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Infection and Immunity, March 2000, p. 1735-1739, Vol. 68, No. 3
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bordetella pertussis Virulence Factors
Affect Phagocytosis by Human Neutrophils
Christine L.
Weingart and
Alison A.
Weiss*
Department of Molecular Genetics,
Biochemistry, and Microbiology, University of Cincinnati,
Cincinnati, Ohio 45267-0524
Received 13 September 1999/Returned for modification 15 October
1999/Accepted 29 November 1999
 |
ABSTRACT |
The interaction between human neutrophils and wild-type
Bordetella pertussis or mutants expressing altered
lipopolysaccharide or lacking virulence factors
pertussis toxin,
adenylate cyclase toxin, dermonecrotic toxin, filamentous hemagglutinin
(FHA), pertactin, or BrkAwas examined. In the absence of antibodies,
the wild-type strain and the mutants, with the exception of
mutants lacking FHA, attached efficiently to neutrophils. The
addition of opsonizing antibodies caused a significant reduction
(approximately 50%) in attachment of the wild-type
strain and most of the mutants expressing FHA, suggesting that
bacterium-mediated attachment is more efficient than Fc-mediated
attachment. Phagocytosis was also examined. In the absence of
antibodies, about 12% of the wild-type bacteria were phagocytosed.
Opsonization caused a statistically significant reduction in
phagocytosis (to 3%), possibly a consequence of reduced attachment.
Phagocytosis of most of the mutants was similar to that of the wild
type, with the exception of the mutants lacking adenylate cyclase
toxin. About 70% of the adenylate cyclase toxin mutants were
phagocytosed, but only in the presence of opsonizing antibody,
suggesting that Fc receptor-mediated signaling may be needed for
phagocytosis. These studies indicate that FHA mediates attachment of
B. pertussis to neutrophils, but adenylate cyclase toxin
blocks phagocytosis.
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TEXT |
Bordetella pertussis is
the causative agent of whooping cough. During infection the bacteria
remain localized to the respiratory tract, causing considerable local
damage. However, whooping cough also has aspects of a
toxin-mediated disease (30). B. pertussis produces two toxins that are essential for virulence (17,
41). Pertussis toxin catalyzes the transfer of an ADP-ribosyl
group to regulatory GTP-binding proteins of mammalian cells,
short-circuiting their ability to regulate cellular processes.
Exposure to pertussis toxin can inhibit several important cells of the
immune system, including neutrophils, macrophages, monocytes, and
lymphocytes (10, 38). A second toxin, the adenylate cyclase
toxin, catalyzes the conversion of ATP to cyclic AMP in mammalian cells
to levels that far exceed what can be achieved by normal cellular
mechanisms. Chemotaxis, phagocytosis, superoxide generation, and
microbial killing are inhibited in neutrophils and monocytes exposed to adenylate cyclase toxin (9, 38). Adenylate cyclase toxin can
also induce apoptosis, or programmed cell death (18). In contrast to pertussis toxin, which is secreted, adenylate cyclase toxin appears to remain on the bacterial surface (23),
and it affects only human cells that come into contact with the bacteria.
B. pertussis also produces several adhesins. Several
antigenically distinct fimbriae are capable of mediating adhesion
(16, 28). The filamentous hemagglutinin (FHA) is a rod-like
structure of about 220,000 Da which mediates attachment using an RGD
(arginine, glycine, and aspartic acid) integrin receptor motif to bind
to mammalian cells and binds to carbohydrate and sulfate groups on lipids and proteins. A family of related outer membrane proteins possessing RGD motifs also promotes adhesion. Pertactin mediates attachment using its RGD motif (26). BrkA also mediates
adherence to cells, and in addition it can protect the bacteria from
the bactericidal activity of complement (11, 12, 43). Other related proteins include tracheal colonization factor and Vag8 (13, 14). Tracheal colonization factor promotes bacterial growth in the trachea, perhaps by acting as an adhesin (13). Currently, no function has been discovered for Vag8 (14). As a result of the redundancy of adhesins, with the exception of BrkA
(39), mutants deficient in the production of a single
adhesin are often as virulent as the wild-type strain in animal models of disease (17, 24, 39), and only mutants lacking more than one adhesin have reduced virulence.
Several studies suggest that pertussis vaccines confer better
protection from severe disease than from infection (1, 7, 21, 22,
31, 34). A study conducted by Storsaeter et al. (34)
showed that 25% of individuals vaccinated with the most efficacious
five-component vaccine (pertussis toxin, pertactin, FHA, and fimbriae 2 and 3) had a persistent cough for 21 days or more. Interestingly, in
this study (34) and another (7), protection
correlated with levels of circulating antibody to pertactin, fimbriae,
and to a lesser extent pertussis toxin but did not correlate with
levels of antibody to FHA. Since infected people with mild disease are
a potential source of infection for susceptible individuals, the ideal
vaccine would promote clearance of the organism and would prevent both
transmission and severe disease. We have begun to examine the role of
bactericidal mechanisms in immunity to pertussis.
Phagocytosis and the subsequent killing of the ingested microorganism
compose an immune mechanism that could clear the bacteria from infected
individuals. Early reports suggested that B. pertussis is
capable of survival and perhaps replication in professional phagocytes
(15, 33, 35), but subsequent reports suggest that its
intracellular survival is only transient (5, 8, 19, 20, 32).
Recently we have shown that only about 1% of B. pertussis
cells phagocytosed by neutrophils remain viable, suggesting that
phagocytosis could be an important immune defense against B. pertussis (26a). In this study we examined the role of
B. pertussis virulence factors and of the presence or
absence of opsonizing antibodies in phagocytosis by human neutrophils.
Phagocytosis assay.
Human neutrophils were purified, and
5 × 105 were allowed to adhere to round glass
coverslips in 24-well plates for phagocytosis assays as previously
described (37). Briefly, green fluorescent protein
(GFP)-expressing bacteria (3 × 106) were harvested
and incubated with human immune serum or buffer at 37°C for 15 min.
Bacterial suspensions were adjusted to 400 µl and were added to
adherent neutrophils for 1 h at 37°C in 5% CO2.
Where indicated below, bacteria were centrifuged (Marathon 8K; Fisher,
Pittsburgh, Pa.) onto the adherent neutrophils at 640 × g for 5 min at room temperature. Phagocytosis was allowed to occur
for 1 h at 37°C in 5% CO2. The cells were washed,
and adherent extracellular bacteria were stained by adding ethidium bromide (100 µg/ml) for 15 min at room temperature. In this
procedure, intracellular GFP-labeled bacteria resist staining with
ethidium bromide and remain green, but extracellular ethidium
bromide-labeled bacteria appear orange by fluorescence microscopy.
Phagocytosis was quantified by fluorescence microscopy.
Role of virulence factors in attachment.
The strains used in
this study are described in Table 1.
Plasmid CW504, which directs high-level expression of GFP from a constitutive B. pertussis promoter (26a), was
introduced into all of the strains by electroporation as described
previously (37). Fimbrial expression undergoes phase
variation, and individual cells within a population may or may not
express fimbriae (27, 28, 39, 42). We have observed temporal
variation in fimbrial expression in parental strain BP338, and even
different single colonies from the same culture can vary (39,
42). Agglutination using monoclonal antibody BPD5 (against
fimbria 2) or BPC10 (against fimbriae 3 and 6) was used to evaluate
fimbrial expression as previously described (39, 42). In
this assay, more than 10% of the cells in the population must express
fimbriae to yield a positive agglutination reaction (39).
None of the strains gave a positive agglutination reaction for fimbriae
3 and 6, and as observed previously, expression of fimbria 2 was highly
variable (Table 1).
The
bvg operon encodes a two-component transcriptional
activator that is required for expression of the
B. pertussis virulence
factors (
4), including pertussis
toxin, adenylate cyclase toxin,
dermonecrotic toxin, FHA, pertactin,
fimbriae, BrkA, and tracheal
colonization factor. An avirulent mutant
with an insertion in
the
bvg operon (BP347) and two mutants
deficient in FHA expression,
but capable of expression of other
bvg-regulated genes, were characterized.
BP353 (FhaA) and
BPM409 (FhaB) are well-characterized FHA mutants
with transposon
insertions in genes required for FHA expression.
The
fhaA
gene is required for secretion of FHA and is contained
in an operon
that also contains genes required for fimbrial secretion
(
27,
28). Polarity from the
fhaA insertion results in a
loss
of expression of both FHA and fimbriae in mutant BP353. The FhaB
mutant has an insertion in the
fhaB gene and lacks
expression
of FHA (
42), but a second promoter distal to the
fhaB gene (
27,
28) could allow this mutant to
express fimbriae. However, fimbrial
expression was not detected in the
parental strain or in any of
the FHA mutants (Table
1).
In previously published studies, bacterial suspensions were added to
adherent cells and were allowed to settle by gravity.
In initial
studies, we examined attachment and phagocytosis using
these
conditions. Approximately 100 wild-type bacteria, or 17%
of the total
bacterial inoculum (Fig.
1), attached per
100 neutrophils.
In contrast, Bvg and FHA mutants bound poorly, with
fewer than
5 bacteria attaching per 100 neutrophils, suggesting that
FHA
is important for attachment to neutrophils. Phagocytosis was also
measured. Only 5 wild-type bacteria per 100 neutrophils, or about
1%
(Fig.
1), were phagocytosed. No internalized bacteria were
observed for
the FhaA and Bvg mutants, and only 0.1 bacterium
per 100 neutrophils
was observed for the FhaB mutant. These numbers
seemed quite low, and
we speculated that these assay conditions,
where bacteria are added to
neutrophils and are allowed to settle
by gravity, do not promote
maximal contact with neutrophils. Further
investigation indicated that
B. pertussis does not settle significantly
within 1 h
(
26a), limiting its ability to interact with the
neutrophils.
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FIG. 1.
Effect of FHA on attachment. One hundred consecutive
neutrophils were examined for the number of intracellular (green [open
bars]) and adherent extracellular (orange [solid bars]) bacteria.
Data were analyzed by the Student t test. Each bar
represents the mean (± the standard error of the mean) from three to
nine independent experiments performed in duplicate. *, significantly
different from wild type (WT) (P < 0.05).
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To overcome this problem, we facilitated contact by gently centrifuging
the bacteria onto the neutrophils for 5 min at 640
×
g. Approximately 700 wild-type bacteria attached to 100 neutrophils,
which corresponds to 100% of the bacterial inoculum (Fig.
2, top
panel, WT-BP338). Attachment of
the strains lacking FHA also was
enhanced by centrifugation, but it
remained at a significantly
lower rate than that of the wild type.
Following centrifugation,
the rate of attachment of the FhaA mutant
increased from 2 to
83 bacteria (
P < 0.0001), the rate
of attachment of the FhaB mutant
increased from 1 to 61 bacteria
(
P < 0.0004), and the rate of
attachment of the
avirulent mutant increased from 0.5 to 6 bacteria
(
P < 0.003). In addition, the rates of attachment of the FhaA
(
P < 0.003) and the FhaB (
P < 0.002)
mutants were significantly
greater than that of the Bvg avirulent
mutant, suggesting that
Bvg-regulated adhesins, such as
fimbriae, pertactin, BrkA, and
tracheal colonization factor, may play a
minor role in attachment
to neutrophils. Since centrifugation
dramatically increased the
efficiency of bacterial attachment to
neutrophils, subsequent
experiments were performed with centrifugation
rather than with
the previously published procedures.
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FIG. 2.
Effects of virulence factors and antibody on attachment.
Bacteria were added to adherent neutrophils (no opsonization) or
incubated with human immune serum prior to being added to adherent
neutrophils (opsonization), centrifugation was performed to promote
contact, and phagocytosis was allowed to occur for 1 h. One
hundred consecutive neutrophils were examined for the number of
adherent extracellular (orange) bacteria. Data were analyzed by the
Student t test. Each bar represents the mean (± the
standard error of the mean) from 3 to 27 independent experiments
performed in duplicate. *, significantly different from parental wild
type (WT) (P < 0.05); #, significantly different from
no opsonization (P < 0.05).
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Attachment of mutants lacking the adhesins BrkA (BPM2041)
and pertactin (Prn) (BBC9) was examined (Fig.
2, top panel). The
rates
of attachment of these mutants were comparable to those
of their
respective parental strains, suggesting that the contribution
of these
adhesins is masked by the presence of FHA. Mutants expressing
altered
forms of the lipopolysaccharide (LPS) O chain would be
predicted to
have significant changes in their surface properties;
however, five
different LPS mutants attached as efficiently as
their parental strains
(Fig.
2, top
panel).
We also examined attachment of mutants lacking the adenylate cyclase
toxin (Cyc) (BPM3183), pertussis toxin (Ptx) (BPRA),
or dermonecrotic
toxin (Dnt) (BPM1809). None of these mutants
was statistically
different from the wild-type strain (Fig.
2,
top panel). These results
show that
B. pertussis efficiently attaches
to neutrophils
via bacterial adhesins, particularly FHA, but other
Bvg-regulated
adhesins may be
involved.
Role of antibody in attachment.
Neutrophils have Fc receptors
that allow them to bind the constant region of the immunoglobulin
molecules immobilized on a bacterial surface. To investigate the role
of opsonization by antibodies in the absence of complement, a serum
sample from an individual with occupational exposure to B. pertussis was heat inactivated at 56°C for 30 min. This serum,
no. 13 (43), has antibodies to B. pertussis LPS as well as to several surface-localized protein
virulence factors. In previous studies, opsonization decreased the rate
of attachment of the wild-type strain (26a, 37), and similar
results were observed in this study (Fig. 2, bottom panel). Opsonization caused a statistically significant decrease in the attachment rates of all three wild-type strains (BP338, BP536, and
BBC8), of mutants lacking FhaB, adenylate cyclase toxin, pertussis toxin, and dermonecrotic toxin, and of four different LPS mutants. Opsonization did not affect attachment of the FhaA, Bvg, LpsL, or
pertactin mutants.
When attachment of the opsonized mutants was compared to attachment of
the opsonized parental strain, only the FhaB mutant
was found to be
statistically different and displayed reduced
attachment rates (Fig.
2,
bottom
panel).
Phagocytosis of mutants without opsonization.
Centrifugation
caused a statistically significant increase (P < 0.002) in ingestion of the wild-type strain, from 5 bacteria per
100 neutrophils (Fig. 1) to 75 bacteria per 100 neutrophils (Fig.
3, top panel), which corresponds to 0.8 to 12.5% of the total bacteria added. Phagocytosis of the bacterial
mutants was also examined following centrifugation (Fig. 3, top panel).
While the mean level of phagocytosis varied, none of the mutants was statistically different from its respective wild-type strain.
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FIG. 3.
Effects of virulence factors and antibody on
phagocytosis. Nonopsonized or opsonized bacteria were incubated with
neutrophils as described in the legend for Fig. 2. One hundred
consecutive neutrophils were examined for the number of intracellular
(green) bacteria. Data were analyzed by the Student t test.
Each bar represents the mean (± the standard error of the mean) from 3 to 27 independent experiments performed in duplicate. *,
significantly different from wild type (WT) (P < 0.05).
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Phagocytosis of mutants with opsonization.
We previously
observed that opsonization caused a statistically significant decrease
in phagocytosis of the wild-type strain with and without centrifugation
(26a, 37), and similar results were observed in this study.
The number of wild-type BP338 bacteria that were phagocytosed decreased
from 75 (Fig. 3, top panel) to 30 (Fig. 3, bottom panel) following
opsonization (P < 0.04).
Phagocytosis of opsonized bacterial mutants was also examined.
Previously, we had shown that fluorescein isothiocyanate-labeled
bacteria expressed reduced levels of adenylate cyclase toxin and
were
efficiently phagocytosed when opsonized (
37). In this study,
phagocytosis of the opsonized adenylate cyclase toxin mutant increased
10-fold compared to that of the wild type (
P < 0.9e
7). Similarly,
phagocytosis of the Bvg mutant (which fails
to express adenylate
cyclase toxin in addition to other virulence
factors) was fourfold
greater than that of the wild-type strain
(
P < 0.02). In addition,
one LPS mutant was
phagocytosed more efficiently than the wild-type
strain (
P < 0.0005).
E. coli mutants with alterations in the core
region of LPS express reduced levels of functional hemolysin
(
6),
a toxin that is highly related to the adenylate cyclase
toxin
of
B. pertussis. LPS mutant MLT7 produces hemolysis on
blood-containing
plates, an activity due to functional adenylate
cyclase toxin.
Western blot analysis was performed as described
previously (
43),
using monoclonal antibodies to adenylate
cyclase toxin (3D1, 5D1,
6E1, and 2A12 [
25]) at a
1/1,000 dilution. BP338, the wild-type
strain, and MLT7, the LPS
mutant, expressed similar levels of
cell-associated antigenic adenylate
cyclase toxin (data not shown),
suggesting that the increased
phagocytosis of this mutant is due
to the LPS
defect.
Effect of adenylate cyclase toxin on phagocytosis.
To confirm
the role of adenylate cyclase toxin in blocking phagocytosis, the
GFP-labeled adenylate cyclase toxin mutant was added directly to
neutrophils or mixed with an equal number of unlabeled wild-type
bacteria. In the absence of opsonization, neither the GFP-labeled
wild-type strain, the GFP-labeled adenylate cyclase toxin mutant, nor
the GFP-labeled adenylate cyclase toxin mutant mixed with the unlabeled
wild-type strain was efficiently phagocytosed (Fig.
4). Opsonization significantly increased
the phagocytosis of the labeled adenylate cyclase toxin mutant but reduced phagocytosis of the wild-type strain as well as of the labeled
adenylate cyclase toxin mutant mixed with the unlabeled wild-type
strain (Fig. 4). These results suggest that the presence of adenylate
cyclase toxin blocks phagocytosis, even when bacteria are not
expressing the toxin.
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FIG. 4.
Effect of adenylate cyclase toxin on phagocytosis.
Phagocytosis studies were performed as described in the legend for Fig.
3, with nonopsonized (open bars) and opsonized (solid bars) bacteria.
One hundred consecutive neutrophils were examined for the number of
intracellular (green) bacteria. WT, wild-type GFP-labeled BP338; Cyc,
GFP-labeled BPM3183; Both, unlabeled BP338 plus GFP-labeled BPM3183.
Data were analyzed by the Student t test. Each bar
represents the mean (± the standard error of the mean) from three
independent experiments. #, significantly different from the Cyc strain
(P < 0.05); *, significantly different from WT
(P < 0.05).
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In this study we examined the influence of
B. pertussis
virulence factors and opsonizing antibodies on phagocytosis by human
neutrophils, an immune response that could play a role in eliminating
the bacteria from the infected individual. FHA is an important
adhesin
for
B. pertussis (
27). Mutants lacking FHA failed
to
attach to neutrophils, demonstrating a major role for this adhesin
in promoting attachment to neutrophils. The lack of efficient
attachment of the FHA mutants suggests that pertactin, BrkA, or
other
putative adhesins, such as tracheal colonization factor,
could not
compensate for FHA. The role of fimbriae in mediating
attachment to
neutrophils is uncertain, since neither the FHA
mutants nor the
parental strain expressed this adhesin. The contribution
of fimbriae to
adherence would best be examined by constructing
mutants that
constitutively express this determinant in an FHA-deficient
background.
While FHA may confer an overall benefit to the pathogen
during disease,
bacteria expressing FHA stick avidly to neutrophils,
and this close
association can promote phagocytosis. We have shown
that opsonization
can reduce the incidence of both attachment
and phagocytosis of
wild-type strains expressing
FHA.
Adenylate cyclase toxin appears to be an important bacterial defense
against phagocytosis, as evidenced by the efficient phagocytosis
of
mutants that fail to express this toxin. However, only opsonized
bacteria were efficiently phagocytosed, suggesting that a signal,
such
as Fc receptor activation, is necessary for neutrophils to
recognize
B. pertussis as foreign and initiate
phagocytosis.
These studies have important implications for vaccine development.
While the current vaccines protect against severe diseases,
they afford
little protection against colonization by the organism
(
1,
7,
21,
22,
31,
34). Developing a vaccine that
prevents bacterial
infection as well as serious disease is an
important goal. In a
previous study, we have shown that
B. pertussis is killed
following phagocytosis by neutrophils (
26a). Identifying
the
antigens that promote phagocytosis and killing by neutrophils
could
improve the pertussis vaccine. While antibodies to FHA may
confer
protection by other means, our studies suggest that antibodies
to FHA
could antagonize phagocytosis by neutrophils. The role
of FHA as a
protective immunogen warrants further investigation.
In contrast,
adenylate cyclase toxin is not included in any of
the acellular vaccine
formulations, and our studies suggest that
neutralizing antibodies to
this toxin could be beneficial in preventing
infection by
B. pertussis. Our studies also suggest that opsonization
is needed
for efficient phagocytosis, and the identity of opsonizing
antibodies
remains to be
determined.
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ACKNOWLEDGMENTS |
This work was supported in part by grant RO1 AI38415 to A.A.W.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Biochemistry, and Microbiology, University of
Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267-0524. Phone: (513)
558-2820. Fax: (513) 558-8474. E-mail:
alison.weiss{at}uc.edu.
Editor:
D. L. Burns
| |
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Infection and Immunity, March 2000, p. 1735-1739, Vol. 68, No. 3
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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