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Infect Immun, June 1998, p. 2922-2927, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vivo Binding of Immunoglobulin M to the Surfaces
of Babesia bigemina-Infected Erythrocytes
Ignacio E.
Echaide,1,2
Stephen A.
Hines,1
Terry F.
McElwain,1
Carlos E.
Suarez,1
Travis C.
McGuire,1 and
Guy H.
Palmer*
Department of Veterinary Microbiology and
Pathology, Washington State University, Pullman, Washington,
99164,1 and
Instituto Nacional
Technologia Agropecuaria, 2300-Rafaela, Santa Fe,
Argentina2
Received 22 July 1997/Returned for modification 18 August
1997/Accepted 2 March 1998
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ABSTRACT |
Babesia bigemina infection of mature bovine
erythrocytes results in new proteins specifically exposed on the
parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a
protein, designated p94, on B. bigemina-infected
erythrocytes but not on either uninfected or B. bovis-parasitized erythrocytes. However, p94 was not encoded by
B. bigemina and was not a parasite-modified erythrocyte
membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following
reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both
infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32
bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine µ heavy-chain MAb, BIg73, reacted specifically with
the surface of infected erythrocytes and bound the 94-kDa molecule in
lysates of infected erythrocytes, normal bovine serum, and purified
IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with
MAb 64/32 depleted the 94-kDa antigen bound by anti-µ MAb BIg73 and
vice versa. Binding of IgM to the infected erythrocyte surface was
detected in vivo early in acute parasitemia and occurred during both
the trophozoite and merozoite stages of intraerythrocytic parasitism.
The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinct B. bigemina strains is suggestive of an
advantageous role in parasite survival in vivo.
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INTRODUCTION |
Apicomplexan parasites in the genera
Babesia and Plasmodium invade and replicate
within erythrocytes, resulting in, respectively, babesiosis and malaria
(13). Invasive stages bind specific receptors normally
expressed on the surfaces of target erythrocytes (15, 27, 28,
40) and enter the cell via formation of a parasitophorous vacuole. The vacuole is later dissolved by babesial but not plasmodial parasites (11, 37). Inside the erythrocytes, the merozoite differentiates into a trophozoite, which undergoes asexual replication to produce daughter merozoites able to exit the host cell and invade
additional erythrocytes (13). During this intracellular replicative cycle, the host erythrocyte membrane is altered
(8). Changes required for intracellular growth are
associated with active transport of nutrients from the serum
(17), as well as elimination of catabolites from the
parasitized erythrocytes (14). In addition to these
metabolic functions, parasite-induced structural changes may alter the
interaction of the infected erythrocyte with other host cells and
molecules (2, 16). Erythrocytes infected with either
Babesia bovis or Plasmodium falciparum are sequestered in the microvasculature as a result of parasite-induced adherence to endothelial cells (4, 48). Erythrocyte adhesion is mediated by parasite-encoded proteins, such as P. falciparum EMP-1, rosettin, and sequestrin (3, 9, 30),
or, alternatively, by parasite modifications of host proteins
(6). Importantly, both parasite-encoded proteins and
modified host proteins may present new epitopes associated exclusively
with infected cells and therefore may serve as targets of immunity as
well as pathogenetic determinants (5, 18).
Similar to B. bovis and P. falciparum
infections, B. bigemina infection results in new
proteins specifically exposed on the erythrocyte surface
(39). However, B. bigemina infection differs in that sequestration of parasitized erythrocytes and the resulting neurological signs do not occur and the clinical signs are referable principally to severe anemia. Correspondingly, we propose that the
structural and functional modifications of the B. bigemina-infected cell surface are likely to be unique. Our
research goal is to identify these modifications associated with
B. bigemina infection and determine their pathogenetic
significance. Using a monoclonal antibody (MAb) developed against a
merozoite fraction (22), we identified a protein exposed on
the surfaces of B. bigemina-infected bovine
erythrocytes. This MAb, designated 64/32, reacts with the surfaces of
erythrocytes infected with B. bigemina strains from Brazil, Mexico, Puerto Rico, and St. Croix but not with either uninfected or B. bovis-infected erythrocytes
(47). Initial results indicated that MAb 64/32 bound a
molecule of 94 kDa that was not metabolically labeled with
[35S]methionine during in vitro culture of B. bigemina. Consequently, we hypothesized that the protein
recognized by MAb 64/32 is not encoded by the parasite but represents a
parasite-dependent modification. In this paper, we describe the
identification of this protein and its binding to B. bigemina-infected erythrocytes.
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MATERIALS AND METHODS |
Parasites.
The Mexico strain of B. bigemina,
its derivative the biological clone JG-29 (29), and the Mo7
clone from the Mexico strain of B. bovis
(35) were maintained as cryopreserved stabilates (31,
47). Parasites were grown in vitro with bovine erythrocytes and
normal bovine serum as described previously (21, 45).
Antibodies.
All MAbs used in this work were from
twice-cloned hybridomas and were of the immunoglobulin G1 (IgG1)
isotype. MAb 64/32 is reactive with the surfaces of B. bigemina-infected erythrocytes and binds a protein initially
designated p94. MAb 14/1 specifically recognizes a 45-kDa glycoprotein,
gp45, on the B. bigemina merozoite surface, and MAb
14/16 is reactive with a 58-kDa B. bigemina rhoptry protein, RAP-1 (24). MAb BIg73 is directed against the heavy chain of bovine IgM, and MAb BIg501 is specific for the bovine Ig 
light chain. MAb 23/8 is specific for a 225-kDa B. bovis spherical-body protein (33) and was used as the
positive control for B. bovis-infected erythrocytes.
MAb Tryp1E1, reactive with the Trypanosoma brucei variable
surface glycoprotein, and MAbs 64/11 and ANA8A, reactive with a 220-kDa
surface protein on normal and infected bovine erythrocytes, were used
as negative controls. A bovine postinfection serum, B240, was obtained
from a calf infected with B. bigemina (44).
A murine polyclonal antibody was obtained by immunizing mice with p94.
The p94 was purified from lysates of infected erythrocytes by MAb 64/32
affinity chromatography (see below).
B. bigemina infection.
A splenectomized
5-month-old calf was inoculated intravenously with 2 × 109 B. bigemina (Mexico strain) cells of a
stabilate cryopreserved with 10% polyvinylpyrrolidone (46).
Blood was collected daily in EDTA, and the reactivity of MAb 64/32 with
infected erythrocytes was evaluated by live immunofluorescence.
IFA.
The fixed indirect immunofluorescence assay (IFA) was
performed as previously described (26) with smears of washed
erythrocytes infected with either B. bigemina JG-29 or
B. bovis Mo7 and fixed with methanol. Smears of
uninfected or Anaplasma marginale-infected erythrocytes were
used as negative controls. Parasite nuclei were stained with 0.025%
ethidium bromide diluted in PBS (pH 3.5). IFA with live parasites was
performed as described previously (24). MAb 14/1, which
specifically binds only the B. bigemina merozoite outer
membrane, was used as a surface specificity control to confirm that the
infected erythrocyte membrane was intact and impermeable to antibody
(24).
Immunoaffinity chromatography.
The B. bigemina-infected erythrocyte surface antigen p94 was purified on
a MAb 64/32 affinity column. Briefly, 25 mg of purified MAb 64/32 was
suspended in 5 ml of 0.1 M phosphate buffer (pH 8.0) and coupled to an
agarose matrix with 0.1 M NaCNBH3. B. bigemina-infected erythrocytes from in vitro cultures (10 ml of
>95% parasitized erythrocytes after concentration on Percoll
gradients) were solubilized in lysis buffer containing protease
inhibitors. The lysate was incubated on the MAb-agarose-coupled column
and eluted as described previously (25). A separate but
identically prepared MAb 64/32 affinity matrix was used to isolate IgM
from normal bovine serum. Eluted proteins were quantitated by the
bicinchoninic acid technique (Pierce, Rockford, Ill.) and were
electrophoresed in polyacrylamide gels with detection by silver
staining or immunoblotting (43).
Electrophoresis and immunoblotting.
Proteins were
solubilized in Laemmli sample buffer, electrophoresed in 7.5 to 17.5%
or 1.5 to 15.0% gradient polyacrylamide gels with sodium dodecyl
sulfate (SDS) (34), and transferred to nitrocellulose
membranes (Schleicher & Schuell, Keene, N.H.). A solution of 3.0%
bovine serum albumin in 10 mM Tris-150 mM NaCl-0.05% Tween 20 was
used to block the membranes and for subsequent washes. The membranes
were incubated at 23°C for 1 h in this buffer containing 2 µg
of MAb per ml (32). Bound MAbs were detected with a 1:6,000 dilution of sheep anti-mouse antibody conjugated to horseradish peroxidase. The conjugate complexes were detected by enhanced chemiluminescence (Amersham Corp., Arlington Heights, Ill.). Protein standards in the range of 7.1 to 208 kDa (kaleidoscope prestained standards; Bio-Rad, Hercules, Calif.) were also electrophoresed and
transferred to the nitrocellulose membranes.
Metabolic and surface labeling.
B.
bigemina-encoded proteins were metabolically labeled, during in
vitro culture, with [35S]methionine (200 µCi/well;
NEG-072 EXPRE35S; Du Pont Co., Wilmington, Del.) or a
mixture of 15 tritiated amino acids as described previously
(10). The radiolabeled samples were immunoprecipitated with
MAbs 64/32 and 14/16, a postinfection serum (from a B. bigemina-infected calf, B-240) (44), and the murine
polyclonal antibody against affinity-purified p94. Proteins located on
the surfaces of infected erythrocytes were labeled with biotin
(19). Briefly, infected erythrocytes were suspended in a
solution of 5.0 mM sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin; Pierce, Rockford, Ill.) in PBS (pH 7.4) to a final concentration of 2.8 mg of NHS-LC-biotin per 5 × 107
erythrocytes. After incubation at 4°C for 1 h, the succinimide esters were neutralized in a solution of 1 mM glycine. For
immunoprecipitation, metabolically radiolabeled or biotin
surface-labeled infected erythrocytes were solubilized as previously
described (25).
In vitro translation of B. bigemina mRNA.
Briefly, RNA was isolated from 9 × 109 B. bigemina-infected erythrocytes by oligo(dT)-cellulose column
chromatography. The mRNA was then translated by using a rabbit
reticulocyte lysate with incorporation of biotin-labeled lysine
(Boehringer Mannheim, Indianapolis, Ind.). Translation products were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected
with streptavidin-horseradish peroxidase.
Depletion of IgM from infected erythrocyte lysate by
immunoprecipitation.
Either MAb 64/32 or BIg73 (anti-bovine µ heavy chain) was incubated with a lysate of biotin-surface-labeled
infected erythrocytes, and bound antigen-antibody complexes were
precipitated with protein G (GammaBind G Plus Sepharose [Amersham]).
This step was repeated once. The depleted supernatant was then
incubated with a second MAb and precipitated with protein G-Sepharose.
The immunoprecipitates were washed, separated by SDS-PAGE under
reducing conditions, and blotted to nitrocellulose (34).
Proteins were detected by using streptavidin-horseradish peroxidase and
enhanced chemiluminescence (47).
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RESULTS |
Specificity of MAb 64/32 binding.
Immunofluorescence of live
B. bigemina-infected erythrocytes from in vitro
cultures showed that the reactivity of MAb 64/32 was localized to the
surfaces of infected erythrocytes (Fig. 1a and
b). Binding occurred on 87% ± 5.0% of
infected erythrocytes and was similar whether the parasite stages were
trophozoites or merozoites. MAb 14/1, which is specific for the
B. bigemina merozoite outer membrane, was unable to
bind, indicating that the infected erythrocyte membranes were intact
and impermeable to antibody. MAb 64/32 showed no reactivity with
B. bovis-infected erythrocytes (Fig. 1c and d) or
uninfected erythrocytes. The isotype control MAb Tryp1E1 was unreactive
with B. bigemina-infected erythrocytes (Fig. 1e and f).
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FIG. 1.
Reactivity of MAbs with the surfaces of B. bigemina-infected erythrocytes assessed by live-cell IFA. (a to f)
In vitro culture. (a) IFA reactivity of MAb 64/32; (b) light microscopy
of the same field; (c) lack of reactivity of MAb 64/32 on the surfaces
of B. bovis-infected erythrocytes; (d) light microscopy
of the same field as in panel c; (e) lack of reactivity of the isotype
control MAb Tryp1E1; (f) light microscopy of the same field as in panel
e. (g and h) In vivo infection with B. bigemina (day
3). (g) IFA reactivity of MAb 64/32; (h) light microscopy of the same
field as in panel g. Parasite nuclei were stained with ethidium bromide
to detect infected erythrocytes.
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To confirm that MAb 64/32 binding was not limited to in vitro-cultured
parasites, a 5-month-old seronegative calf was infected
with the Mexico
strain of
B. bigemina. Infected erythrocytes were
initially observed on day 2 postinoculation, and their surfaces
were
bound by MAb 64/32 in the same pattern (Fig.
1g and h) as
observed
above on infected cells from in vitro culture (Fig.
1a
and b). The
binding pattern was unchanged on the subsequent 5
days of acute,
increasing parasitemia (data not shown). Interestingly,
in the infected
calf, MAb 64/32 bound only 45% ± 4.6% of infected
erythrocytes
containing merozoites or trophozoites.
Immunoprecipitation of labeled proteins from infected
erythrocytes.
To identify the protein recognized on the
surfaces of B. bigemina-infected erythrocytes by MAb
64/32, biotin-surface-labeled infected erythrocytes were lysed
and immunoprecipitated and the proteins were separated by
SDS-PAGE. MAb 64/32 specifically recognized an approximately
94-kDa protein (initially designated p94) from infected erythrocytes
(Fig. 2, lane 2) but not from uninfected erythrocytes. Labeled proteins of about 50 and 25 kDa were also nonspecifically precipitated (lane 4).
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FIG. 2.
Immunoprecipitation of antigens from the surfaces of
B. bigemina-infected erythrocytes. Biotin-labeled
surface proteins from infected erythrocytes were immunoprecipitated
with MAb 64/32 (lane 2, arrow) or the negative control MAb 64/11 (lane
4). Surface-labeled uninfected erythrocytes were immunoprecipitated
with either MAb 64/32 (lane 1) or MAb 64/11 (lane 3). Molecular size
standards are designated on the left.
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To test whether the polypeptide recognized by MAb 64/32 was
B. bigemina encoded, proteins synthesized by the
parasite during
in vitro cultures were labeled with
[
35S]methionine, immunoprecipitated, and separated by
SDS-PAGE. Autoradiography
showed that neither the polyclonal antibody
to p94 (from mice
immunized with affinity-purified p94) nor MAb 64/32
was able to
precipitate metabolically labeled p94 from uninfected (data
not
shown) or infected (Fig.
3, lanes 2 and 4) erythrocytes. The positive
control MAb 14/16 precipitated
labeled RAP-1 (lane 5), and the
postinfection serum B240 (from a
B. bigemina-infected calf) precipitated
multiple
labeled proteins including RAP-1 (lane 3). To test whether
the failure
to detect
35S-labeled p94 was attributable to a paucity of
methionine residues,
a mixture of 15 tritiated amino acids was used in
a second in
vitro labeling experiment. Neither MAb 64/32 nor the
polyclonal
antibody against p94 was able to precipitate any labeled
protein
(Fig.
4, lanes 1 and 5). The
positive control MAb 14/16 immunoprecipitated
3H-RAP-1
(lane 3). To exclude the possibility that p94 translation
was blocked
in the in vitro cultures, purified mRNA from
B. bigemina JG-29 was added to a reticulocyte translation system
which included
biotin-labeled lysine. The biotin-labeled
translation products
were transferred to a nitrocellulose membrane and
detected with
streptavidin-horseradish peroxidase. The lack of p94
translation
(data not shown) supported the results of the
metabolic labeling
experiment.
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FIG. 3.
Immunoprecipitation of 35S-labeled
B. bigemina proteins. Proteins labeled with
[35S]methionine during in vitro growth of B. bigemina were immunoprecipitated with MAb 64/32 (lane 4), MAb
14/16 (anti-p58, RAP-1) (lane 5), murine polyclonal antiserum to p94
(lane 2), bovine postinfection serum B240 (lane 3), or preinoculation
murine serum (lane 1). Molecular size standards are designated on the
left.
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FIG. 4.
Immunoprecipitation of 3H-labeled
B. bigemina proteins. Proteins labeled with a mixture
of 15 tritiated amino acids during in vitro growth of B. bigemina were immunoprecipitated with MAb 64/32 (lane 1), MAb
14/16 (lane 3), murine polyclonal antiserum to p94 (lane 5), isotype
control MAb Tryp1E1 (lane 2), or preinoculation murine serum (lane 4).
Molecular size standards are designated on the left.
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|
Host-encoded proteins on the infected erythrocyte surface.
To
identify host proteins on the surfaces of the erythrocytes, lysates of
uninfected erythrocytes and normal bovine serum were separated by
SDS-PAGE and probed with the polyclonal anti-p94 antibody. This
polyclonal antibody was used in an attempt to identify p94 as a
normal erythrocyte protein by binding multiple epitopes, since
the single epitope recognized by MAb 64/32 could be a
parasite-generated modification restricted to infected erythrocytes
(6). Neither the polyclonal anti-p94 antibody nor MAb 64/32
was able to bind any protein from the uninfected erythrocytes (data not
shown). However, the polyclonal antibody bound a protein from normal
bovine serum which was abundant and had a molecular size similar to
that of the p94 identified on the surfaces of infected erythrocytes (data not shown). Moreover, MAb 64/32 also recognized this
approximately 94-kDa serum protein. This suggested that p94 was
not an erythrocyte membrane protein modified by the parasite but
could be a normal serum protein bound to B. bigemina-infected erythrocytes. Based on the abundance in serum
and the molecular size, p94 was hypothesized to be the reduced µ chain of bovine IgM. When proteins from infected erythrocytes and
normal bovine serum, both eluted from MAb 64/32 affinity chromatography
columns, and purified bovine IgM were electrophoresed under reducing
conditions and immunoblotted, MAb 64/32 bound an approximately 94-kDa
protein in each preparation (data not shown). Subsequently, infected
erythrocytes, normal bovine serum, purified IgM, and, as a negative
control, purified IgG were electrophoresed and immunoblotted with
either MAb 64/32 or MAb BIg73 (anti-bovine µ heavy chain). Both MAbs
bound comigrating proteins from the infected erythrocytes, normal
bovine serum, and purified IgM but not from purified IgG (Fig.
5). This strongly suggested that p94 was
the IgM heavy chain.
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FIG. 5.
Reactivity of MAb 64/32 with infected erythrocytes,
normal bovine serum, and purified IgM. B. bigemina-infected erythrocytes (lanes 1, 5, and 9), normal bovine
serum (lanes 2, 6, and 10), purified bovine IgM (lanes 3, 7, and 11),
or purified bovine IgG (lanes 4, 8, and 12) were separated by SDS-PAGE
under reducing conditions. The immunoblotted proteins were probed with
MAb 64/32 (lanes 1 to 4), MAb BIg73 (anti-bovine µ heavy chain)
(lanes 5 to 8), or isotype control MAb Tryp1E1 (lanes 9 to 12). The
87-kDa molecular size standard is indicated on the left.
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Confirmation of MAb 64/32 reactivity with bovine IgM.
MAb
64/32 immunoprecipitation of biotin-labeled B. bigemina-infected erythrocyte proteins (Fig.
6, lane 11) depleted IgM from the
lysate as detected with anti-µ heavy-chain MAb BIg73 (lane 12).
As expected if both MAbs bound to the same antigen, immunoprecipitation of infected erythrocyte lysate with MAb BIg73 (lanes 7 and 9) also
depleted all reactivity for MAb 64/32 (lane 10), as well as for itself
(lane 8). Therefore, MAb 64/32 has the same antigen specificity as the
anti-µ heavy-chain MAb, BIg73. Initial depletion by
immunoprecipitation with an isotype control MAb (lane 3) did not block
subsequent reactivity of MAb 64/32 with infected erythrocytes (lane 4).
MAb 64/32 was unreactive with biotin-labeled uninfected erythrocytes
(lane 2).
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FIG. 6.
Binding of IgM from B. bigemina-infected
erythrocytes by MAbs 64/32 and BIg73. Biotin-labeled infected
erythrocyte lysates were specifically depleted by immunoprecipitation
with the first MAb; the proteins remaining in the supernatant were then
immunoprecipitated by the second MAb. The following six pairs of MAbs
were used in a "first-second" order for the immunoprecipitations:
for uninfected erythrocytes, ANA8-64/32 (lanes 1 and 2); for infected
erythrocytes; 64/11-64/32 (lanes 3 and 4), 64/32-64/11 (lanes 5 and 6),
BIg73-BIg73 (lanes 7 and 8), BIg73-64/32 (lanes 9 and 10), and
64/32-BIg73 (lanes 11 and 12). BIg73 is against the bovine µ heavy
chain; MAbs ANA8 and 64/11 are isotype controls. The 87-kDa molecular
size standard is indicated on the left.
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| |
DISCUSSION |
The antigen defined by MAb 64/32, exposed on the surfaces of
B. bigemina-infected erythrocytes, is present among
otherwise antigenically and genetically distinct B. bigemina strains isolated from Mexico, Brazil, and the Caribbean
(12, 23, 25, 36, 47). This conservation and reactivity
during both trophozoite and merozoite stages is unique among the
antigens previously identified on Babesia-infected
erythrocytes (39, 47). Initial experiments involving MAb
64/32 immunoprecipitation of metabolically radiolabeled B. bigemina proteins failed to identify a parasite-encoded protein. In this study, polyclonal antibody to p94, produced in mice, also failed to precipitate either 35S- or 3H-p94,
confirming the previous results obtained with MAb 64/32 (47). In addition, MAb 64/32 was unable to bind in vitro
translation products of B. bigemina mRNA. These
techniques, which take advantage of the inability of mature
erythrocytes to synthesize proteins or mRNA, have been used
successfully to identify parasite-encoded proteins of
Babesia and Plasmodium (1, 10, 20,
24). Consequently, after these initial results, we directed
our approach to the identification of host proteins specifically
associated with intraerythrocytic B. bigemina
parasitism.
Erythrocyte membrane proteins can be modified during invasion and
intracellular parasitism, resulting in specific changes on the
surfaces of infected erythrocytes. For example, P. falciparum cleaves the integral membrane protein band 3, a
modification generating new surface-exposed epitopes (6). To
determine whether MAb 64/32 bound to a new epitope resulting from
parasite-mediated modification of a normal host protein, polyclonal
anti-p94 was reacted with uninfected erythrocytes in an attempt to
detect the normal unmodified host protein. However, this polyclonal
antiserum retained specificity for infected erythrocytes. Although this did not totally preclude the possibility of a host protein modification as the source of infected erythrocyte specificity, we proceeded to
address whether a host serum protein could specifically bind infected
erythrocytes. In other parasitic infections, host antibodies bind the
surface of either the parasite or the infected cell. Schistosoma
mansoni parasites express tegumental IgG-Fc receptors in both
schistosomula and adult stages (41, 42). Similarly, sequestered P. falciparum-infected erythrocytes have
IgM bound between the erythrocyte membrane and the endothelial cells
(38). Based on this rationale, we investigated and
determined that MAb 64/32 recognizes bovine µ heavy chain, which
consistently binds to the surfaces of B. bigemina-infected erythrocytes. The evidence includes the
following: MAb 64/32 bound a reduced molecule of 94 kDa in both
infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32
bound a 94-kDa molecule in reduced preparations of purified IgM; (iii)
an anti-bovine µ heavy-chain MAb, BIg73, reacted specifically with
the surfaces of infected erythrocytes and bound the 94-kDa molecule in
lysates of infected erythrocytes, normal bovine serum, and purified
IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with
MAb 64/32 depleted the 94-kDa antigen bound by anti-µ MAb BIg73 and
vice versa. The 94-kDa apparent molecular size of the reduced bovine µ heavy chain is similar to the 85 kDa previously reported
(7).
Importantly, both MAb 64/32 and the anti-bovine µ heavy-chain MAb
BIg73 bound the surfaces of infected erythrocytes obtained from a calf
during early acute B. bigemina infection. This
indicated that IgM binding occurs in vivo and before the induction of a specific immune response. This observation is consistent with the
hypothesis that the IgM binding is not antigen specific and is mediated
by a receptor for the Fc region. This is supported by the binding of
IgM from uninfected cattle to the surfaces of B. bigemina-parasitized erythrocytes. In addition, purified
Fc5µ but not Fab fragments were able to bind
B. bigemina-parasitized erythrocytes after removal
of whole IgM by acid elution (data not shown). However, this
rebinding of Fc5µ may not necessarily involve the same
receptor used to bind whole IgM in vivo, since the acid elution
procedure could alter the integrity of the receptor or the erythrocyte
membrane.
IgM binding may be useful for parasite growth or survival, as indicated
by its conservation among the otherwise antigenically and genetically
distinct B. bigemina strains. Interestingly, not all
infected erythrocytes observed in vivo had bound IgM, but the binding
was not associated with any particular B. bigemina stage. These results are in contrast to those obtained with two other
MAbs, specific for a B. bigemina-encoded protein of
~200 kDa expressed on the erythrocyte surface, which bound 45%
of B. bigemina-infected erythrocytes with
reactivity determined by the parasite stage (39). Whether
the variable IgM binding to B. bigemina-infected
erythrocytes reflects variations among the host erythrocytes or in the
parasite is unknown. Identification of the receptor on the infected
erythrocytes to which the µ heavy chain binds is the next step in the
determination of the significance of IgM binding during B. bigemina infection.
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ACKNOWLEDGMENTS |
This work was supported by U.S. Department of Agriculture
NRICGP grant 96-35204-3667, U.S. Department of Agriculture BARD grant US-2496-94C, Washington State University, and the Instituto Nacional de Tecnologia Agropecuaria.
We appreciate the excellent technical assistance of Debra Alperin,
Beverly Hunter, Emma Karel, and Carla Robertson.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Microbiology and Pathology, Washington State University,
Pullman, WA 99164-7040. Phone: (509) 335-6033. Fax: (509) 335-8529. E-mail: gpalmer{at}vetmed.wsu.edu.
Editor: P. J. Sansonetti
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Infect Immun, June 1998, p. 2922-2927, Vol. 66, No. 6
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
