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Infection and Immunity, March 2001, p. 1895-1901, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1895-1901.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Alginate O Acetylation in Resistance of
Mucoid Pseudomonas aeruginosa to Opsonic
Phagocytosis
Gerald B.
Pier,1,*
Fadie
Coleman,1
Martha
Grout,1
Michael
Franklin,2 and
Dennis E.
Ohman3,4
Channing Laboratory, Department of Medicine,
Brigham and Women's Hospital, Harvard Medical School, Boston,
Massachusetts 02115-58041; Department of
Microbiology, Montana State University, Bozeman, Montana
597172; Department of Microbiology and
Immunology, Medical College of Virginia Campus of Virginia Commonwealth
University, Richmond, Virginia 23298-06783; and
McGuire Veterans Affairs Medical Center, Richmond, Virginia
232494
Received 31 July 2000/Returned for modification 2 October
2000/Accepted 22 November 2000
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ABSTRACT |
Establishment and maintenance of chronic lung infections with
mucoid Pseudomonas aeruginosa in patients with cystic
fibrosis (CF) require that the bacteria avoid host defenses.
Elaboration of the extracellular, O-acetylated mucoid
exopolysaccharide, or alginate, is a major microbial factor in
resistance to immune effectors. Here we show that O acetylation of
alginate maximizes the resistance of mucoid P.
aeruginosa to antibody-independent opsonic killing and is the
molecular basis for the resistance of mucoid P.
aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients.
O acetylation of alginate appears to be critical for P.
aeruginosa resistance to host immune effectors in CF patients.
| |
TEXT |
The predominant bacterial pathogen
in chronic pulmonary infection in cystic fibrosis (CF) patients is the
mucoid variant of Pseudomonas aeruginosa, which is
encapsulated by and overproduces mucoid exopolysaccharide (MEP), or
alginate. That alginate is the major virulence factor of P. aeruginosa in CF lung infection is evident from the epidemiology
of this disease. The pulmonary function of patients with CF declines
only when mucoid P. aeruginosa is isolated and associated
lung pathology develops (9, 32, 33). The growth of mucoid
P. aeruginosa as a biofilm in the lungs of CF patients has
been suggested to be a major factor in long-term bacterium survival.
Biofilm formation by P. aeruginosa has been linked to genes
involved in quorum sensing (7) and motility
(31), with a recent demonstration that the acyl-homoserine lactone molecules involved in the quorum-sensing system
(8) can be detected in the sputa of CF patients
(42). However, the genes controlling alginate production
appear to be independent of control by the known quorum-sensing genes
of P. aeruginosa, including lasR and
rhlR (8, 44, 45). Therefore, the question of
whether there is a regulator or environmental cue common to both
alginate production and quorum-sensing systems has not yet been answered.
The conversion of P. aeruginosa to the mucoid state in CF
patients is often associated with mutations at the mucA
locus (23). MucA and MucB (also called AlgN) act as
anti-sigma factors for the alternative sigma factor 
E
(47), encoded by algT (25), also
known as algU (22). Increased activity of this
sigma factor results in hyperexpression of the alginate biosynthetic
operon located at 34 min on the P. aeruginosa genome
(25). Conversion of P. aeruginosa to the mucoid
state is often associated with the loss of production of the
lipopolysaccharide (LPS) O side chains that normally render
strains serum resistant (18, 30, 34).
MEP/alginate is a high-molecular-weight polysaccharide of
1-4-linked residues of mannuronic and guluronic acids
(40, 41). The ratio of mannuronic acid to guluronic acid
varies from strain to strain, on the order of 10:1 to 1:1 (40,
41). Acetylation occurs on the C-2 and C-3 hydroxyl
groups of the mannuronic acid residues. The products of
algI, algJ, and algF, located on the alginate biosynthetic operon, are required for the O acetylation of
alginate (14, 15). Much research has been published on the
biosynthesis of alginate (5, 6, 26, 46, 48) as well as on
the control of synthesis by both genetic (2-4, 11, 13,
29) and environmental (10-12, 24, 25) factors.
Despite this wealth of information, the exact molecular mechanisms by which alginate promotes the survival of bacteria in the lungs of
otherwise immunocompetent hosts for years to decades have not been
fully elucidated.
Defining the molecular properties of alginate that mediate the
resistance of mucoid P. aeruginosa to host immune effectors is key to understanding the role of this material in pathogenesis. A
property of MEP/alginate previously reported to be involved in the
inability of CF patients to clear mucoid P. aeruginosa from
their lungs is its elicitation during chronic infection of specific
antibodies that fail to mediate the opsonic killing of mucoid P. aeruginosa growing either in suspension (32, 38) or
in biofilms (27). Another characteristic of MEP/alginate that may confer bacterial resistance to host phagocytes and complement, particularly in the presence of the loss of production of the LPS O
side chains that normally render strains serum resistant (18, 30, 34), is the presence of acetate substituents.
Acetate residues are bound via ester linkages to hydroxyl groups that, when unsubstituted, can serve as acceptors for covalent linkage of the
complement opsonins C3b and C4b to the bacterial surface (19). In addition, the presence of acetate residues may
affect the activation of complement in an antibody-independent fashion. Thus, by linking acetate to hydroxyl groups, mucoid P. aeruginosa may be able to escape phagocytic killing by dampening
the activation of complement. We therefore evaluated the susceptibility
of P. aeruginosa FRD1153 (14, 15), an
algJ mutant derived from mucoid P. aeruginosa
strain FRD1, to opsonic killing by antibody-free human complement and
by human complement with added MEP-specific opsonic and
nonopsonic antibodies. These studies were performed to define further
the role of acetate substituents in the long-term persistence of mucoid
P. aeruginosa in the lungs of CF patients.
Comparative susceptibilities of strains to opsonic killing mediated
by complement and leukocytes only.
We initially assessed whether
two components of the innate immune system
phagocytes and
complementcould mediate the opsonic killing of parental,
O-acetylation-deficient, and trans-complemented mucoid
P. aeruginosa strains in the absence of antibody by using a
well-established opsonophagocytic assay (1). The strains used were mucoid P. aeruginosa FRD1, a clinical isolate that
has been extensively studied (2, 4, 5, 17, 29); mucoid P. aeruginosa FRD1153, which contains a point mutation
generated in algJ as described previously (14,
15) and which produces only 7% of the parental level of O
acetylation on alginate; strain FRD1153 complemented with plasmid pMF52
(15), which contains the algI, algJ,
and algF genes under the control of the Ptrc
promoter and which provides full restoration of parental levels of
alginate acetylation in strain FRD1153; and strain FRD1153 complemented with plasmid pMF54, the vector control (15).
Strains with plasmid pMF52 or pMF54 were routinely cultured in
Trypticase soy broth or on Trypticase soy agar plates containing 300 µg of carbenicillin/ml. Human serum was used as a source of complement; the serum was diluted 1:10 in RPMI medium-15% fetal calf
serum and adsorbed twice with 1 to 2 mg of lyophilized P. aeruginosa strain FRD1 cells at 4°C for 30 min to remove
specific antibody. Bacterial cells were removed by centrifugation, and the serum was filter sterilized and then diluted further for studies involving different concentrations of complement.
As shown in Fig.
1, 80 to 100% of cells
of the parental strain, FRD1, and the fully complemented strain,
FRD1153(pMF52), survived
when up to 100 µl of a 10% concentration of
adsorbed normal human
serum was added to an opsonic killing
assay with a final volume
of 400 µl. Higher concentrations of human
serum could not be used
because mucoid strains from CF patients produce
rough LPS (
18),
rendering the organisms sensitive to
killing by complement alone
at higher serum concentrations
(
35). In contrast, a maximum
of 37% of cells of
acetate-deficient strains FRD1153 and FRD1153(pMF54)
survived when 100 µl of a 1.25% concentration of adsorbed normal
human serum was added
to the assay (Fig.
1). The survival of FRD1153
was reduced at higher
concentrations of serum, with less than
10% survival at a 10% serum
concentration.
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FIG. 1.
Susceptibility of variously acetylated mucoid P.
aeruginosa strains to opsonic killing by human peripheral blood
leukocytes (PBL) and complement at the indicated concentrations. Strain
FRD1153 is an algJ mutant strain, unable to O acetylate
alginate. Strain FRD1 is the parental strain of FRD1153. Plasmid pMF52
contains the algI, algJ, and
algF genes under the control of the Ptrc
promoter and restores the O-acetylation phenotype to FRD1153. Plasmid
pMF54 is the cloning vector. Bars represent the mean CFU surviving, and
error bars indicate the standard deviation. Asterisks indicate that at
all complement concentrations tested, the percentage of bacteria
surviving was significantly lower in both of the
O-acetyl-deficient strains than in the
O-acetyl-sufficient strains (P < 0.001, as determined by ANOVA and Fisher's PLSD test for pairwise
comparisons).
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|
Complementation in
trans of FRD1153 with
algJ
from plasmid pMF52 restored the parental level of resistance to
complement-mediated
opsonic killing, whereas FRD1153 with the vector
control, pMF54,
was susceptible to complement-mediated opsonic killing.
At all
concentrations of human complement tested, the rate of survival
of the strains producing acetylated MEP/alginate was significantly
greater than that of strains with deficient levels of acetylation
of
MEP/alginate (
P < 0.001, as determined by analysis of
variance
[ANOVA] and Fisher's probable least-significant-difference
[PLSD]
test). The data shown in Fig.
1 were reproduced four
additional
times with different adsorbed normal human sera as sources
of
complement (data not shown), with essentially identical
results.
The above results indicate that acetylation of MEP/alginate is likely
essential to the development of resistance of mucoid
P. aeruginosa to basic host defenses. The very low level of
complement
that effectively opsonized the acetylation-deficient
derivatives
would make such strains prone to elimination by host
defenses
in the lungs. However, it is difficult to know what the actual
level of complement activity is in CF lungs. Since most mucoid
P. aeruginosa cells isolated from the lungs of CF patients produce
rough LPS (
35) and are susceptible to bactericidal killing
at
serum concentrations of
10%, it can be assumed that the levels
of
complement needed in chronically infected CF lungs to form
the membrane
attack complex capable of killing
P. aeruginosa cells
are
<10% the levels in serum. It is not known whether the observed
phagocytic killing of strains deficient in acetylation of alginate
in
vitro with complement concentrations as low as 1.25% is indicative
of
an inability of such strains to survive in CF lungs. Nonetheless,
the
ability of very small amounts of human serum to mediate opsonic
killing
of nonacetylated mucoid
P. aeruginosa suggests that
acetylation
of MEP may be critical for bacterial resistance to host
defenses
during chronic lung infection in
CF.
Effect of acetate substituents on antibody-independent complement
activation.
To determine the effect of acetate substituents on
complement activation at serum concentrations lower than 10% that
mediate phagocyte-dependent opsonic killing of the nonacetylated
mutant, we compared the consumption of the activity of the alternative pathway of complement by the P. aeruginosa
acetylase-deficient strains and by strains with wild-type levels of
acetate. This goal was accomplished by dilution of human sera 1:10 in
Veronal-buffered saline, adsorption as described above with lyophilized
cells of P. aeruginosa strain FRD1 to remove specific
antibody, and incubation with 107 CFU of the
various strains for 30 min at 37°C. Bacteria were removed by
centrifugation, and 108 rabbit red blood cells
were added to the residual sera. After 30 min at 37°C, the samples
were centrifuged to remove intact red blood cells, 100 µl of the
supernatant was added to 96-well plates, and the amount of hemoglobin
released into the supernatant was measured at 405 nm. Controls included
serum samples treated with zymosan to consume all of the alternative
pathway components and samples with no bacteria, whose
hemoglobin release value represented 100% of the complement activity.
The percentage of residual activity of the alternative pathway of
complement left after incubation with each strain was calculated as
follows: 100 × (optical density at 405 nm of test sample/optical
density at 405 nm of sample showing 100% lysis of red blood
cells). At a concentration of adsorbed, intact human serum of
6.25%, 76% of the alternative pathway activity remained after
incubation with O-acetylated mucoid P. aeruginosa strain
FRD1 (Fig. 2). In contrast, the poorly
O-acetylated strain, FRD1153, consumed essentially all of the
alternative pathway activity at this serum concentration; 96% of the
alternative pathway activity remained when serum was incubated with
FRD1153 containing algJ in trans, but 100% of
the activity was consumed by incubation with strain FRD1153 containing
only the vector control. These results are indicative of a role for the
acetate substituents in abrogating the activation of the alternative
pathway, an event that could lead to deposition of opsonic fragments of
C3 and C4 and phagocytic killing. Therefore, at the molecular level, it appears that acetylation of alginate, particularly when it is expressed
on a rough-LPS P. aeruginosa strain, dampens complement activation, leading to resistance to antibody-independent phagocytic killing.
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FIG. 2.
Acetate residues on MEP/alginate inhibit activation of
the alternative pathway of complement. Exposure of 6.25% adsorbed
normal human serum to 107 CFU of each mucoid P.
aeruginosa strain for 30 min followed by the removal of the
bacteria and the evaluation of the residual complement-activating
capacity of the serum showed that the fully O-acetylated strains, FRD1
and FRD1153(pMF52), activated <25% of the available complement,
whereas the poorly O-acetylated strains, FRD1153 and FRD1153(pMF54),
activated >98% of the available complement. Values of residual
activity below 0% are due to experimental variation. Data are reported
as mean and standard deviation. Asterisks indicate a significant
difference between the value shown and that obtained with 100%
residual activity (i.e., 100% red blood cell lysis;
P <0.0001, as determined by ANOVA and Fisher's PLSD
test).
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Effect of acetate substituents on the functional activity of
MEP-specific opsonic and nonopsonic antibodies.
Another factor
involved in the pathogenesis of chronic mucoid P. aeruginosa
infection in CF lungs is the lack of elicitation of an immune response
that is effective at controlling infection. In other studies, we
attributed this situation, in part, to the production of MEP-specific
antibodies incapable of mediating opsonic killing of either suspended
or biofilm-grown P. aeruginosa (27, 32, 38).
Immunogenicity studies using MEP and mice have indicated that in the
presence of preexisting nonopsonic antibodies to MEP, opsonic
antibodies cannot be readily elicited, even with doses of MEP that do
elicit opsonic antibodies in naive mice (16). Nonopsonic
antibodies to MEP occur naturally in all human sera examined to date
and are present in the sera of young CF patients prior to colonization
with P. aeruginosa (38). These nonopsonic MEP-specific antibodies mediate high levels of complement activation in
the presence of mucoid P. aeruginosa (37), but
opsonic complement fragments derived following activation fail to bind
efficiently to mucoid P. aeruginosa cells (37).
In contrast, opsonic MEP-specific antibodies of the same immunoglobulin
isotypes both activate complement and deposit opsonically active C3b
and C3bi fragments onto the bacterial surface (37).
To determine whether the acetate substituents on MEP/alginate form the
molecular basis for the lack of opsonic killing by
nonopsonic
antibodies, we carried out phagocytic assays using
complement along
with the following: (i) normal human serum containing
naturally
occurring antibodies to MEP that fail to mediate opsonic
killing
(
36,
38), (ii) immunization-induced nonopsonic mouse
antibodies obtained from mice immunized three times at 5-day intervals
with purified MEP at a high dose (50 µg/dose), or (iii) an
MEP-specific
murine immunoglobulin G2a (IgG2a) monoclonal antibody that
does
not mediate opsonic killing of mucoid
P. aeruginosa
(
37,
43).
To determine if MEP-specific opsonic
antibodies recognized acetylated
epitopes, we used the following: (i)
human sera with MEP-specific
opsonic antibodies obtained from
individuals vaccinated with purified
MEP (
36) (which
already contained preexisting nonopsonic antibodies);
(ii) sera from
mice immunized three times at 5-day intervals with
purified MEP at a
low dose (10 µg), which elicits both opsonic
and nonopsonic
antibodies (
16); or (iii) an opsonic IgG2a monoclonal
antibody (
43). To avoid phagocytic killing of the poorly
acetylated
strains in an antibody-independent manner, we used human
serum
at a concentration of 0.625%, which does not on its own opsonize
nonacetylated mucoid
P. aeruginosa strains for phagocytic
killing.
For the fully acetylated strains, we used human serum at a
concentration
of 10% as a source of complement. As shown in Fig.
3, even at
a very low complement
concentration, the usually nonopsonic antibodies
readily mediated
phagocytic killing of the poorly O-acetylated
strains, FRD1153
and FRD1153(pMF54). In contrast, at a complement
concentration of
10%, the fully O-acetylated mucoid
P. aeruginosa strains,
FRD1 and FRD1153(pMF42), were resistant to phagocytic
killing by the
nonopsonic antibodies. Data are shown for sera
pooled from five
immunized mice and for one normal human serum
sample. The assays were
repeated with four other normal human
serum samples, with essentially
identical results (data not shown).
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FIG. 3.
Opsonic killing of nonacetylated or acetylated mucoid
P. aeruginosa strains in the presence of a nonopsonic
monoclonal antibody (MAb), nonopsonic antibody to MEP in a normal human
serum sample, and nonopsonic antibody in pooled sera from mice
immunized with a high dose (50 µg) of MEP. Bars represent mean CFU
killed, and error bars show the standard deviation. The percentage of
the two nonacetylated strains killed by each of the three antibodies
was significantly higher than the percentage of the acetylated strains
killed by the corresponding antibodies (P < 0.001, as determined by ANOVA and Fisher's PLSD test for pairwise
comparisons). C', complement.
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Effect of O-acetyl substituents on the functional
activity of MEP-specific opsonic antibodies.
Sera obtained from
mice or humans vaccinated with MEP and developing specific opsonic
antibodies killed both poorly and fully acetylated mucoid P. aeruginosa strains in the phagocytic assay (Fig.
4). Again, data are shown for sera pooled
from five immunized mice and for one immunized human serum sample, but
the assays were repeated with four other immunized human serum samples,
with essentially identical results (data not shown). The poorly
acetylated strains were phagocytosed due to the concomitant presence of
MEP-specific nonopsonic antibodies in the sera from the vaccinated mice
and humans. However, when a murine IgG2a monoclonal antibody with opsonic killing activity was used, only the fully acetylated strains were opsonized for phagocytic killing (Fig. 4), a result indicating that acetate substituents form the epitope recognized by opsonic antibodies in vaccinated mouse and human sera and by the murine opsonic
monoclonal antibody.
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FIG. 4.
Opsonic killing of nonacetylated or acetylated mucoid
P. aeruginosa strains in the presence of opsonic
monoclonal antibody (MAb 9/5/23) to MEP, opsonic antibody in serum from
a person immunized with MEP, or opsonic antibody in sera pooled from
mice immunized with a low dose (10 µg) of MEP. Bars represent the
mean CFU killed, and error bars show the standard deviation. The
percentage of the two nonacetylated strains killed by the opsonic MAb
was significantly lower than the percentage killed by the other
antibody preparations (P < 0.01, as determined by
ANOVA and Fisher's PLSD test for pairwise comparisons). The immune
mouse and human sera killed the nonacetylated strains because of the
concomitant presence of nonopsonic antibody induced by immunization in
mice (a 10-µg dose of MEP elicits both opsonic and nonopsonic
antibodies) or occurring naturally in humans.
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Overall, our results indicate one potential molecular basis for the
pathogenesis of chronic mucoid
P. aeruginosa infection
in CF
lungs. O acetylation of MEP/alginate prevents activation
of the
alternative pathway of complement; the result is resistance
to
antibody-independent phagocytosis. In addition, acetylation
of
MEP/alginate precludes phagocytic killing by the nonopsonic
antibodies
to MEP found both in normal human sera and at high
titers in the sera
of infected CF patients (
38). Acetate residues
appear to
be key factors in the epitope that is recognized by
protective human
and murine opsonic antibodies (
39), and complement
activation by these antibodies leads to phagocytic killing via
deposition of C3b and C3bi (
37). Phagocytic killing of
mucoid
P. aeruginosa correlates with the resistance of
older, uninfected
CF patients to infection with mucoid
P. aeruginosa (
32,
38)
as well as with protective
efficacy in rodent models of endobronchial
infection with mucoid
P. aeruginosa (
39). In addition, acetate
residues on MEP/alginate have been shown to be able to scavenge
hypochlorite produced by activated phagocytes, potentially protecting
mucoid
P. aeruginosa from this host defense
(
20), as well as
to suppress neutrophil and lymphocyte
antibacterial functions
and responses (
21). Thus,
O-acetyl substituents are likely one
of the important
components of MEP/alginate protecting mucoid
P. aeruginosa
from host
defenses.
| |
ACKNOWLEDGMENTS |
We thank Brian Hyett, Denise DesJardins, and Elizabeth Kieff for
contributions to this work.
Support was obtained from NIH grants AI 22836 (to G.B.P.), AI46588 (to
M.F.), and AI 19146 (to D.E.O.) and from Veterans Administration Medical Research Funds (to D.E.O.).
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FOOTNOTES |
*
Corresponding author. Mailing address: Channing
Laboratory, 181 Longwood Ave., Boston, MA 02115-5804. Phone: (617) 525 2269. Fax: (617) 731-1541. E-mail:
gpier{at}channing.harvard.edu.
Editor:
V. J. DiRita
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Infection and Immunity, March 2001, p. 1895-1901, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1895-1901.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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