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* Department of Cell Biology, Neurobiology, and Anatomy and
Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of Medicine, and
Research Division, Veterans Administration Medical Center, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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). To identify the ligand(s) on Hc yeasts that is recognized by
M, purified human complement receptor type 3 (CR3, CD11b/CD18) was
used to probe a Far Western blot of a detergent extract of Hc cell wall
and cell membrane. CR3 recognized a single 60-kDa protein, which was
identified as heat shock protein 60 (hsp60). Biotinylation of viable
yeasts, followed by precipitation with streptavidin-coated beads, and
Western blotting with anti-hsp60 demonstrated that hsp60 was on the
surface of Hc yeasts. Electron and confocal microscopy revealed that
hsp60 resided on the yeast cell wall in discrete clusters. Recombinant
hsp60 (rhsp60) inhibited attachment of Hc yeasts to M. Recombinant
hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese
hamster ovary cells transfected with CR3 (CHO3). Polystyrene beads
coated with rhsp60 bound to M, and attachment was inhibited by Abs
to CD11 and CD18. Freeze/thaw extract (F/TE), a preparation of Hc yeast
surface proteins that contained hsp60, inhibited the attachment of Hc
yeasts to M. Depletion of hsp60 from F/TE removed the capacity of
F/TE to block binding of Hc to M. Interestingly, rhsp60 did not
inhibit binding of Hc yeasts to dendritic cells (DC), which recognize
Hc via very late Ag 5. Moreover, F/TE inhibited attachment of Hc to DC
even when depleted of hsp60. Thus, Hc hsp60 appears to be a major
ligand that mediates attachment of Hc to M CD11/CD18, whereas DC
recognize Hc via a different ligand(s). |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) recognize unopsonized Hc yeasts and
microconidia via the CD18 family of adhesion-promoting
glycoproteins (LFA-1 (CD11a/CD18), complement receptor 3 (CR3;
CD11b/CD18), and CR4 (CD11c/CD18)) (2, 3). The bound Hc
are rapidly phagocytosed and are contained within a phagocytic vacuole
in the M cytoplasm.
Upon ingestion, the yeasts subvert the normally hostile intraphagosomal
environment and replicate (4). Presumably, dividing yeasts
destroy the alveolar M and are phagocytosed by neighboring alveolar
M and inflammatory M recruited to the site of infection.
Repetition of this cycle results in the dissemination of the yeasts
from the lung to other organs. Thus, the infection of M by Hc is a
crucial step in the pathogenesis of histoplasmosis.
In the present study we sought to identify the ligand(s) on the surface
of Hc yeasts that binds to macrophage CD11/CD18 by probing cell
wall/cell membrane (CW/M) and freeze/thaw extract (F/TE) preparations
of Hc yeasts with purified CR3 (CD11b/CD18). The results presented
herein demonstrate that Hc yeasts use heat shock protein 60 (hsp60) to
bind to M.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hc strain G217B was maintained as previously described
(3). Yeasts were grown in histoplasma M medium
(5) at 37°C with orbital shaking at 150 rpm. For binding
assays, log-phase yeasts were heat-killed (HK) at 65°C for
1 h and stored at 4°C in PBS containing 0.05% sodium azide
(3). HK yeasts were labeled with fluorescein, washed, and
resuspended in HBSS containing 20 mM HEPES and 0.25% BSA (HBSA) as
described previously (3).
Preparation of Hc CW/M
Hc yeasts killed by incubation in PBS containing thimerosal were suspended in PBS containing 1 mM PMSF, 5 µM leupeptin, and 50 mM disodium EDTA and were disrupted in a bead beater (Biospec Products, Bartlesville, OK). The homogenate then was used to obtain a soluble preparation of yeast CW/M as described previously (6). The protein concentration was quantified by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).
Preparation of Hc F/TE
An F/TE of Hc yeasts was prepared as described by Klein et al. (7) for Blastomyces dermatitidis. Pellets of yeasts were frozen and thawed twice over 48 h. The yeasts were removed by centrifugation, and the supernatant containing crude surface ligand(s) was sterile-filtered and stored at 4°C. The protein concentration was quantified by the BCA protein assay. Preparations of the F/TE extract did not contain cytosolic proteins as determined by assay for lactate dehydrogenase (8), and <5% of the yeasts were lost during the freeze/thaw procedure.
Preparation of human M and dendritic cells (DC)
Human monocytes were purified from buffy coats obtained from
Hoxworth Blood Center (Cincinnati, OH) via sequential centrifugation on
Ficoll-Hypaque and Percoll gradients (Amersham Pharmacia Biotech,
Piscataway, NJ). M were obtained by culture of monocytes for 5–7
days in suspension in Teflon beakers as described previously
(3). DC were derived from purified monocytes by culture at
6.5 x 105/ml in medium containing human
rGM-CSF (115 ng/ml) and human rIL-4 (50 ng/ml; PeproTech, Rocky Hill,
NJ) for 6–8 days as described previously (9).
Isolation of human neutrophils and purification of CR3 (CD11b/CD18)
Human neutrophils were purified from buffy coats by centrifugation on Ficoll-Hypaque. Erythrocytes and PMN were separated via dextran sedimentation, and the remaining contaminating RBCs were removed by hypotonic lysis (10). The PMNs were washed in HBSS, and the cell pellet was snap-frozen in liquid nitrogen and stored at -70°C until used.
CR3 was purified from neutrophil lysates by a modification of the
procedure of Diamond et al. (11). PMNs were solubilized
for 1.5 h at 4°C in cold lysis buffer (100 mM Tris-HCl (pH 8.0),
150 mM NaCl, 2 mM MgCl2, 1% Triton X-100, 5 mM
iodoacetamide, 0.025% NaN3, 1 mM PMSF, 1 mM
di-isopropylfluorophosphate, and 0.2 U/ml of aprotinin), with gentle
stirring. After removal of insoluble components, CR3 was purified by
affinity chromatography over tandem columns of mouse IgG and LM2/1 Ab
(anti-CD11b, IgG1) coupled to cyanogen bromide-activated Sepharose
(CNBr-Sepharose). CR3 was eluted with 50 mM triethylamine (pH 10.0),
300 mM NaCl, 2 mM MgCl2, and 1%
n-octyl-
-D-glucopyranoside into
tubes with neutralizing buffer (10% by volume 1 M Tris-HCl, pH 7.4).
Peak protein fractions were pooled, aliquoted, and stored at -70°C.
The purified CR3 appeared as a single band on SDS-PAGE (data not
shown).
Antibodies
mAb TS-1/22 (12), specific for CD11a (LFA-1); MN-41
(13), specific for CD11b (CR3); Leu-M5 (14),
specific for CD11c (CR4); and IB4 (15), specific for CD18
(-chain), were described previously (3). LM2/1
(anti-CD11b) coupled to Sepharose was a gift from Dr. C. Parkos
(Emory University, Atlanta, GA).
A polyclonal monospecific Ab to Hc hsp60 was prepared from immune serum obtained from a rabbit immunized with native Hc hsp60 (16). Partially purified IgG was obtained by precipitation with saturated ammonium sulfate to a final concentration of 50%. The IgG then was affinity-purified on a column of recombinant hsp60 (rhsp60) coupled to CNBr-Sepharose. After elution, the affinity-purified Ab was dialyzed against PBS and concentrated, and the protein concentration was determined by the BCA protein assay. The monospecificity of the Ab was verified in two ways. First, both a total cell lysate and a cytosolic preparation of Hc yeasts yielded a single band after separation by SDS-PAGE and analysis by Western blot with the Ab. Further, incubation with a 10-fold molar excess of rhsp60 completely competed out the bands. Second, by flow cytometric analysis, a 10-fold molar excess of rhsp60 completely blocked the binding of the Ab to Hc yeasts (data not shown).
For use as a control in flow cytometry studies, the natural Abs to
common fungal cell wall carbohydrates found in naive rabbit IgG were
removed by absorbing once with -glucan (5 mg), and twice with
zymosan (1 x 109 particles/ml). These
absorptions removed 
90% of the cross-reactivity and allowed the
naive rabbit IgG to be used as a control for flow cytometry, electron
microscopy, and confocal microscopy analysis. The affinity-purified
anti-hsp60 IgG was treated in an identical manner without any loss
of binding activity to Hc yeasts.
Preparation of Hc hsp60-coated beads
Recombinant Hc hsp60 and native Hc hsp60 (nhsp60) were prepared as described previously (16, 17). Polystyrene fluorescent beads (Sigma-Aldrich, St. Louis, MO) were washed with 0.1 M borate buffer, pH 4.0, and then resuspended in 1 mg/ml of rhsp60 or BSA and incubated for 24 h at 25°C. The beads were blocked by incubation in BSA (10 mg/ml). Approximately 50% of the protein was adsorbed to the beads, and bound hsp60 was confirmed by flow cytometric analysis (data not shown).
Chinese hamster ovary cells transfected with CR3 (CHO3) culture
CHO3 cells and nontransfected CHO cells (a gift from Dr. G. Ross, University of Louisville, Louisville, KY) were cultured in DMEM (Life Technologies, Rockville, MD) containing 15% heat-inactivated FCS, 16 µM thymidine, 0.1 µM methotrexate, fungizone, and 10 µg/ml of gentamicin (18). Cells were harvested by scraping after reaching 80% confluence and were passaged or used in binding experiments with Hc yeasts.
Binding of Hc yeasts to M, DC, and CHO3 cells
M, DC, and CHO3 cells were suspended to 2.5 x
105/ml in HBSA containing 2% aprotinin. For
binding assays with M or CHO3 cells, the cells were adhered in the
wells of a Terasaki tissue culture plate (Miles, Naperville, IL) that
was coated with 1% human serum albumin. After adherence, 5 µl of
test protein or HBSA as a control was added to the monolayers and
incubated for 30 min (M) or 1 h (CHO3) at 37°C. Five
microliters of FITC-labeled HK Hc yeasts (5 x
107/ml) were added to each well and incubated for
30 min (M) or 2 h (CHO3) at 37°C. Unattached yeasts were
removed by washing, and the monolayers were fixed in 1%
paraformaldehyde. Binding of the yeasts was quantified via phase and
fluorescence microscopy on an inverted microscope (Diaphot; Nikon,
Melville, NY). The data are presented as the attachment index (AI), the
total number of attached yeasts per 100 cells, or the percent
inhibition of binding (1 - experimental AI/control AI
x 100).
For binding assays with DC, 50 µl DC (4 x
106/ml) and 50 µl of the test protein, or HBSA
as a control, were mixed in polypropylene tubes for 30 min at 37°C.
Subsequently, 50 µl of Hc yeasts (2 x
107/ml) were added to each tube and incubated for
an additional 30 min. At the end of the incubation period, 10 µl of
each sample was mounted on a clean glass slide and coverslipped for
immediate quantitation of the attachment index as described above.
Binding assays with rhsp60- or BSA-coated latex beads were performed in
suspension with human M in an identical manner.
Far Western blot analysis with purified CR3
One-dimensional Far Western immunoblotting studies of Hc proteins were performed by electrophoresis of Hc CW/M, F/TE, Hc nhsp60, or rhsp60 in a 12% SDS-PAGE gel. Separated proteins were electroblotted onto nitrocellulose membranes (Bio-Rad, Richmond, CA), blocked with 5% nonfat dried milk in TBS, and incubated for 3 h at 37°C with 25 µg of purified CR3. After washing, the membranes were incubated with anti-human CD11b, followed by HRP-conjugated, goat anti-mouse IgG. The membranes were developed with ECL substrate (Amersham Pharmacia Biotech) and exposed on Hyperfilm ECL.
For two-dimensional electrophoresis, 100 µg CW/M was electrofocused on 11-cm Immobiline isoelectric focusing strips, with a linear pH of 3–11 (Amersham Pharmacia Biotech). After focusing was complete, the isoelectric focusing strips were electrophoresed in a 10% SDS-PAGE gel. Separated proteins on one gel were stained with Coomassie Brilliant Blue R-250, and a duplicate gel was electrotransferred to a nitrocellulose membrane and analyzed by Far Western blot analysis as described above.
Biotinylation and precipitation of surface proteins on Hc yeasts
Viable Hc yeasts (1 x 109/ml) were suspended in PBS or in PBS containing 10 mg/ml of biotin (EZ-Link sulfo-NHS-LC-biotin; Pierce) and incubated overnight at 4°C. After washing, yeast pellets were resuspended in lysis buffer and mixed with an equal volume of glass beads. After sonication, the beads were removed by centrifugation, and insoluble components were removed by centrifugation. Biotinylated proteins were precipitated by incubation with streptavidin (SA) agarose beads (Pierce), and 50 µg of each SA-bead fraction and 0.25 µg of rhsp60 as a positive control were separated on a 10% SDS-PAGE gel. Biotinylated Hc hsp60 was visualized on a Western blot probed with the affinity-purified anti-hsp60 Ab.
Immunolabeling and electron microscopy of hsp60 on Hc yeasts
Hc yeasts were incubated for 1 h at 4°C with either 40 µg of absorbed, affinity-purified anti-hsp60 or absorbed normal rabbit IgG. The yeasts then were incubated with biotinylated goat anti-rabbit IgG, followed by SA-conjugated 5 nm-colloidal gold. The samples were fixed in 4% paraformaldehyde/2.5% glutaraldehyde and processed for electron microscopy (19). Samples were viewed on a 100CX electron microscope (JEOL, Peabody, MA) operating at 80 kV. In identical experiments Hc yeasts were prepared using an anti-hsp60 mAb or an isotype control Ab.
To estimate the number of hsp60 antigenic sites on the surface of Hc yeasts, we analyzed 81 experimental and 30 control electron micrographs using the standard stereological methods of planimetry and point counting (20, 21, 22).
Flow cytometric analysis of hsp60 on Hc yeasts
Hc yeasts (1 x 106/ml) were incubated for 1 h at 4°C with the affinity-purified anti-hsp60 or absorbed rabbit IgG as a control. After washing, yeasts were incubated with FITC-labeled, goat anti-rabbit IgG and then fixed overnight in 1% paraformaldehyde. Yeasts were analyzed by flow cytometry on a Coulter EPICS XL flow cytometer, and the acquired data were analyzed with Easy 2 software (Beckman Coulter, Brena, CA).
Immunodepletion of hsp60 from Hc F/TE
Hc F/TE was immunodepleted of hsp60 by absorption onto an affinity column prepared with the affinity-purified anti-hsp60 IgG. Two hundred microliters of F/TE was loaded onto the column and incubated overnight at 4°C. The F/TE was run through the column, and the column was washed extensively. All fractions containing protein were pooled, dialyzed against PBS, and concentrated to the original volume.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the early stages of infection, unopsonized Hc yeasts attach
to M via the CD11/CD18 family of adhesion-promoting glycoproteins
(2). As an initial step to identify a putative ligand(s)
on Hc yeasts that is recognized by M CD11/CD18, we prepared CW/M
from Hc yeasts and found that it inhibited the attachment of yeasts to
M by >90% (data not shown). We then used purified CR3 (CD11b/CD18)
to probe a Far Western blot of proteins from the CW/M preparation. CR3
recognized a single protein band at 60 kDa (data not shown). To
determine whether the 60-kDa band was comprised of one or more
proteins, Hc CW/M was resolved by two-dimensional SDS-PAGE and was
re-analyzed by Far Western blotting with CR3. Fig. 1
B shows a single protein spot
that migrated to an isoelectric point of pH 5.9 and had a
Mr of 60 kDa. Fig. 1A shows the
Coomassie-stained gel.
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C, CR3 avidly bound to
both nhsp60 and rhsp60. Detection and localization of hsp60 on the surface of Hc yeasts
Hsp60 is reported to be predominantly in the cytosolic fraction of cells (23, 24). However, to act as a ligand for host cell CD11/CD18, hsp60 would have to be localized to the surface of Hc yeasts. That hsp60 was indeed on the surface of Hc yeasts was confirmed by four complementary techniques. In one experiment yeast cell surface proteins were biotinylated with a membrane-impermeant reagent, and biotinylated and untreated control yeasts were simultaneously lysed and solubilized. The insoluble fraction was removed by centrifugation, and the supernatants were incubated with SA-agarose-coated beads to precipitate the biotinylated proteins. The SA-coated beads were resolved by SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed with affinity-purified rabbit anti-hsp60. Hsp60 was detected in the fraction of precipitated biotinylated proteins, but not in untreated controls (data not shown).
The surface expression of hsp60 on Hc yeasts was further confirmed by
flow cytometric analysis using the absorbed, affinity-purified
anti-hsp60. Both viable and HK-yeasts contained equivalent amounts
of hsp60 (Fig. 2, A and
B).
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A), whereas control Ab
labeling was virtually undetectable. Both monoclonal (data not shown)
and polyclonal Ab demonstrated aggregated surface hsp60 labeling. Fig. 3B shows the quantitation of hsp60 expressed as the number
of gold particles per yeast. Analysis by fluorescent confocal
microscopy also revealed punctate labeling over the surface of the
yeasts (data not shown). Using the standard stereological methods of
planimetry and point counting (20, 21, 22), and both
monoclonal and polyclonal anti-hsp60, we observed that there were
approximately three antigenic sites per thin section per yeast. We
calculated that each thin section represented 1.2% of the surface area
of a yeast cell. Accordingly there are 250 hsp60 sites/Hc yeast
(three sites per section/1.2% of surface area per section).
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and CHO3 cells and
binds to CD11/CD18
As hsp60 bound to CR3 and clearly was expressed on the surface of
Hc yeasts, it was an excellent candidate as a ligand for M
CD11/CD18. Therefore, we next tested the ability of Hc rhsp60 to
inhibit the binding of Hc yeasts to M or to CHO cells transfected
with CR3 (CHO3 cells). M or CHO3 cells were incubated with varying
concentrations of rhsp60 or rH-Ag as a control and then
incubated with FITC-labeled Hc yeasts. Recombinant hsp60, but not
rH-Ag, inhibited the attachment of Hc yeasts to both M (Fig. 4A) and CHO3 cells (Fig. 5A) in a concentration-dependent
manner. The rH-Ag was chosen as a control
because, like hsp60, immunization with rH confers protection in a
murine model of pulmonary histoplasmosis (25), but, unlike
hsp60, H-Ag resides in the cytosol (26). Fig. 4B shows that M bound viable and HK Hc yeasts equally
well, and that binding was inhibited by rhsp60, but not by rH-Ag. The
attachment of Hc yeasts to CHO3 cells also was inhibited by mAbs to
CD11b and CD18, but not by mAbs to CD11a or CD11c (Fig. 5B).
Untransfected CHO cells did not bind Hc yeasts at all (data not shown).
Finally, rhsp60 also inhibited the binding of Hc microconidia to M
(Table I), but not quite to the extent
that it inhibited the binding of Hc yeasts. Microconidia contained
levels of hsp60 on their surface equivalent to viable and HK yeasts
(Fig. 2C).
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CD11/CD18, hsp60- or
BSA-coated fluorescent polystyrene beads were incubated with M in
the presence or the absence of mAbs to CD11 and CD18. Fig. 6 shows that there was a 4-fold increase
in the binding of rhsp60-beads to M compared with BSA-beads.
Preincubation of the M with mAbs to CD11b and CD11c inhibited the
binding of rhsp60-beads by 50%, whereas a mAb to CD11a had no
affect on the binding of beads to M. An mAb to CD18 or a mixture of
mAbs to all three 
-chains inhibited the binding of rhsp60-beads to
the background level of control BSA-beads.
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We next sought to determine whether hsp60 was the only ligand on
Hc yeasts that mediated binding to M. An F/TE prepared from viable
Hc yeasts was highly enriched for surface proteins, as we detected no
lactate dehydrogenase activity in the F/TE, and <5% of the yeasts
were lost during the freeze/thaw procedure (data not shown). Further,
hsp60 was detected in a Western blot of F/TE probed with the
affinity-purified rabbit anti-hsp60 (Fig. 7A). Yeasts frozen and thawed
to prepare the F/TE bound minimally to M, even though they contained
about half the amount of hsp60 as viable and HK yeasts (Fig. 2D). We depleted hsp60 from F/TE using a column of
anti-hsp60 Ab immobilized on CNBr-activated Sepharose. An affinity
column prepared with rabbit IgG was used for mock depletion. The F/TE
flow-through was concentrated to its original volume and tested for its
ability to inhibit the binding of Hc yeasts to M. Western blotting
of the immunodepleted F/TE demonstrated that 90% of the hsp60
protein was removed by affinity chromatography (Fig. 7A).
Untreated F/TE inhibited the binding of Hc yeasts to M by 70%,
whereas F/TE immunodepleted of hsp60 had no inhibitory activity (Fig. 7B).
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Recently, we demonstrated that immature human DC recognized Hc
yeasts via the fibronectin receptor very late Ag 5 rather than
CD11/CD18 (9). These data suggest that DC also might
recognize Hc yeasts via a different ligand than does M CD11/CD18. To
test this hypothesis, DC were preincubated with F/TE, hsp60-depleted
F/TE, mock-depleted F/TE, rhsp60, or rH-Ag and then incubated with
FITC-labeled Hc yeasts. Although F/TE was found to inhibit the binding
of Hc to DC, rhsp60 and rH-Ag were without effect. Moreover, F/TE
depleted of hsp60 was just as inhibitory as undepleted F/TE, indicating
that a different component of the F/TE inhibited the attachment of Hc
yeasts to DC (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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serve as a critical interface in the pathogenesis of
histoplasmosis. Initially these cells provide a permissive
intracellular environment that allows Hc yeasts to gain a foothold in
the host and to disseminate from the lung to other organs. After the
induction of Th1-type immunity, M become activated and kill the
invading fungus. The ability of Hc and other intracellular pathogens to
subvert the normally hostile confines of the M phagosome to a
"friendly" environment may in part depend on which receptors the
pathogen uses to gain entrance into M.
Hc yeasts and conidia are recognized and ingested by M via
2 integrin receptors (CD11/CD18) (2, 3). The CD18 family consists of three distinct -chains
noncovalently linked to a common -chain (CD11a/CD18 (LFA-1),
CD11b/CD18 (CR3), and CD11c/CD18 (CR4)) (27). CR also are
involved in the recognition and phagocytosis of other intracellular
pathogens, including Legionella pneumophila
(28), Mycobacterium tuberculosis
(29), Leishmania major (30, 31),
and Klebsiella pneumonia (32). Thus, under
certain circumstances CR may act as a safe portal of entry into
M.
As the initial contact of Hc yeasts with M may be crucial to its
ability to survive intracellularly, we sought to identify the ligand(s)
on the surface of yeasts that is recognized by M CD11/CD18. The
results presented herein demonstrate that Hc hsp60 is a major surface
molecule recognized by human M. As hsp60 generally is considered a
cytoplasmic molecule, we used four separate techniques to demonstrate
that hsp60 was indeed expressed on the surface of Hc yeasts. First,
surface proteins on viable Hc yeasts were biotinylated, precipitated
with SA agarose beads, and analyzed by SDS-PAGE and Western blotting.
Anti-hsp60 Ab detected hsp60 on biotinylated yeasts, but not on
untreated control yeasts. Surface expression of hsp60 on Hc yeasts also
was demonstrated by flow cytometry with the absorbed, affinity-purified
anti-hsp60 Ab. Finally, immunoelectron microscopy with both the
affinity-purified anti-hsp60, and an mAb to hsp60 showed that hsp60
was present on the outer surface of the electron translucent cell wall
in distinct clusters; this topology was confirmed by confocal
microscopy.
Using standard stereological methods, we calculated that there are
250 hsp60 antigenic sites. This low number of sites aligns with our
previous observation that disruption of M actin microfilaments with
cytochalasin D inhibits binding of Hc yeasts to M (3).
Thus, M CD11/CD18 receptors must be mobile within the plane of the
membrane to efficiently make contact with the relatively few hsp60
antigenic sites that are on the surface of the yeasts. Despite the
relative paucity of hsp60 on Hc yeasts, once the yeasts bind to M,
they are rapidly ingested, thus achieving their goal of gaining
entrance into a safe environment in which to replicate.
Hsp60 is a 60-kDa hsp homologous to GroEL of Escherichia coli (33). A general role ascribed to hsps is to chaperone nascent or aberrantly folded proteins. Hsps also have been shown prevent protein aggregation, promote proper folding of nonnative proteins, assist in the assembly of multisubunit protein complexes, as well as target proteins for membrane translocation (23, 24). From an immunological standpoint, hsps are interesting in that they are immunogenic for a remarkable number of microbial pathogens, including Borrelia burgdorferi (34, 35), L. pneumophila (36), Chlamydia trachomatis (37, 38), Coxiella burnetti (39), Neisseria meningitidis (40), Rickettsia tsutsugamushi (41), M. tuberculosis (42), Helicobacter pylori (43), Yersinia entercolitica (44), Plasmodium yoelii (45), Pseudomonas aeruginosa (46), and Coccidioides immitis (47). In addition, adoptive transfer of hsp60-reactive T cell clones protects mice from a normally lethal infection of Y. enterocolitica (44), and vaccination of mice with hsp60 DNA from P. yoelii is partially protective (45). Finally, vaccination of mice with Hc rhsp60 is protective against pulmonary histoplasmosis (16). Thus, hsp60 serves a dual function in the immunopathology of histoplasmosis.
In addition to being a potent Ag, hsp60 has been implicated in the attachment and invasion of nonphagocytic host cells by pathogenic microorganisms. Thus, H. pylori uses cell surface hsp60 to promote attachment to gastric epithelia (48), and hsp60 on the surface of Clostridium difficile is involved in the attachment to various epithelial cell lines (49). L. pneumophila hsp60 is important for invasion of HeLa cells (50, 51). Of particular interest is that latex beads coated with L. pneumophila hsp60 were more efficiently taken up by HeLa cells than BSA-coated beads, and phagosomes that contained hsp60-beads did not exhibit phagolysosomal fusion, while phagolysosomal fusion occurred in HeLa cells that had ingested BSA-coated beads (51). Whether Hc hsp60 is involved in the capacity of Hc to inhibit phagolysosomal fusion (52) is unknown.
The mechanism by which Hc hsp60 reaches the surface of the cell wall is unclear, as the gene does not contain a classical secretory leader sequence (GenBank accession no. P50142). Indeed, the Hc hsp60 gene possesses a unique leader sequence upstream from the amino terminus of the mature polypeptide that has high homology with the leader sequence of two other pathogenic fungi, Coccidiodes immitis (GenBank accession no. O660008) and Paracoccidiodes brasiliensis (GenBank accession no. U81786). This leader sequence has not been found in the hsp60 sequence of higher organisms such as mammals, in nonpathogenic yeasts such as Saccharomyces cerevisiae (GenBank accession no. M33301), or in commensal opportunists such as Candida albicans (GenBank accession no. AF085694). However, we have found that the hsp60 associated with the CW/M of Hc is N-glycosylated, while the cytosolic form is not, suggesting that the surface form of hsp60 traffics through the Golgi apparatus. RACE analysis of the hsp60 message suggests an alternative exon with a canonical leader sequence that may direct the export of an hsp60 subpopulation to the surface of the yeasts. Additional experiments are aimed toward confirming these preliminary findings.
Several lines of evidence demonstrated that Hc hsp60 served as a ligand
for M CD11/CD18. First, rhsp60 inhibited the attachment of Hc yeasts
to M and CHO cells transfected with CR3 (CHO3). Second, mAbs to
CD11b and CD18 inhibited the binding of Hc yeasts to CHO3 cells. Third,
polystyrene beads coated with Hc hsp60 bound to M, and binding was
partially inhibited by mAbs to CD11b and CD11c. Further, a mixture of
anti-CD11-specific mAbs and an mAb to CD18 inhibited the binding of
hsp60 beads to the background level of control BSA-coated beads.
Fourth, immunodepletion of hsp60 from Hc F/TE completely removed the
capacity of the F/TE to inhibit binding of Hc yeasts to M. Hsp60
also blocked the attachment of Hc microconidia to M. Thus, both Hc
conidia and yeasts appear to enter M using the same ligand-receptor
combination (3)
It is intriguing that Hc hsp60 failed to block the attachment of Hc
yeasts to DC, and that removal of hsp60 from F/TE did not abrogate its
ability to inhibit the binding of yeasts to DC. Thus, DC not only
recognize Hc yeasts via very late Ag 5 rather than CD11/CD18
(9), but also recognize a different ligand on Hc yeasts
than M. This utilization of different receptor-ligand combinations
by M and DC may in part account for the ability of DC to kill Hc
yeasts (9), while M provide a permissive intracellular
environment (4)
Besides the present study, to our knowledge there is only one other
report of a microbial hsp mediating attachment to M. Thus, the
68-kDa protein of M. avium, which exhibits strong homology
with the 65-kDa hsp of M. tuberculosis, M.
leprae, and M. bovis, binds to the vitronectin
receptor, v3, on
human monocyte-derived M (53). Further, binding through
the v3 receptor
enhances the expression of CR3 (CD11b/CD18). Since CR3 also plays a
role in the phagocytosis of M. avium (54), the
data suggest a complex interaction between these receptors.
It also has been reported that human hsp60 activates human M via
CD14 in a manner similar to LPS (55), and others have
reported that human hsp60 stimulation of mouse M to produce NO and
secrete TNF- is dependent on Toll-like receptor 4 (56).
Finally, human and chlamidial hsp60 have been shown to activate the
stress protein kinases and mitogen-activated protein kinases via
Toll-like receptors 2 and 4 in transfected human fibroblasts
(57). Overall, it is clear that both self and non-self hsp
may have a profound effect on the host immune response. In addition,
the current data suggest that the mode of entry of Hc into host cells,
as determined by its microbial ligand-to-host cell receptor
interaction, determines its intracellular fate. Further studies of
receptor-ligand interactions on M and DC with Hc, and the different
signal transduction pathways that are activated, should provide
additional knowledge of the mechanism(s) employed by Hc to promote
pathogenesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Simon L. Newman, Division of Infectious Diseases, University of Cincinnati College of Medicine, P.O. Box 670560, Cincinnati, OH 45267-0560.
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; BCA, bicinchoninic acid; CHO3, Chinese hamster ovary cells transfected with CR3; CNBr-Sepharose, cyanogen bromide-activated Sepharose; CR, complement receptor; CW/M, cell wall/cell membrane; DC, dendritic cell; F/TE, freeze/thaw extract; HK, heat-killed; hsp60, heat shock protein 60; M, macrophage; nhsp60, native hsp60; rhsp60, recombinant hsp60; SA, streptavidin.
Received for publication August 7, 2002. Accepted for publication November 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J. Immunol. 156:1235.[Abstract]
-glucosidase enzymatic function of the Histoplasma capsulatum H antigen using a native expression system. Gene 247:191.[Medline]
-subunits and a common -subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158:1785.-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol. 152:743.[Abstract]
v3 on human monocyte-derived macrophages enhances complement receptor type 3 expression. Infect. Immun. 65:1211.[Abstract]
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