBS containing 0.01 mg/ml trypsin at 37˚C for 0, 5,
10, 15, and 20 min. At each time point, aliquots (15 ml) were withdrawn
and the reactions were inhibited with soybean trypsin inhibitor (1:1). The
samples were thereafter separated by SDS-PAGE (10%) and visualized by
Coomassie blue staining.
Analysis of C3a generation
For activation of the alternative pathway, NHS (5%) was incubated with
zymosan (100 mg/ml) with or without increasing concentrations (0-50 mg/
ml) of recombinant UspA150–770or UspA230–539in a buffer only allowing
alternative pathway activation (PBS containing 3 mM MgCl2and 0.01 M
EGTA) for 30 min at 37˚C. This MgEGTA-containing buffer inhibits the
classical and lectin pathways and therefore allows separate analysis of the
alternative pathway. The incubations were kept in a final volume of 100 ml.
After incubation, the supernatants (7.5 ml/lane) were subjected to 12%
SDS-PAGE. Electrophoretical transfer of protein bands from the gel to
a nitrocellulose membrane was done in semidry conditions. After transfer,
the Immobilon-P membrane was blocked in PBS-Tween containing 1%
BSA and 4% milk powder (Carl Roth, Karlsruhe, Germany). After several
washings in PBS-Tween, the membrane was incubated with rabbit anti-
C3a pAb, followed by a HRP-conjugated swine anti-rabbit pAb (Dako-
patts). Development was performed with ECL Western blotting detection
reagents (Applichem, Darmstadt, Germany).
To estimate which fragments of C3 were involved in the binding to M.
catarrhalis, microtiter plates (F96 Maxisorb; Nunc-Immuno Module) were
coated with live M. catarrhalis or purified recombinant full-length UspA1
(5 mg/ml) or UspA2 (5 mg/ml) in 0.1 M Tris-HCl (pH 9.0) overnight at
4˚C. Plates were washed with PBS-0.05% Tween and blocked for 1 h at
room temperature with AppliChem blocking buffer. After washings, the
wells were incubated for 1 h at room temperature with C3 (5 mg/ml), C3b
(5 mg/ml), C3c (5 mg/ml), or C3d (5 mg/ml) in PBS. Thereafter, the plates
were washed and incubated with goat anti-human C3 pAb or rabbit anti-
human C3d pAb for 1 h. After additional washings, HRP-conjugated anti-
rabbit pAb or HRP-conjugated anti-goat pAb were added and incubated at
room temperature for 40 min. The wells were washed, developed, and the
absorbance was measured at OD492 nmwith a Multiscan Ascent (Thermo
Labsystems, Helsinki, Finland).
To determine which part of UspA1 and UspA2 was responsible for the
C3d binding, UspA1 and UspA2 fragments (20 mM) were immobilized on
microtiter plates overnight at 4˚C. The wells were blocked using Appli-
Chem blocking buffer and thereafter washed four times. C3d (5 mg/ml)
was added to the wells and incubated for 1 h at room temperature.
Thereafter, the plates were washed and incubated with rabbit anti-human
C3d pAb for 1 h followed by HRP-conjugated anti-rabbit pAb. The wells
were washed, developed, and the absorbance was measured at OD492 nm
with a Multiscan Ascent (Thermo Labsystems).
Direct binding assay
Recombinant C3d was labeled with 0.05 mol iodine (GE Healthcare,
Buckinghamshire, U.K.) per mol protein, using the chloramine-T method
(66). The M. catarrhalis wild type (RH4) and UspA1/A2H-deficient
mutants were grown on solid medium overnight and washed in PBS
containing 1% BSA (Saveen Werner). Bacteria (2 3 107) were incubated
with [125I]-labeled C3d at 37˚C for 1 h. After incubation, the bacteria were
centrifuged (10,000 3 g) through a 20% sucrose column. The tubes were
frozen and cut, and radioactivity in the pellet supernatant was measured in
a gamma counter. Binding was calculated as amount of bound radioactivity
(pellet) versus total radioactivity (pellet plus supernatant).
Statistical analysis
Most results were analyzed by a Student t test (*p # 0.05; **p # 0.01;
***p # 0.001) where p # 0.05 was considered to be statistically signifi-
cant. C3met binding and UspA1/A2/A2H expression were assessed by
Pearson correlation analysis.
The Journal of Immunology3
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Binding of C3 is related to UspA expression of M. catarrhalis in
clinical isolates and correlates with serum resistance
We have previously shown that M. catarrhalis binds and neu-
tralizes C3 at the bacterial surface, which protects against the
complement-mediated attack and contributes to serum resistance
(27). UspA1 and UspA2/A2H are the main outer membrane pro-
teins that are involved in the interaction between M. catarrhalis
and C3 (27). To analyze whether the UspA expression of different
M. catarrhalis strains correlated with C3 binding, bacteria were
incubated with a rabbit anti-UspA pAb recognizing all UspAs or
C3met, followed by FITC-conjugated swine anti-rabbit or rabbit
anti-human C3 pAb, and analyzed by flow cytometry. A correla-
tion between UspA expression and C3met binding (Pearson cor-
relation coefficient, 0.57; p # 0.01) was found (Fig. 1A). Thus,
most of the UspA-expressing M. catarrhalis interacts with C3.
To further analyze whether there was a correlation between
serum resistance of different clinical M. catarrhalis isolates and
the capacity to bind C3, C3met binding to M. catarrhalis was
analyzed by flow cytometry and correlated with the ability of the
different strains to survive in NHS. Native C3 was purified from
human serum and treated with methylamine, which converts C3 to
a C3met molecule equivalent to C3b without the capacity to co-
valently bind to microbes. In the bactericidal assay, the different
M. catarrhalis isolates were incubated in the presence of NHS for
30 min of incubation and surviving bacteria (CFU) were de-
termined. Serum-resistant strains bound more C3met; that is, the
high C3met-binding strains were more resistant to NHS than were
the low C3met-binding strains (Fig. 1B). More than 75% survival
after 30 min of incubation was classified as serum-resistant strains.
Additionally, the serum-sensitive M. catarrhalis isolates bound
less C3met as compared with the more serum-resistant strains.
Less than 25% survival after 30 min of incubation was classified
as serum-sensitive strains.
Binding of C3met to M. catarrhalis wild type and the Usp
double mutant was analyzed by flow cytometry using a polyclonal
anti-C3 antiserum. The M. catarrhalis RH4 wild type bound
C3met in a dose-dependent manner at C3met concentrations
ranging from 1 to 750 mg/ml (Fig. 1C). In contrast, no significant
binding of C3met was observed with the UspA1/A2H-deficient
double mutant (RH4 DuspA1/A2H) even at the highest C3met
concentration (750 mg/ml) used. The UspA2H of M. catarrhalis
RH4 was formerly known as UspA2, but the C3met binding ca-
pacity of UspA2 and UspA2H is similar (26, 28, 62). The
experiments shown in Fig. 1C were performed using purified
C3met in the absence of serum. To analyze whether the M.
catarrhalis RH4 wild type also bound C3 directly from serum, we
incubated bacteria with increasing concentrations of NHS with
addition of EDTA (0–60%) and analyzed C3 binding using
a polyclonal anti-C3 antiserum and flow cytometry. EDTA inhibits
all complement pathways by absorbing divalent cations (Mg2+and
Ca2+). When the complement system was inactivated in the
presence of EDTA, M. catarrhalis still bound C3 (Fig. 1D). Thus,
M. catarrhalis bound C3 regardless of complement activation. The
RH4 wild type bound C3 in a dose-dependent manner and the
binding was saturated at 50% NHS-EDTA, which corresponded
to a C3 concentration of ∼500 mg/ml (Fig. 1D). As expected, the
UspA1/A2H-deficient Moraxella mutant did not show any sig-
nificant binding to C3 when incubated with NHS-EDTA. Aweaker
intensity of the C3 binding was detected when serum was used as
a source of C3 (mean fluorescence intensity, ∼15) compared with
the purified C3met (mean fluorescence intensity, ∼40) when
binding at 500 mg/ml C3met was analyzed. Taken together, M.
catarrhalis dose-dependently bound C3met or C3 directly from
serum via the UspAs.
UspAs inhibit the hemolytic activity of the classical pathway
Recombinant UspA1 and UspA2 inhibit the alternative pathway
when analyzed in a hemolytic assay using rabbit erythrocytes and
a buffer only allowing the activation of the alternative pathway
(27). To establish the role of UspA1 and UspA2 as inhibitors of
the classical pathway, we included a classical hemolytic assay
consisting of NHS and amboceptor-coated sheep erythrocytes
(64). NHS was preincubated with recombinant UspA150–770or
UspA230–539followed by addition of erythrocytes. These two
UspAs were derived from the UspA1/A2-expressing strain M.
catarrhalis Bc5. After 1 h incubation, the amount of erythrocyte
lysis was measured. Maximum hemolysis (NHS without inhibitor)
in each experiment was defined as 100%. Significantly decreased
hemolysis was observed when NHS was preincubated with
UspA150–770or UspA230–539as compared with untreated NHS
(Fig. 2). UspA1 and UspA2 (both at 10 mg/ml) inhibited hemo-
lysis by 82 and 50%, respectively. Thus, in addition to inhibit the
alternative pathway (27), the UspAs also reduced the classical
pathway-mediated hemolysis of sheep erythrocytes.
UspAs inhibit complement activation at the C3 level
To confirm that UspA1 and UspA2 inhibit both the alternative and
classical pathways and to determine at what level UspAs inhibit
complement activation, analysis of complement activation was
performed with ELISA. The alternative and classical pathways
binding, and serum resistance of different Moraxella catarrhalis strains
corresponds to C3met binding. A, Bacteria were incubated with an anti-
UspA pAb, followed by a FITC-conjugated swine anti-rabbit IgG pAb and
flow cytometry analysis. To determine C3 binding, M. catarrhalis was
incubated for 60 min with 40 mg/ml C3met (A, B) or increasing concen-
trations of C3met (1–750 mg/ml) (C). C3 bound to the bacteria was ana-
lyzed by a FITC-conjugated anti-C3 pAb and flow cytometry. In B, the
clinical M. catarrhalis isolates were incubated in the presence of 10%
NHS for 30 min. Thereafter, the suspensions were spread on BHI agar
plates to allow determination of surviving bacteria. The number of bacteria
(CFU) at the initiation of the experiment was defined as 100%; .75%
survival and ,25% survival was classified as serum-resistant or serum-
sensitive strains, respectively. D, M. catarrhalis binds C3 directly from
NHS via UspAs, and the binding is dose-dependent. Moraxella was in-
cubated with increasing concentrations of NHS-EDTA (5–60%) and ana-
lyzed by flow cytometry. The mean values out of three independent
experiments are shown. Error bars indicate SD. **p # 0.01, ***p # 0.001.
UspA expression of M. catarrhalis correlates with C3
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were activated with LPS and IgM, respectively, NHS was pre-
incubated with UspA1 and UspA2, and activation was followed by
detection of C3b and MAC (C5b–9) deposition. As can be seen in
Fig. 3, UspA1 and UspA2 inhibited activation of the alternative
pathway. In the alternative pathway, recombinant UspA2 at low
concentrations was more efficient in inhibiting C3b and C5b–9
deposition compared with UspA1 (Fig. 3A, 3B), but at higher
concentrations both UspA1 and UspA2 showed an efficient in-
hibition. At a concentration of 50 mg/ml, UspA1 lowered both
C3b and C5b–9 deposition by 50% and UspA2 by . 70%, re-
spectively, when the alternative pathway was analyzed (Fig. 3A,
3B). Thus, both bacterial proteins were efficient inhibitors of the
alternative pathway and already inhibited on the level of the C3
convertase. In parallel to inhibiting the alternative pathway, the
microbial proteins also inhibited the activation of the classical
pathway. At 50 mg/ml, UspA1 and UspA2 inhibited the C3b de-
position by 61 and 48%, respectively (Fig. 3C). As expected, when
the bacterial proteins blocked complement at the C3 level, the
C5b–9 deposition was also inhibited (Fig. 3B, 3D). The positive
controls factor H (alternative pathway inhibitor) and C4BP
(classical pathway inhibitor) inhibited C3b and/or C5b–9 de-
position. In contrast to a C4BP-dependent blockage of C5b–9
deposition (Fig. 3D), C4BP did not block C3b deposition (Fig.
3C). The reason for this is presently unknown. In conclusion,
UspA1 and UspA2 inhibited activation of both the alternative and
classical pathways. Our results further proved that the bacterial
outer membrane proteins inhibited the C3b deposition and there-
fore acted at the C3 level in the complement cascade.
UspA1 and UspA2 do not induce conformational changes
of C3
Protease sensitivity assays can be used to determine whether
a protein induces changes in the conformation of a particular
protein (57). Interestingly, it has been shown that the C3d-binding
Efb of S. aureus changes the conformation of C3, and that C3
bound to Efb is degraded into several low-molecular mass frag-
ments in the presence of trypsin (57). To determine whether
binding of UspA1 and A2 to C3 would give similar result as Efb,
a trypsin sensitivity assay was performed. C3 was incubated with
UspA1, UspA2, or Efb in the presence of trypsin. At different time
points, aliquots were removed and inhibited by soybean trypsin
inhibitor, separated by SDS-PAGE, and visualized by Coomassie
blue staining. When a bacterial protein changes the conformation
of C3 and induces trypsin sensitivity, the intensity of the C3 a9-
chain is reduced and low-molecular mass fragments appear (57).
No significant decrease in the intensity of the a9-chain in the
presence of UspA1 or A2 could be detected compared with C3
alone (Fig. 4). However, additional bands with a molecular mass
between 40 and 50 kDa appeared in the presence of UspA1 or
UspA2. Western blot analysis using anti-human C3 pAbs was
performed to determine whether these extra bands were degra-
dation products of C3. The anti-C3 pAb did not detect these
bands, suggesting that the extra bands were degradation products
of UspA1 and UspA2 (data not shown). Thus, UspA1 and UspA2
did not induce a conformational change in C3, making it sus-
ceptible to trypsin. Since it previously has been shown that Efb
makes C3 susceptible to proteolysis in the presence of trypsin
(57), Efb was included as a positive control. A reduced intensity of
sheep erythrocytes by inhibiting the classical pathway. NHS was incubated
with or without recombinant UspA150–770(2.5–10 mg/ml) or UspA230–539
(2.5–10 mg/ml) for 15 min at 37˚C and thereafter added to the amboceptor-
coated erythrocytes. After incubation for 60 min, the suspension was
centrifuged and the supernatants were measured by spectrophotometry at
414 nm. Maximum hemolysis in each experiment was defined as 100%.
The mean values out of three separate experiments are shown and error
bars correspond to SD. *p # 0.05, **p # 0.01, ***p # 0.001.
Recombinant UspA1 and UspA2 decrease hemolysis of
ways. A and C, C3b deposition via the alternative (A; 20% NHS) and the
classical (C; 2% NHS) pathways. B and D, C5b–9 deposition via the al-
ternative (B; 20% NHS) and the classical (D; 2% NHS) pathways. NHS
was incubated with UspA1 (5–50 mg/ml) (:), UspA2 (5–50 mg/ml) (n),
BSA (5–50 mg/ml) (gray shaded circle), and factor H (5–50 mg/ml) (s), or
C4BP (5–50 mg/ml) ()), and activation was recorded by using an anti-
C3b mAb or anti-C5b–9 mAb and HRP-conjugated rabbit anti-mouse pAb.
Maximum complement activation (NHS without inhibitor) in each ex-
periment was defined as 100%. The mean values out of three separate
experiments are shown and error bars correspond to SD. *p # 0.05, **p #
0.01, ***p # 0.001.
UspA1 and UspA2 inhibit classical and alternative path-
of C3 in the presence of trypsin. C3 was incubated with UspA1, UspA2,
Efb, or buffer alone in the presence of 0.1% trypsin. At different time
points aliquots were removed, inhibited by soybean trypsin inhibitor,
separated by SDS-PAGE, and proteins were stained with Coomassie blue.
C3 without trypsin was used as a control (lane 2, 0 min). A typical ex-
periment out of three is demonstrated.
UspA1 and UspA2 do not induce a conformational change
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the a9-chain of C3 and the appearance of additional degradation
fragments were found in the presence of recombinant Efb (Fig. 4).
UspA1 and UspA2 are efficient inhibitors of C3a generation
Complement activation results in generation of the anaphylatoxins,
including C3a and C5a, and these proteins are an important part of
the inflammatory process (37). As UspA1 and UspA2 bound to
C3b and inhibited complement activation at the level of the C3
convertase, we wanted to analyze whether the bacterial proteins
would affect the C3a formation and thereby the inflammatory
response. The C3 convertase cleaves C3 into C3b and C3a. The
effect of UspA1 or UspA2 upon the formation of C3a was ex-
amined in zymosan-activated NHS. Zymosan was used to activate
NHS with or without recombinant UspA1 and UspA2, and ali-
quots of the supernatants were analyzed for C3a production by
SDS-PAGE and Western blotting. When C3a was detected with an
anti-human C3a pAb, detectable levels of C3a were observed in
the control samples without recombinant UspA1/A2 (Fig. 5, lane
1). In contrast, 25 mg/ml both UspA1 and UspA2 reduced the
amount of C3a generated, and UspA1 and UspA2, both at 50 mg/
ml, completely inhibited the C3a generation (Fig. 5, lanes 4–5).
Factor H (Fig. 5, lane 6) and BSA (Fig. 5, lane 7) were used as
positive and negative controls, respectively. Thus, both UspA1 and
UspA2 inhibited the C3a generation that may lead to a decreased
inflammatory reaction.
M. catarrhalis binds to the C3d domain of the C3 molecule
C3 is comprised of one a-chain and one b-chain, which are
connected by disulfide bonds and associated by noncovalent forces
(35). During complement activation, C3 undergoes multiple
cleavage events that generate various fragments, including C3a,
C3b, iC3b, C3c, C3dg, and C3g. To analyze which part of the C3
molecule mediates the interaction between M. catarrhalis and C3,
a series of different experiments were performed. To identify the
binding fragment of C3, binding of C3, C3b, C3c, or C3d to M.
catarrhalis RH4 wild type was analyzed in a whole-cell ELISA.
M. catarrhalis significantly bound C3d (Fig. 6A), whereas no
binding of the C3c fragment was seen. Binding of C3 and C3b,
which both contain the C3d fragment, was also demonstrated.
Binding of C3d to the wild type and the different UspA mutants
was analyzed by a direct binding assay with [125I]-labeled C3d
(Fig. 6B). Interestingly, the binding of C3d correlated with
UspA1/A2H expression; both of the M. catarrhalis DuspA1 and
DuspA2H isogenic mutants bound less C3d, whereas C3d binding
to the M. catarrhalis DuspA1/A2H mutant was further decreased
and nearly abolished. To further analyze the interaction between
C3 and UspA1/A2, recombinant UspA150–770and UspA230–539
were immobilized in microtiter plates, followed by incubation
with different C3 fragments as indicated in Fig. 6C and 6D. The
ELISA demonstrated that both UspA1 and UspA2 bound C3d and
the C3d-containing fragments (i.e., C3 and C3b). Addition of C3c,
which does not contain the C3d region, showed a much weaker
binding to both recombinant UspA1 and UspA2. Our experiments
revealed that M. catarrhalis bound C3d via the outer membrane
proteins UspA1 and UspA2/A2H.
The C3d binding domains are located within UspA1299–452and
To define the specific C3d-binding domains of UspA1 and UspA2,
a series of recombinant proteins spanning the entire UspA150–770
and UspA230–539molecules were manufactured. C3d was in-
cubated with equimolar amounts of the immobilized full-length
proteins and the truncated UspA1 and UspA2 fragments. There-
after, the binding was quantified by ELISA. UspA1299–452and
UspA2165–318efficiently bound C3d, suggesting that a binding
domain was located within these amino acid residues (Fig. 7).
However, UspA150–321was most likely also involved in the C3d
binding, albeit this sequence had a lower binding capacity com-
pared with UspA1299–452.
UspAs interact with C3 derived from mice
To determine whether C3 binding of UspA1/A2H-expressing M.
catarrhalis is species specific, the binding of C3 from murine
serum was analyzed. Bacteria were incubated with increasing
concentrations of EDTA-treated mouse serum (0–10%). When the
mouse complement system was inactivated in the presence of
activation assay. Human serum was incubated with or without recombinant
UspA150–770(A) and UspA230–539(B) and activated by zymosan. The
supernatants were analyzed by Western blots using anti-human C3a pAb
and HRP-conjugated swine anti-rabbit pAb. A typical experiment out of
three is demonstrated. Factor H (20 mg/ml) that inhibits C3a formation and
BSA (50 mg/ml) were included as positive (+) and negative (2) controls,
UspA1 and UspA2 inhibit C3a formation in a zymosan
type (A), UspA150–770(C), and UspA230–539(D) were immobilized on
microtiter plates and incubated with C3, C3b, C3c, or C3d, followed by
incubation with goat anti-human C3 pAb or rabbit anti-human C3d pAb.
HRP-conjugated anti-goat pAb or anti-rabbit pAb was used as a secondary
layer. Because two different pAbs were used, the absolute absorbance
obtained is not directly comparable. B, The wild type and UspA1-,
UspA2H-, and UspA1/A2H-deficient Moraxella mutants were incubated
with [125I]-labeled C3d. Binding was determined as percentage of bound
radioactivity versus added radioactivity measured after separation of free
and bound [125I]-labeled protein over a sucrose column. The mean values
of these experiments with duplicates are shown with error bars indicating
SD. *p # 0.05, **p # 0.01.
M. catarrhalis UspAs interact with C3d. M. catarrhalis wild
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EDTA, M. catarrhalis still bound murine C3 in a dose-dependent
manner (Fig. 8A). In contrast, murine C3 showed a significantly
decreased (∼80%) binding to the RH4DuspA1/A2H double mu-
tant. Both the UspA1- and UspA2H-deficient mutants displayed
a reduced binding; UspA1- and UspA2H-deficient mutants bound
∼20 and 40% less C3, respectively, as compared with the wild
type when incubated with 10% mouse serum (Fig. 8A). Thus, M.
catarrhalis bound murine C3 regardless of complement activation,
and both UspA1 and UspA2 contributed to the binding.
To analyze whether the inhibition of the alternative pathway
was species specific, a hemolytic assay with murine serum was also
performed. Serum was preincubated with recombinant UspA150–770
or UspA230–539, followed by addition to rabbit erythrocytes. After
3 h incubation, the amount of erythrocyte lysis was measured. In-
terestingly, significantly decreased hemolysis was observed when
mouse serum was preincubated with UspA230–539compared with
untreated mouse serum (Fig. 8B). However, when the mouse serum
was preincubated with UspA150–770, no inhibition was detected.
Taken together, in parallel with the experiments comprising NHS,
M. catarrhalis UspA2 inhibited the alternative pathway in mouse
serum by binding mouse C3.
The pathogenesis of M. catarrhalis relies on its capacity to resist
the human host defense, including complement (1). The comple-
ment system is very harmful for Gram-negative pathogens, in-
cluding M. catarrhalis, and bacterial complement resistance is one
of the most important virulence mechanisms (67). M. catarrhalis
has thus developed several efficient strategies to circumvent
complement. It has been demonstrated that UspA1 and UspA2
interact with and inhibit the alternative pathway of complement
by noncovalently binding C3 (27). In the present study we show
a correlation between C3met binding and Moraxella-dependent
UspA expression. Furthermore, C3met binding corresponded with
serum resistance of M. catarrhalis. Because C3 is a key molecule
in the complement system, the binding of C3 most likely results in
the regulation of all three pathways and may contribute to serum
resistance. In fact, UspAs inhibit the activation of the alternative
and the classical pathways and block generation of C3b and MAC
(C5b–9) deposition. The reduction of the C3b deposition shows
that UspAs act early in the complement cascade and at the C3
level by binding C3. Because the bacterial proteins act and in-
terfere with this early level of complement activation, the in-
hibitory capacity of UspAs is not restricted to one single pathway.
By blocking the C3b deposition, the downstream steps, including
formation of the C5 convertase and generation and deposition of
MAC, are also inhibited. In fact, Moraxella UspAs inhibit both
MAC deposition and hemolysis of erythrocytes when activated via
the alternative and classical pathways (27). Inhibition of the
classical pathway is most likely caused by simultaneous re-
cruitment of both C4BP and C3 (26). By inhibition of the different
complement pathways resulting in increased survival, M. catar-
rhalis can colonize, infect the human host, and eventually cause
A very common evasion mechanism used by most pathogens
involves the acquisition and utilization of human complement
inhibitors, such as C4BP, factor H, and vitronectin (38). The dif-
ferent complement inhibitors are functionally active when bound
to the surface of the pathogens and are capable of inhibiting dif-
ferent steps of the complement cascade. In addition to binding the
complement inhibitor C4BP, M. catarrhalis binds and utilizes
vitronectin for protection against the terminal pathway (25, 28).
These interactions contribute to serum resistance and most M.
catarrhalis isolates from patients with lower respiratory tract
infections are resistant to complement-mediated killing (68). It is
a well-known fact that both complement factors and inhibitors are
present in the respiratory tract (69–71). Therefore, respiratory
pathogens must protect themselves from complement in similarity
to blood-borne pathogens, and consequently these microbes also
have access to complement inhibitors. The surface-expressed
and UspA2165–318. Truncated proteins derived from UspA1 and UspA2 are
shown. All fragments were tested for binding to C3d by ELISA; 20 mM
each fragment was immobilized on microtiter plates and and incubated
with 5 mg/ml C3d. Bound C3d was detected with rabbit anti-human C3d
pAb followed by HRP-conjugated anti-rabbit pAb. Mean values are shown
with error bars indicating SD. **p # 0.01, ***p # 0.001.
Active C3d binding regions are located within UspA1299–452
decreases hemolysis of rabbit erythrocytes by inhibiting the alternative
pathway in mouse serum. A, M. catarrhalis bound C3 from mouse serum.
The wild type and corresponding UspA1/A2H-deficient mutants were in-
cubated with increasing concentrations of mouse serum (0–10%), followed
by detection with a FITC-conjugated rabbit anti-mouse C3 pAb and flow
cytometry analysis. B, Mouse serum was incubated with or without 10–100
mg/ml UspA150–770or UspA230–539at 37˚C for 30 min. The preincubated
mouse serum was added to the erythrocytes. After incubation for 3 h, the
suspension was centrifuged and the supernatants were measured by
spectrophotometry. The mean values out of three separate experiments are
shown and error bars correspond to SD. *p # 0.05, **p # 0.01.
Moraxella UspAs interact with mouse C3, and UspA2
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UspA1 and/or UspA2/A2H of M. catarrhalis have been shown to
be responsible for C4BP and vitronectin binding and thus are
important for bacterial survival in human serum (10, 22, 25, 26,
28). The use of more than one strategy to evade the complement
system is common and shared by several pathogens (38–40). One
pathogen can express multiple proteins contributing to the escape
of the complement attack. It has been shown that M. catarrhalis is
equally resistant to both the classical and alternative pathways
(27). What exact mechanism that is the most important in the
clearance of M. catarrhalis in the human host is at present un-
known. However, M. catarrhalis uses several evasion strategies
for protection against the complement attack involving the ex-
pression of different surface proteins, including the UspAs, CopB,
protein CD, and protein E (13, 26–28, 72, 73). The contribution of
the different complement evasion mechanisms to the pathogenesis
of M. catarrhalis needs to be further studied and clarified. How-
ever, every mechanism that provides protection from complement
is certainly beneficial for bacterial survival. By interfering with
the complement system at the C3 level, which is common for all
pathways, C3d-dependent complement inhibition of M. catar-
rhalis is a very efficient mechanism that contributes to serum
resistance and thereby the virulence of the bacteria.
During complement activation the C3 convertases cleave C3
into the opsonin C3b and the anaphylatoxin C3a. UspA1 and
UspA2 bind C3 and inhibit C3b deposition, and by inhibiting
C3b deposition M. catarrhalis may prevent opsonization and
phagocytosis. Furthermore, the UspAs inhibited the generation of
the potent anaphylatoxin C3a when human serum was used as
a source of the C3 convertase. C3a belongs to the group of the
most powerful immunopharmacological substances known and is
an important part of the inflammatory process. This group of small
peptides causes histamine release from mast cells, smooth muscle
contraction, and increases vascular permeability (74, 75). Addi-
tionally, C3a recruits inflammatory cells to the site of inflam-
mation and activates their effector mechanisms. Thus, UspA1 and
UspA2 both inhibit the C3b deposition and C3a generation, sug-
gesting that these interactions may inhibit phagocytosis and re-
duce the inflammatory response.
During complement activation, C3 undergoes multiple cleavage
events that generate various fragments, including C3a, C3b, iC3b,
C3c, C3dg, and C3g. We show that M. catarrhalis interacts with
the C3d fragment of C3. By analyzing the UspA1 mutant, the
UspA2H mutant, and the double mutant (M. catarrhalis DuspA1/
A2H) in a direct binding assay, we found the UspAs to be the
major C3d-binding proteins of M. catarrhalis. Additionally, the
C3d-containing fragments C3, C3b, and C3d bound both re-
combinant UspA1 and UspA2 in ELISA, whereas C3c, which
lacks the C3d region, did not bind. Defining the M. catarrhalis and
UspA-binding site to the C3d fragment is important since this
fragment has binding sites for both factor H and the complement
receptor 2 (CD21) (35). CD21 is expressed on B cells and, to-
gether with CD19 and CD81, forms the B cell coreceptor com-
plex, which lowers the threshold for B cell activation substantially
(76). Binding of pathogen-bound C3d is therefore regarded as an
essential link between the innate and adaptive immune responses.
In addition to inhibiting complement, UspA1 and UspA2 may also
affect the interaction of C3d with its major receptor and to at-
tenuate the initiation of downstream immune responses. Several
pathogens have been shown to bind C3 and its fragments, in-
cluding C. albicans, S. pneumoniae, and S. aureus (52, 57, 77, 78).
S. aureus Efb binds native C3 and some of its fragments (i.e., C3b
and C3d), and binding of C3 induces a conformational change of
C3 so that the protein is unable to participate in successful acti-
vation of the complement cascade (57). In contrast to Efb, neither
UspA1 nor UspA2 induced a conformational change in C3 when
analyzed in a trypsin sensitivity assay. Therefore, UspA1 and
UspA2 inhibit the complement system in a different way com-
pared with Efb from S. aureus. Additionally, Efb and Ehp, which
is another complement inhibitory protein of S. aureus, both bind to
C3 and reduce its availability at the bacterial surface, thus re-
ducing the opsonization of the bacteria (51, 56, 57, 79). Further-
more, S. aureus Sbi binds C3d and inhibits the alternative pathway
of complement (55, 80). Interestingly, Efb, Ehp, and Sbi bind the
C3d fragment and inhibit the interaction between C3d and its
receptor CD21, which plays an important role in B cell activation
and maturation (55, 81, 82). In addition to binding human C3,
UspAs also bind C3 from mouse serum. UspA2 inhibits the al-
ternative pathway of mouse complement, suggesting that the in-
teraction with C3 and inhibition of complement activation are not
species specific.
The C3d-binding regions UspA1299–452and UspA2165–318from
the clinical isolate M. catarrhalis Bc5 were found to contain the
C3d binding sites. Intriguingly, UspA1299–452and UspA2165–318
share a sequence of 31 identical amino acids residues, including
the 23-aa NNINNIYELAQQQDQHSSDIKTL (NNINNIY se-
quence) (24). This sequence contains the epitope for the protective
mAb 17C7, for which there is universal reactivity (16, 83). In
a mouse model, passive immunization with mAb 17C7 provided
protection and improved pulmonary clearance of M. catarrhalis
(83). Additionally, these two fragments (UspA1299–452and
UspA2165–318) also contain the fibronectin-binding domains of
UspA1/A2 from M. catarrhalis and they have been shown to be
important in adhesion to epithelial cells (24). The capacity of the
UspAs to interact with several human proteins shows that they
indeed are multifunctional adhesins.
In conclusion, M. catarrhalis UspAs are highly important vir-
ulence factors that interfere with complement in several ways to
protect the species from the complement-mediated attacks (1, 38).
To our knowledge, this is the first report describing that M.
catarrhalis does not only avoid killing by complement but also
modulates complement activation at the level of ther C3 con-
veratse and thus may also inhibit the inflammatory response by
neutralizing C3 and reducing C3a generation.
We thank Michael Reuter and Sascha Bo ¨hm from the Department of In-
fection Biology of the Leibniz Institute for Natural Product Research and
Infection Biology, Hans Kno ¨ll Institute, for providing the recombinant Efb
from S. aureus.
The authors have no financial conflicts of interest.
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Passion flower won't bloom outside
I planted my passion flower outside last summer and it grew like crazy. Maybe 9 - 10 feet but it didn't flower. I had it growing on the wall of my garage which faces east on the south side of my house. I have cutting overwintering again this year and I would like to put them out again but how do I get them to flower.
Hi jo-smo,
I'm probably not the best person to address your problem, but since nobody has gotten back to you I'll ask a couple questions:
Do you know what species/hybrid it is? This may be crucial. If you don't know, if you can find a picture of it that may also be useful.
Do you fertilize? Apparently sometimes this can produce growth at the expense of flowers.
How old was the plant? It's possible it needs more than one summer to go from rooted cutting (if that's what it was) to blooming size. If that's a problem you may want to start it in a pot for a year or two before putting it in the ground.
I'm sure there are other obvious questions; those are just the ones off the top of my head.

subset: commoncrawl


text:
Do resistance debuffs stack?
I am wondering if things such as fire at my mark and sensor scan stack? They're both resistance debuffs. Any input would be appreciated. Thanks!
Yes, but all resistances in STO operate on diminishing returns. So the more resistance or -resistance you have applied the less effective it is.
Around what % does the DR become not worth it?
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text:
The privacy of personal information will be a routine part of the catalog of security controls used to protect federal information and information systems if a draft version of such controls that the National Institute of Standards and Technology realeaed on July 20 is adopted.
The controls that would become a part of the basic security controls in the latest guidelines for the Federal Information Security Management Act.
Protecting personally identifiable information, whether in paper or electronic form, is a fundamental responsibility of agencies, the draft version of the new guidelines says.
The proposed privacy controls include administrative, technical, and physical safeguards and are included as a separate appendix in the latest version of NIST's Security Controls for Federal Information Systems and Organizations (Special Publication 800-53 Revision 4).
The privacy guidelines have been released separately by NIST for public comment.
Related coverage:
Can government and industry solve the security/privacy equation?
"Information security and privacy are taking on new levels of importance in the public and private sectors," the guidelines say. "Privacy, with respect to personally identifiable information, is a core value that can be achieved only with appropriate legislation, policies, and associated controls to ensure compliance with requirements," it adds.
SP 800-53, now in revision 3, is a foundational document for FISMA compliance. The current version is a suite of publications released in 2009 and was a collaboration effort between NIST, the military and intelligence communities to establish a standard set of security controls for all government systems, including national security systems. The document, with new privacy controls, is being updated and revision 4 is expected to be released in December.
The proposed controls in Appendix J would supplement security controls in Appendix F of 800-53, and provide a roadmap for identifying and implementing privacy controls over the PII life cycle for both electronic and paper records.
The publication emphasizes the relationship of privacy and security.
"Privacy and security controls in federal information systems, programs, and organizations are complementary and mutually reinforcing in trying to achieve the privacy and security objectives of organizations," it says.
But although the two are complementary, they are separate. "Organizations cannot have effective privacy without a solid foundation of information security. However, privacy is more than security and confidentiality and includes the principles of transparency and notice and choice, for example," the guideline say.
The objectives of the Privacy Appendix are:
There is a similarity in the structure of the proposed privacy controls in Appendix J and the security controls in Appendix F, and the use of privacy plans in conjunction with security plans should help organizations select the appropriate set of controls to meet mission and business requirements. NIST also plans to develop assessment for evaluating the effectiveness of the controls. The privacy controls in Appendix J are being released for comment separately from the body of SP 800-53 because of the importance and special nature of the material, NIST announced. Comments should be sent by September 2 to email@example.com.
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William Jackson is freelance writer and the author of the CyberEye blog.
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Unisys Federal Systems' Venkatapathi Puvvada, this year's Eagle award winner for industry, stresses the importance of community.
Experts tell committee that business models, not technology, are the main obstacle to better sharing across systems.
Protests have delayed SEWP V, but industry experts say the contract vehicle offers agencies service and value that are hard to find.
The power of the individual informs FCW's coverage each and every day, but with the Federal 100, we take time to really spotlight and celebrate it. Get to know each of this year's winners.
The latest version of the Cybersecurity Information Sharing Act purportedly protects companies from liability and individuals from government intrusion.
Popular CIO is headed for the private sector.
The Census Bureau hasn't established a time frame for its cloud computing plans, including testing for scalability, security, and privacy protection, as well as determining a budget for cloud services.
President Obama's proposal to boost government coordination with the private sector got a warm welcome in the House Homeland Security Committee.
The spy agency wants to better integrate cybersecurity into its traditional human intelligence operations.
Huh - being that we already protect PII, this sounds redundant...
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|Events Calendar
JEvents v1.5.3 Copyright © 2006-2009
|Events Calendar
JEvents v1.5.3 Copyright © 2006-2009
|Cole Library Children's Story Time
Thu Mar 26 @09:30AM - 10:00AM
|Lisbon Public Library Story Hour
Thu Mar 26 @10:30AM - 11:00AM
|Public Lecture: "My Life as a Commercial Artist"
Thu Mar 26 @11:30AM - 12:30PM
|Live Music at Baxa's Sutliff Store & Tavern: Doug Ducey & Frank Zahorik
Thu Mar 26 @06:00PM - 09:00PM
|Live Music at the Lincoln Winebar: The Tornadoes
Thu Mar 26 @07:00PM - 09:00PM
|Movie: Insurgent
Thu Mar 26 @07:15PM -
|Teen DIY Workshop
Fri Mar 27 @03:30PM - 04:30PM
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Fri Mar 27 @05:00PM - 07:30PM
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Senior Prom – Oh, what a night!
Oh, what a night! 300 folks, dressed in their finery, celebrated Older Americans Month in May at the Roaring 20s Prom
The Flapper or "Queen" of the evening was Marjorie Factor and the Sheik or "King" of the evening was Gary Shoemaker. Both received PNC bank cards. The queen received a floral bouquet from Kroger Marketplace of Lewis Center and the king oil changes from AutoMax. The evening consisted of photos with a 1929 Dodge Roadster owned by Mr. and Mrs. T. Jack Carpenter and a 2014 Charger courtesy of AutoMax. A lovely dinner, prepared by All Occasions was enjoyed, followed by music and dancing by The Blue Sultans. Corsages were provided by the Delaware Career Center. Brent Carson was the emcee for the evening. A good night treat of a cookie was provide by Richwood Bank and a carnation from Kroger Marketplace Lewis Center as folks left for the evening. We thank all of our sponsors for an evening to remember.


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Tomlin has control over his staff
Also, surrounding yourself with former head coaches isn't a guy fearing for his job, that's just surrounding yourself with great football people.
No, Tomlin doesn't hire assistants like a coach afraid for his job.
He doesn't seem to coach like a coach afraid for his job, either. A little of the latter might be a good thing, IMO.
I think he looks like a coach who's growing frustrated over his lack of control, as he should be.
Last edited by WindyCitySteel; 10-11-2014 at 10:00 AM.
Start with Arians, the York High graduate. Tomlin said Monday after the playoff loss that he anticipated both of his coordinators would return for the 2012 season. Art Rooney later said some coaches might "retire."
The Post-Gazette reported that not only did Arians want to come back but that Tomlin told him he wanted him back.
Then, the Steelers issued a statement that Arians had "retired" with a comment from Tomlin wishing him well in his retirement. A week later, Arians took a job as offensive coordinator of the Indianapolis Colts.
A couple of weeks after that, Tomlin said that it was his decision to make a change at OC.
Do I think he was getting some pressure to make that change? Very likely. But I believe the final decision was his...I don't think he's lying about that.
It wouldn't be the only time Tomlin made an abrupt personnel change. Remember when he fired the STs coach (?Everest) after indicating that his staff was set for the season?
tomlin might be allowed to hire some low level coaches/assistants
1) First ballot HOF, ROTY
2) All Pro first year, first round talent that fell to us in second
3) Starter for years to come, cant believe he fell
4) Absolute steal of draft, 2nd round grade
5) Back up at first then starter for 10 years
6) Incredible depth and ST demon
7) Amazing value, shoulda went 3 rounds earlier


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New Native American Cuisine
Product Description
Product Information
|Format: Hardcover
Vendor: Globe Pequot
Publication Date: 2009
Dimensions: 9.75 X 8.50 (inches)
Availability: Usually ships in 24-48 hours.
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Publisher's Description
The New Native American Cuisine is the first book to make this cuisine available to home cooks everywhere. Beautifully illustrated with rich full-color photographs of the resort and its restaurant and dishes, it presents more than fifty recipes for cocktails; small plates and main courses; soups and salads, fish, meat, game, vegetables, and desserts—from grilled elk chop with truffles and sweet corn panacotta with venison carpaccio, to buffalo tartare with prairie quail egg.
Author Bio
Marian Betancourt is the author or coauthor of more than a dozen books, including The Texas Hill Country Cookbook: A Taste of Provence (Globe Pequot).
Michael O'Dowd, the award-winning Executive Chef of Sheraton Wild Horse Pass Resort, previously worked in New York City's finest restaurants and hotels.
Jack Strong is Chef de Cuisine at Kai. Hailing from the Confederated Tribes of Siletz Indians in Oregon.


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2421 Bissonnet
Houston, TX 77005
Category: Retail
Region: Kirby-West U
Houston-based Zepeda is well-known for her ability to create sparkling, three-dimensional characters and compelling storylines. She's done the same again with this novel. She'll be discussing and signing Better with You Here today at the book's launch party at 7 p.m. Brazos Bookstore, 2421 Bissonnet.
Fri., July 20, 7 p.m., 2012
Sponsor Content


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One could talk about Venezuela's economic policy in Shakespearean terms. To devalue or not to devalue; to converge foreign exchange rates or not to converge; to raise the price of the world's cheapest gasoline or not to raise; to sell Citgo or not to sell; to default or not to do so – these are the questions.
The distortions created by the government's foreign exchange and price controls – covering even Barbie dolls – keep playing a treacherous role in Venezuela's unfolding tragedy. Why is this happening instead of not happening? To some analysts, that is the question. Read more


text:
Sunday, January 1 1995
Something in the Air: An Interview with the Warlocks
Two drummers? A sound reminiscent of Swinging London? An evasive lead singer? PopMatters sets out to determine the myths and truths about mystery-rockers the Warlocks.
Singing Along With Your Walkmen
These Brooklyn softies are in great danger every day.
What Goes Around Comes Around: A Conversation With Patricia Vonne
Don't mess with Texas or a green-eyed girl. Whatever it is, Patricia Vonne has got it going on. But PopMatters is taller and wears bigger shoes.
The Slow-Burning Songs of Vetiver: An Interview with Andy Cabic
Devendra Banhart's favorite songwriting partner, Andy Cabic, is back with a darker, more percussive second album that will take more than a couple of spins to sink in.
Make It Beautiful and Trash It: An Interview with John Vanderslice
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Karl Hyde's Dirty Epic
Throughout their career, techno kingpins Underworld never bothered looking back. Does the release of their first greatest hits compilation mean they've entered their golden years? That's not the case, according to Karl Hyde; things are just getting interesting.
Superstar Tourism: An Interview with Carrie Underwood
'I keep my camera with me everywhere I go,' says American Idol winner Carrie Underwood. 'I try not to be too annoying with it.'
Ugly Duckling
The group formed in 1992-93 with members Dizzy Dustin, Young Einstein and a guy named Clout.
True Fictions: An Interview with Two Gallants' Adam Stephens
Already a master storyteller and folklorist at 23, Stephens brings us to the frontier, Thomas Edison, and the drunk tank.
Pumpin' the Brakes
PopMatters gets answers from Olly Knights and Gale Paridjanian of British rock duo Turin Brakes.
Crash Landing in Los Angeles: British Songwriter Alex Troup Soldiers on in an Unfamiliar Place
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The "C" Word: The Trash Can Sinatras Are Back
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The Ship Is Sinking: An Interview With ...And You Will Know Us By the Trail of Dead
Trail of Dead's Conrad Keely talks about the fate of the world, the course of history, and other minor matters.
A Family Room Affair: The Trachtenburg Family Slideshow Players
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Tower of Power
TOWER OF POWERSoul with a Capital 'S': The Best of Tower of PowerEpic/LegacyU.S. Release Date: 26 March 2002 Imagine the old days as the San
A Lazarus Taxon: the Definition of "Tortoise" Continues to Change Before Our Eyes
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A Conversation with Steve Bertrand, formerly of The Tories
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Lest we forget
|UrbanToronto News - the latest headlines
|Design of The Well Development at Spadina and Front Evolving
ALSO• Tarion Awards Recognize New Home Builders For Customer Service
• ZIGG Condos Offering Mid-Rise Mid-Town Living
• Casey House Expanding To Better Meet Needs
• Tower Floors Rising at The Madison on Eglinton
• Daily Headlines: News From the Internet for March 27, 2015
• Photo of the Day: Galleria Stairs
Pape Ave., one block above Danforth, at Lipton - 1927
Original house is still visible behind new storefronts - 2012
Subway station under renovation (behind green barrier).
We don't see many Then and Nows of houses turned into storefronts. It's always interesting visually to see what was lost and at the same time what is still there looming out from the back. I have an interest in naval history and a similar analogy would be those battleships converted into aircraft carriers.
Hey it's the weekend again.
This link is somewhat related to Then and Now. Cindy Sherman is a visual artist and photographer who uses herself as the subject in surreal and fantasy photographic compositions. I say somewhat related because she has been doing this for over thirty years. Be warned, some of her work can be upsetting.
Yes, Sherman has been playing her game for a long, long time.
Much more of her work is at this site:
Last edited by Goldie; 2012-Mar-24 at 21:59.
"In Vibrant Color, Vintage Celebrity Portraits."
Emma Stone reminds me so much of the young Lucille Ball...
thecharioteer, wwwebster, junctionist:
Thank you for adding to our body of knowledge of about Merchants Bank.
We need to have a UT picnic or gathering at the Guild Inn some day.


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Reynolds brings Cardinals' bench a surge of power and a dash of humor
ST. LOUIS -- Mark Reynolds did not take long to show off a sense of humor with his new team. As the media kept piling into his introductory presser at the Cardinals Winter Warm-Up, Reynolds stood at the podium wondering about the fuss being made. "Hey guys," he said with a smile. "Bench player, come on." He better get used to the attention. Reynolds might have been signed to a one...
Via Fox Sports Midwest
January 27, 2015 | Discuss
Reynolds Replaces A Missing Component
If you listened around St. Louis this afternoon after the Cardinals signed veteran bat Mark Reynolds to a one year deal, there wasn't much optimism in the air. One would think the Cards just signed a guy to a long term deal and dumped a ton of money into him. Many are overthinking this smart [...]
Via The Cardinal Conclave
December 12, 2014 | Discuss
Keeping Perspective With Mark Reynolds Deal
The Cardinals pulled their first fish out of this year's free agent waters on Thursday, by inking first baseman Mark Reynolds. Reynolds, most recently of the Milwaukee Brewers, was a specific target for the Cardinals this winter and his acquisition came to fruition finally today. He agreed to a one-year deal with financial terms yet to be disclosed. The club entered the off...
Via i70baseball
December 11, 2014 | Discuss
WATCH: Fan makes great catch of HR ball, tosses back other ball
65-year-old Cubs fan Mike Pullin caught Milwaukee Brewers first baseman Mark Reynolds' home run ball at the wall, escaped certain death...and then pulled the old switcharoo -- tossing back a 2nd ball. Keeping the game used one. [FOX]Your browser does not support iframes.Bam.ahhhhhhhhhhhhhhh pic.twitter.com/bCZErptoDT— Amanda Kaschube (@amandakaschube) August 12, 2014Follow us on...
Via Bob's Blitz
August 12, 2014 | Discuss
Reynolds not feeling pressure to make Opening Day roster
PHOENIX -- Spring training has been business as usual for Mark Reynolds, but there's nothing normal about this camp for the veteran. Ever since his rookie season of 2007, Reynolds has come to camp knowing he's going to be on the Opening Day roster. There's no certainty this spring. "It's definitely different, but I'm not going to approach it any different," Reynolds said. "I'm still...
Via Fox Sports Wisconsin
March 12, 2014 | Discuss
Mark Reynolds agrees to deal with Brewers
According to a report from RealGM.com, the Milwaukee Brewers have agreed to a minor league deal with slugger Mark Reynolds. Reynolds was reportedly weighing offers from five different teams earlier this season, but the Brewers were not on that list. The deal will pay Reynolds $2 million if added to the 40-man roster. Mark Reynolds played with both the Cleveland Indians and New York...
Via Sports-Glory
January 18, 2014 | Discuss
Former Indians 1B Mark Reynolds signs with Brewers
The Milwaukee Brewers have signed former Indians first baseman Mark Reynolds to a minor league deal with an invitation to spring training. Reynolds is expected to be on the Brewers Opening Day roster and will handle first base against lefties. Reynolds, 30, batted .220 with 21 home runs and 67 runs batted in over 135 games with Cleveland and the New York Yankees last year. He was...
Via Fox Sports Ohio
January 17, 2014 | Discuss
Will Mark Reynolds be the Yankees Ichiro of 2014?
There are whispers across the Yankiverse that the Retrieval Empire will try to move Ichiro Suzuki this winter. Ahhh, as if it were so easy! But such a move requires a lackey. In a perfect Yankee universe - sort of like 1959, when Kansas City functioned as our Waylan Smithers - we would trade Ichiro to the Rakuten Golden Eagles in exchange for the rights to Masahiro Tanaka.�...
Via IT IS HIGH, IT IS FAR, IT IS caught
December 24, 2013 | Discuss
Yankees Rumors: New York considering re-signing Mark Reynolds
With their infield in a state of flux, the New York Yankees are mulling the idea of re-signing infielder Mark Reynolds. According to Jon Heyman of CBS Sports, the Yankees have shown interest in a reunion but are not without a fair share of projected competition for his services. #yankees interested in return for mark reynolds. need INF help. twins, brewers, angels, #rays among other...
Via Sports Injury Alert
December 16, 2013 | Discuss
Yankees could bring back Mark Reynolds
Mark Reynolds could return to the New York Yankees for the 2014 season. The Yankees have joined half the American League in sending out feelers for the infielder. Reynolds could offer the Yankees protection at third base as well as first base with the status of Alex Rodriguez still uncertain. #yankees interested in return for mark reynolds. need INF help. twins, brewers, angels,...
Via Tireball.com
December 15, 2013 | Discuss
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DME Series Radial Polyester Film Capacitors
± 10% self-healing capacitors for general purpose AC or DC applications
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21-1046 - Lead Free Rosin Core Solder - Tin / Copper - 6OZ.
Lead–Free Solder
Ideal for general purpose electronic repair and assembly use. Tin/copper formulation meets current requirements for lead free solder use.
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31-11457 - Capacitor Radial 35V 5600uF 22 x 36mm Snap-In
Voltage: 35, uF: 5600, Dimensions: 22 x 36mm, Type: Radial Snap-In
50-14795 - Universal TV Stand, For Televisions 32" ~ 60"
Universal Television Stand
This universal TV stand is perfect for flat panel televisions that were mounted to the wall, but are to be moved to tabletop use. Universal mounting plate attaches to all VESA standard sets from 200x200 through 600x400, and provides a ...
55-2517 - 10'' Pyramid Pro Audio Woofer
These high-efficiency, high power capacity drivers are perfect as replacements in stage speakers, guitar amps, and monitors. Paper cones, treated accordion cloth surrounds and heavy duty stamped baskets combine to provide characteristics very similar to most OEM d...
Requires Adobe Acrobat Reader.
Smooth flawless transaction. Quality products for great prices. I compared my item to other stores before buying and was blown away by the price difference.


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I don't know how I lived without this CD before! I also read Jon Kabat-Zinn's books!Published 1 day ago by Julie Harnisch
WTH? It was missing one of the 4 CDs. Oh well, Sometimes you do what you overpaid for.Published 1 month ago by Manuela Mage
Kabat-Zinn has made a really fine guided meditation. His is basically a "how to" for deep meditation. I have series 1, 2 and 3. Read morePublished 1 month ago by David Hardesty, author of Electronic Commerce: Taxation and Planning (firstname.lastname@example.com)
I was so impressed when I saw Anderson Cooper's interview on this I bought it right away. I am so disappointed in this program. There isn't anything I can truly relate to. Read morePublished 1 month ago by Emma Feix Alberts
I have used these and really like them. HE speaks very slow and quietly so it lulls you into a relaxed state.Published 2 months ago by T. Priddle
I don't like posting negative reviews and I really wanted to like this CD, but the truth needs to be heard when peoples' health and well-being are at stake. Read morePublished 2 months ago by fademusic