Download Contribution of Choline

Contribution of Choline-Binding Proteins to
Cell Surface Properties of Streptococcus
pneumoniae
Edwin Swiatlo, Franklin R. Champlin, Steven C. Holman, W.
William Wilson and James M. Watt
Infect. Immun. 2002, 70(1):412. DOI:
10.1128/IAI.70.1.412-415.2002.
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INFECTION AND IMMUNITY, Jan. 2002, p. 412–415
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.1.412–415.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 1
Contribution of Choline-Binding Proteins to Cell Surface
Properties of Streptococcus pneumoniae
Edwin Swiatlo,1* Franklin R. Champlin,2 Steven C. Holman,3 W. William Wilson,3
and James M. Watt2
Department of Medicine, University of Mississippi Medical Center and VA Medical Center, Jackson, Mississippi 39216,1
and Departments of Biological Sciences2 and Chemistry,3 Mississippi State University, Starkville, Mississippi 39762
Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs)
which have been associated with adhesion and virulence. The purpose of this study was to determine the
contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may
affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting
bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to
their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic
hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis.
Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with
a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule.
Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs
possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocytelike cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that
CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and
may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.
Pneumococci express a family of choline-binding proteins
(CBPs) which are surface exposed and possibly function as
specific adhesins for glycoconjugates on eukaryotic host cells
(9, 17). Some members of this family specifically bind the
secretory component of immunoglobulin A (10) and complement components (20, 22). Much information about the interaction of CBPs with host proteins or receptors has recently
become available, but little is known about how this family of
surface proteins contributes to the physical properties of the
pneumococcal cell surface. This study was undertaken to examine the contribution of CBPs to the overall hydrophobicity
and surface charge of pneumococci. The ability of pneumococci, with or without CBPs, to adhere to human monocytelike cells was also studied. The results suggest that CBPs contribute significantly to the chemistry of the subcapsular
compartment of pneumococci and enhance interaction with
eukaryotic cells. A possible additional mechanism for the protective effect of capsule polysaccharide is implied.
Bacterial strains and media. Pneumococcal strains used in
this study have been previously described. Pneumococcal strain
D39 is a capsule type 2 clinical isolate (2), and strain Rx1 is a
functionally unencapsulated derivative of D39 (19). WU2 is a
clinical type 3 isolate (5), and JD908 is an isogenic mutant
which produces no detectable extracellular capsule (7). Strain
A66 is a capsule type 3 clinical isolate (2), and A66R2 is an
unencapsulated mutant (14). All strains were grown at 37°C in
Todd-Hewitt broth with 0.5% yeast extract (THY) and frozen
at ⫺80°C after supplementation with glycerol to a 20% final
concentration. Unadapted pneumococci were grown in chemically defined medium (CDM; JRH Biosciences, Lenexa,
Pathogenic bacteria interact with eukaryotic cells by a variety of mechanisms, most notably the specific binding of bacterial adhesins to ligands on the host cell surface. In addition to
these specific interactions, the physicochemical properties of
the bacterial cell surface can significantly influence the outcome of bacterium-host cell contact. Net cell surface charge
and hydrophobicity are two properties of bacterial surfaces
which are known to be important in this interaction (3, 13).
The polysaccharide capsules of pathogens such as Streptococcus pneumoniae (pneumococcus) are, generally, negatively
charged polymers which are thought to protect the bacterial
cell from humoral immune mechanisms (11). The net negative
charge of capsule polysaccharide may also function to electrostatically repel pneumococci from phagocytic cells, which typically have a net negative charge of ⫺18 to ⫺12 mV at their
surface (13, 24). This has been shown for encapsulated group
A streptococci (25). In addition to electrostatic interactions,
cells may associate by nonspecific hydrophobic interactions.
Using adherence to hexadecane droplets, it has been shown
that surface proteins of group A streptococci confer a hydrophobic character to the bacterial surface in the absence of
capsule (15). Bacterial surfaces with a high degree of hydrophobicity may adhere more efficiently to phagocytes (12), host
tissue cells (8), and biomedical materials such as polyethylene
(23).
* Corresponding author. Mailing address: VA Medical Center, Research and Education (151), 1500 Woodrow Wilson Dr., Jackson, MS
39216. Phone: (601) 984-5560. Fax: (601) 984-5565. E-mail: swed
@sprintmail.com.
412
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Received 3 August 2001/Returned for modification 13 September 2001/Accepted 25 September 2001
VOL. 70, 2002
413
FIG. 1. GelCode Blue (Pierce, Rockford, Ill.) stain of SDS-polyacrylamide gel electrophoresis of concentrated culture supernatants
from mid-log-phase Rx1 grown in CDM with either choline or ethanolamine. Growth in CDM-EA results in most of the CBPs free in the
extracellular medium. Lanes: 1 and 5, molecular size markers (in
kilodaltons); 2, CDM-C with no bacterial inoculum, 3, growth in
CDM-EA; 4, growth in CDM-C.
Cells which were adapted to grow in EA were washed in a 2%
choline solution, and the eluate was concentrated 30-fold and
was analyzed by SDS-polyacrylamide gel electrophoresis with
silver staining. This method has been used as an alternative for
eluting CBPs off the pneumococcal surface (28). After adaptation to EA, no stainable proteins could be detected by a
choline wash (data not shown).
Removal of CBPs results in a significant alteration in the
hydrophobic character of the pneumococcal cell surface, which
is seen primarily when capsule has been removed (Fig. 2). For
strains D39 and A66, removal of CBPs had little effect on the
hydrophobicity of the cell surface. Either with or without
CBPs, D39 and A66 have very hydrophilic surfaces as observed
by their weak adherence to a nonpolar hydrocarbon. In contrast, removal of CBPs from WU2 results in a cell surface
which is significantly more hydrophobic, suggesting a contribution of CBPs to the highly hydrated surface of this type 3
strain. Unencapsulated mutants of pneumococci readily demonstrate the contribution of CBPs to cell surface hydrophobicity. For each unencapsulated strain, tested removal of CBPs
resulted in greater adherence to hydrocarbon; thus, a more
hydrophobic cell surface. The differences between cells with
and without CBPs were significant for Rx1 and JD908 (P ⬍
0.01) but not quite so for A66R2 (P ⫽ 0.064).
Cell surface charge for selected strains was examined by
measuring zeta potential. The electronegative surface of D39
was mostly unchanged by removing CBPs (Table 1). As was the
case in the hydrocarbon adherence assay, removing CBPs in
the face of an intact capsule had little effect on the cell surface
charge. The exception was WU2, which had significantly more
electronegative charge when CBPs were removed. When capsule is removed, the effect of CBPs on cell surface charge is
marked. The overall surface charge became more electronegative in the absence of CBPs, as seen by a decrease in surface
voltage of more than 10 mV for Rx1 (Table 1).
The effect of the full complement of CBPs on adhesion to a
human monocyte-like cell line was measured by flow cytometry
Downloaded from http://iai.asm.org/ on February 27, 2014 by PENN STATE UNIV
Kans.) which was supplemented with choline chloride to a final
concentration of 0.02% (CDM-C). Adaptation to ethanolamine (CDM-EA) was done by serially transferring aliquots of
pneumococcal cultures growing in CDM in which the choline
concentration was decreased in a stepwise manner to a final
concentration of ⬍0.0001%, and the ethanolamine concentration was concurrently increased to a final concentration of
0.5% (4). Culture supernatants of pneumococci growing in
CDM-C or CDM-EA were passed through a 0.45-␮m-poresize, low-protein-binding filter, concentrated 30-fold, and analyzed by electrophoresis in 10% polyacrylamide gels after boiling in sodium dodecyl sulfate (SDS) (1).
Hydrocarbon adhesion assay. Cell surface hydrophobicity
was measured by bacterial adherence to hexadecane with slight
modifications to a previously described procedure (16). Bacteria were grown in 5 ml of THY to approximately mid-log
phase, collected by centrifugation, and washed twice in phosphate-buffered saline (PBS, pH 7.0). Washed cells were resuspended in 1 ml of PBS, and optical density at 620 nm (OD620)
was measured. This measurement served as the control (C0).
Subsequently, 200 ␮l of hexadecane was added and the mixture
was vortexed for 1 min. After the phases were allowed to
separate, the OD620 of the lower aqueous phase was measured
(CH). The percent hydrocarbon adherence was determined by
the following formula: [(C0 ⫺ CH)/C0] ⫻ 100.
Cell surface charge measurement. Net bacterial cell surface
charge was measured as the electrical potential of the interface
between the bacterial surface and the aqueous environment
(zeta potential) (26). The zeta potential was estimated with an
automated Zetasizer apparatus (Malvern Instruments, Southborough, Mass.). Exponentially growing pneumococcal cultures
in THY were washed twice in PBS (pH 7.0) and resuspended
in PBS to an OD620 of 1. Samples were run at 25°C and a field
strength of 19.2 V/cm. Other parameters of the Zetasizer were
adjusted according to the manufacturer’s specifications.
Cell adhesion. Unadapted and EA-adapted pneumococcal
cells were washed twice in PBS (pH 7.0) and incubated at 37°C
in 10 ␮M 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes, Inc., Eugene, Oreg.) for 60 min in PBS. Cells
were then washed twice in dye-free PBS. Fluorescein-labeled
pneumococci were incubated with U937 cells (21) at a ratio of
103:1 (bacteria to cells) for 5 min at 37°C in RPMI medium
supplemented with 10% fetal calf serum. After incubation the
U937 cells were collected by centrifugation and were then
washed and resuspended in Hanks balanced salt solution for
analysis by flow cytometry (FACScan; Becton Dickinson, San
Jose, Calif.). Differences between means were determined by
two-sample t tests. A P value of ⬍0.05 was considered significant.
Growth of pneumococci in a defined medium which contains
ethanolamine (CDM-EA) as the sole amino alcohol effectively
removes most surface-exposed CBPs. These proteins can be
detected in concentrated culture supernatants (Fig. 1) from
exponentially growing pneumococcal cultures. Bacteria grown
in CDM-C release no detectable protein in concentrated culture supernatants, although the limit of sensitivity of the protein stain (10 ng) does not entirely preclude this possibility.
Culture medium which contains EA still has an exceedingly
small concentration of choline (⬍0.0001%) which may be sufficient for some CBPs to remain attached to the cell surface.
NOTES
414
NOTES
INFECT. IMMUN.
TABLE 2. Mean fluorescence intensity of U937 cells incubated
with CMFDA-labeled pneumococci grown in either
CDM-C or CDM-EAa
Strain
Peak channel
2,018.0
2,310.3
30.8
2,017
2,307
29
Rx1
Rx1(A)
U937
888.6
14.9
27.2
1,176
11
28
WU2
WU2(A)
U937
236.3
84.9
33.6
294
79
32
JD908
JD908(A)
U937
2,280.8
479.9
19.0
2,227
478
19
D39
Rx1
U937
480.6
1,267.1
24.8
429
1,186
24
a1
of fluorescently labeled pneumococci incubated with U937
cells for 5 min. This short incubation was necessary to minimize the number of CBPs which would be surface expressed
when pneumococci were reintroduced into a choline-containing medium. As seen in Table 2, for most strains grown in
CDM-EA the mean fluorescence intensity was significantly less
than for the corresponding strain grown with choline. D39
attachment to human cells was unchanged whether bacteria
were grown in CDM-C or -EA. When D39 was compared
TABLE 1. Net cell surface charge as represented by zeta potentiala
Strain
Zeta potential (mV)
D39 ...........................................................................................⫺11.3
D39(A)1....................................................................................⫺12.3
Rx1............................................................................................⫺13.3
Rx1(A) .....................................................................................⫺23.6
WU2 .........................................................................................⫺33.4
WU2(A) ...................................................................................⫺42.9
a
All values are the mean of three independent experiments. 1, Adapted to
grow in CDM-EA.
, Adapted to grow in CDM-EA; 2, U937 cells with no added bacteria.
directly to Rx1 in the same experiment with the same U937
culture, the mean fluorescence with Rx1 was more than 2.5fold greater than that of U937 cells with D39 (Table 2, bottom).
Bacteria that colonize or infect humans may simultaneously
utilize different mechanisms to maintain close association with
host cells. In addition to specific receptor-ligand interactions,
the physicochemical properties of bacterial surfaces can modulate the affinity of pathogens for host cells (8, 12, 16). There
are more than 90 pneumococcal serotypes, each defined by a
chemically distinct polysaccharide capsule. Nearly all of these
polymers impart a strong electronegative charge to the cell
surface. Pneumococci also express a number of surface-exposed proteins which are noncovalently attached to phosphorylcholine moieties of teichoic and lipoteichoic acids (9). Certainly, some of these proteins may have cognate ligands on
eukaryotic cells and function as adhesins. However, data presented here also show that the full complement of CBPs has a
profound effect on the physical characteristics of the pneumococcal cell surface.
Bacterial cells which do not display CBPs on their surface
are significantly more hydrophobic than those which display
normal amounts of CBPs. This effect is greatest in pneumococci which lack a capsule, except for strain WU2, in which the
CBPs measurably contribute to the hydrophilic character of
the cell surface. Pneumococci which lack CBPs also have a
more negatively charged cell surface, as measured by zeta
potential. Again, this is best measured in unencapsulated
strains but is also seen for the fully encapsulated strain WU2.
The contribution of the full complement of CBPs to surface
electrochemistry is a net positive charge. At least one CBP,
PspA, is known to have an overall positive charge in the animoterminal domain which is surface exposed (27). The net positive charge of CBPs may serve to stabilize the negatively
charged polysaccharide polymers. Alternatively, the positive
charges may promote the surface exposure of CBPs by reducing electrostatic repulsion which would otherwise keep func-
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Geometric mean (FL1-H)
D39
D39(A)1
U9372
FIG. 2. Adherence of pneumococcal strains to hexadecane. The
percent adherence was determined as described in Materials and
Methods. The diagonally striped bars are unadapted (CDM-C), and
stippled bars represent adapted cultures (CDM-EA). Standard error
bars are calculated from the means of at least three independent
measurements in each group.
Fluorescence intensity
VOL. 70, 2002
We thank David Briles and Janet Yother for providing strains and
Brenda Chapman for assistance with cell culture. We also appreciate
many insightful discussions with Larry McDaniel.
This work was supported by a grant from the Department of Veterans Affairs.
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tional domains inaccessible below the capsular surface. Overall, capsule is the major determinant of pneumococcal surface
chemistry and surface proteins appear to have minor effects in
its presence. An exception is the type 3 strain WU2, in which
CBPs have an effect on surface hydrophobicity and charge. The
surface of the other type 3 strain tested, A66, was essentially
unchanged when CBPs were removed.
The CBPs have a particularly large effect on adherence of
pneumococci to human monocytes. For capsule type 2 strain
D39, the effect is only seen when capsule is removed. For
WU2, the effect of CBPs on adherence is measurable both in
the presence and absence of capsule. This result is in agreement with the studies of hydrophobicity and surface charge for
WU2. It is possible that WU2 represents a unique phenotype
for type 3 strains or that type 3 strains as a group are heterogeneous in the manner by which CBPs contribute to cell surface chemistry and adhesion.
Certainly, some of the effect of CBPs on cell adhesion is
related to the loss of specific adhesins from the pneumococcal
cell. However, given the striking effect that CBPs have on the
physical characteristics of pneumococcal cells, it is plausible
that the full complement of CBPs may promote bacterium-host
cell interaction by electrostatic and/or hydrophobic interactions as well. An alternative explanation for these results is that
the presence of ethanolamine in teichoic acids may be responsible for the alterations in surface chemistry. Phosphorylethanolamine is incorporated into teichoic acids in the same location as phosphorylcholine via a phosphodiester linkage to
N-acetyl-D-galactosamine. The result of this substitution would
not be predicted to have any net effect on surface charge or
hydrophobicity.
The observations presented here suggest an additional function for the polysaccharide capsule of pneumococci. In addition to protection from host humoral immune components, the
pneumococcal capsule may inhibit interactions of surface proteins with phagocytes during invasive disease. In the context of
colonization, where inflammation and phagocytes are minimal
at the mucosal surface, the CBPs may provide a favorable
surface chemistry for adhesion in addition to receptor-ligand
interactions. In this case pneumococci may down-regulate the
quantity of capsular polysaccharide to potentiate adhesion. In
some model systems it has been shown that bacterial adherence to cultured mammalian cells is impeded by capsule (6,
18). Further work on the regulation of pneumococcal capsule
expression in different in vivo environments will be necessary
to address this question.
NOTES