| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B and Nitric Oxide1
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, New South Wales, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activation
in host splenocytes than infections with untreated parasites. Neither
proliferative ability nor conversion from tachyzoites to bradyzoites
was affected by lack of HSP70 in avirulent strains of T.
gondii. Furthermore, avirulent T. gondii strains
induced high levels of host iNOS expression and NO production,
regardless of HSP70 expression in these parasites, and inhibition of
HSP70 had no significant effects on translocation of NF-B to the
nucleus. Therefore, the 70-kDa parasite stress protein may be part of
an important survival strategy by which virulent strains down-regulate
host parasiticidal mechanisms. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, which stimulates macrophage production of TNF
(4, 5, 6). These cytokines act synergistically to initiate
signal transduction of the transcription factor NF-B.
NF-B performs an integral role in the regulation of immune response
genes (7); it is an essential step in the innate immune
response to pathogens (8). In their constitutive form,
NF-B dimers are sequestered in the cell cytoplasm by the inhibitor
protein, IB, which suppresses the nuclear localization signal of
NF-B (9). In response to the appropriate stimuli, IB
undergoes a process of phosphorylation, ubiquitination, and
degradation, leading to the release and nuclear translocation of
NF-B, where it mediates transcription (10). NF-B
proteins activate gene transcription by binding to sites in the target
promoters of a range of genes including those encoding cytokines,
growth factors, and immunoreceptors. Of particular note is NF-B’s
effect on inducible NO synthase
(iNOS)3, which
produces the antimicrobial radical, NO (11), one of the
key parasiticidal mechanisms used in intracellular protozoan
infections.
In T. gondii infection, NF-B (RelB) has been shown to be
essential for the induction of innate NK and adaptive T cell responses
that lead to the development of host resistance (12).
However, a recent paper by Butcher et al. (13) has
demonstrated the ability of this pathogen to invade cells without
triggering proinflammatory responses. Indeed, T. gondii
seems to be able to suppress NF-B activity. The molecular basis for
this suppression is not clear, but a potential explanation may rest
with a 70-kDa heat shock protein (HSP70) of T. gondii
associated with virulent phenotypes of the parasite (14, 15). Thus, HSP70s are associated with protection against
NO-induced cellular pathology and inhibition of NF-B activity in a
range of eukaryotic cells. Kim et al. (16) demonstrated
that HSP70 antisense oligonucleotides abrogate NO-induced protection
against TNF in rat hepatocytes, Bellmann et al. (17)
showed that transfection of the human gene encoding HSP70 into rat
insulinoma cells is protective against NO-induced cell lysis, and
Feinstein et al. (18) found that stress-induced HSP70
reduces NF-B nuclear uptake and subsequent downstream iNOS
expression and activity in astroglial cells. Thus, in this study we
tested the hypotheses that the HSP70 of T. gondii inhibits
iNOS expression, NO production, and NF-B activity, and that such
effects are confined to virulent strains of the parasite.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RAW 264.7 murine macrophages were a gift from Dr. G. Chaudry (Department of Pathology, University of Sydney, Sydney, New South Wales, Australia) in 1997. Since then, the cells have been passaged twice weekly and maintained in DMEM containing 5% FBS and 100 IU ml-1 penicillin and 100 µg ml-1 streptomycin (PS).
Primary splenocytes were prepared from spleens of 6–8 wk old female BALB/c mice. Briefly, mice were killed by CO2 overdose and cervical dislocation, and their spleens removed and retained in an excess of wash medium (DMEM and PS) and kept on ice. Under sterile conditions, the organs were poured onto a 70 µm-nylon mesh (Falcon; BD Biosciences, Mountain View, CA) over a sterile 50-ml polypropylene tube (Falcon) and homogenized to produce a single-cell suspension. The homogenate was rinsed well with wash medium and centrifuged at 1500 rpm at 4°C for 5 min. The supernatant was discarded and the cell pellet gently resuspended in RBC lysis buffer (0.85% NH3Cl in 0.01 M Tris-HCl, pH 7.2) and left for 2 min at room temperature. Additional wash buffer was added to the lysis mix before centrifugation. Splenocytes were resuspended in complete medium (DMEM, 10% FBS, PS). Viable cells were assessed using the trypan blue dye exclusion test and counted using a Neubauer chamber.
Parasites and infection
Parasites used in these experiments were two virulent strains (RH, ENT) and two avirulent strains (ME49, C) of T. gondii. Parasites were cultured twice weekly in vitro in RAW 264.7 cells and grown in DMEM containing 5% FBS and PS.
Parasites were diluted in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, PBS) containing PS to a concentration of 1 x 106 tachyzoites/ml. BALB/c mice were infected via i.p. injection with 1 x 105 parasites. Mice were sacrificed at various intervals postinfection by CO2 overdose and cervical dislocation and parasites harvested from the peritoneal cavity using a PBS wash.
Transient transfection
Expression of T. gondii HSP70 was reduced by transiently transfecting HSP70 antisense oligonucleotides into parasites, then treating parasites with quercetin, a bioflavonoid that specifically reduces HSP expression (19, 20, 21).
Phosphorothioate antisense oligonucleotides were designed to anneal to the T. gondii HSP70 ATG start codon. The antisense effect of the active oligonucleotide (AntiA: CAC AGC AGG AGA GTC CGC CAT, Tm = 71°C) was compared with a number of control oligonucleotides with varying degrees of target sequence homogeneity; AntiB: four sequence mismatches, CAC ATC ATG CGA GTA CGC CAT, Tm = 72°C; AntiC: two sequence mismatches, CAC ATC AGG AGA GTA CGC CAT, Tm = 71°C (underlined bases correspond to antisense sequence mismatches), and orientation; SenseA: active oligonucleotide in the sense orientation, ATG GCG GAC TCT CCT GCT GTG, Tm = 71°C.
Oligonucleotides were introduced into T. gondii tachyzoites via lipid-mediated transient transfection. Briefly, to each well of a 6-well plate (Costar, Cambridge, MA), 6 µl of lipid reagent DMRIE-C (Life Technologies, Rockville, MD) was added to 500 µl serum-reduced medium (Optimem; Life Technologies). To this was added 500 µl Optimem containing 4 µg DNA (the antisense effect was observed to be dependent on oligonucleotide concentration with 4 µg of DNA proving optimal for transfection-induced HSP70 inhibition, as determined by repeated concentration optimization experiments). The total sample was mixed by gently swirling the plate then incubated for 30 min at room temperature to allow formation of DNA-lipid complexes. A total of 2 x 106 tachyzoites in 200 µl Optimem was added and the whole solution mixed gently and incubated for 5 h at 37°C in 5% CO2. Posttransfection, 2 ml of antibiotic-free medium was added to each sample and the parasites were left to recover overnight. Parasites were then resuspended in complete medium containing 50–100 µM quercetin (Sigma-Aldrich, St. Louis, MO) and incubated for 5 h at 37°C in 5% CO2.
HSP70 assay
Quercetin-treated parasites were centrifuged and resuspended in complete medium before being subjected to heat stress (43 ± 0.5°C for 2 h). Parasites were assessed for cell viability (typically >90%) and counted, then collected and assayed for expression of HSP70 as described previously (22, 23). Briefly, protein concentration was measured in a Bradford assay and 10 µg of sample was loaded onto an 8% SDS-PAGE gel (Gradipore, Sydney, Australia). Samples were transferred to a nitrocellulose membrane and HSP70 was detected by Western blot using an avian polyclonal chicken Ab targeting the GGMPGGM repeat at the 3' end of T. gondii HSP70 (Chick29; Nerang Biotechnology, Melbourne, Australia). Chemiluminescent detection of signal (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) was performed and densitometric analysis (MultiAnalyst; Bio-Rad, Hercules, CA) of signal was used to quantify differences between samples.
Parasite burden
BALB/c mice were infected via s.c. injection with
106 tachyzoites. Spleens, livers, and lungs
from infected mice were collected at 4 days postinfection to track
parasite dissemination from the site of infection and assess parasite
burdens. Each treatment group comprised four mice. Organs were
sectioned using a scalpel blade and multiple imprints made by lightly
pressing against a glass slide. Slides were then fixed and stained as
described below. The mean and SE were calculated from the numbers of
parasites in spleens from eight mice per condition.
Stage conversion
To promote tachyzoite-bradyzoite conversion, host RAW 264.7
cells (2.5 x 103) were cultured in sterile
2-well Labtek chamber slides (Nalge Nunc International, Rochester, NY)
in the presence of 100 ng/ml recombinant mouse IFN- (R&D Systems,
Minneapolis, MN) and 10 µg/ml LPS (Sigma-Aldrich), then infected with
samples (1 host cell: 100 parasites). Each condition was duplicated and
the experiment was repeated three times. After 48 h, medium was
removed from the chamber slides and cells washed in PBS before being
fixed and analyzed for stage differentiation events.
Immunocytochemical procedures
For immunochemistry analysis, imprint slides were washed in PBS before being fixed in 3% paraformaldehyde for 10 min. Samples were incubated in methanol at 4°C for 10 min then blocked with 2.5% goat serum in 5% low fat milk in PBS for 30 min. For ease of parasite counting in dissemination experiments, slides were stained with anti-T. gondii goat polyclonal Ab in PBS with 2.5% goat serum for 45 min, followed by anti-goat IgG TRITC-conjugated secondary Ab (Sigma-Aldrich), both at a dilution of 1/50. For histochemical detection of tachyzoites in imprints, slides were fixed in methanol for 20 min, then stained in freshly diluted May-Grunwald stain for 5 min. Excess stain was removed then slides immersed in fresh Giemsa stain for 10 min followed by three rapid washes in buffered dH2O. Slides were allowed to dry before mounting with a coverslip. Fluorescent staining tachyzoites were counted from 20 fields of view per slide for four mice in each of two experiments.
Differentiation of T. gondii parasites was assessed by expression of tachyzoite- and bradyzoite-specific markers SAG1 and BAG1, detected using DG52 anti-SAG1 polyclonal Ab and 7E5 anti-BAG1 mAb at dilutions of 1/100 (kindly provided by Prof. J. Boothroyd, Stanford University, Stanford, CA, and Prof. U. Gross, Georg-August-University, Goettingen, Germany, respectively). Parasites were subsequently reacted with anti-mouse IgG FITC conjugate secondary Ab (Sigma-Aldrich) at a dilution of 1/64. As a control for cross-reactivity, some samples were stained with secondary Ab only. The percentage of parasites undergoing stage conversion, as assessed by BAG1 and SAG1 expression, was evaluated using fluorescent microscopy. Twenty fields of view per chamber were counted in duplicate in three separate experiments.
iNOS analysis
RAW 264.7 cells (2.5 x 107) were
cultured in sterile 75 cm2 flasks (Falcon) in the
presence of 100 ng/ml recombinant mouse IFN- (R&D Systems) and 10
µg/ml LPS (Sigma-Aldrich), then infected with 1 x
105 tachyzoites. After a 3-h incubation,
extracellular parasites were removed and the cells washed in cold PBS
before being harvested for RNA isolation. TRIzol reagent (Life
Technologies) was added directly to culture flasks and RNA extracted
from cells according to the manufacturer’s instructions.
mRNA was isolated from total RNA using Dynabeads (Dynal Biotech, Great Neck, NY) and reverse transcribed with oligo (dT) primers (Life Technologies) and Superscript II RNase H reverse transcriptase (Life Technologies). The resultant cDNA was amplified with specific primers in a semiquantitative PCR. The iNOS-specific forward primer had the sequence CAT GGC TTG CAC CTG GAA GTT TCT CTT CAA AA, while the reverse primer was GCA GCA TCC CCT CTG ATG GTG CCA TCG; the expected product size was 754 bp (GenBank accession no. M84373). To verify that equal amounts of RNA were added to each reaction and confirm uniform amplification, hypoxanthine phosphoribosyltransferase (HPRT) was transcribed and amplified for each sample in a multiplex PCR. The HPRT-specific forward primer used was GTT GGA TAC AGG CCA GAC TTT GTT G, and the reverse was GAT TCA ACT TGC GCT CAT CTT AGG C; the expected product size was 162 bp (GenBank accession no. J00423). After initial incubation at 94°C for 3 min, temperature cycling was initiated with 30 cycles of 94°C for 30 s, 45°C for 1 min, and 72°C for 3 min. The product was separated on a 1.5% agarose gel in the presence of ethidium bromide. Laser densitometry (MultiAnalyst; Bio-Rad) was used to quantify Fuji negative film and the amount of cDNA was standardized according to background and control HPRT message.
NO analysis
Nitrite, the stable byproduct of NO, was analyzed in the culture
supernatant of macrophages primed with 100 ng/ml IFN- and 10 µg/ml
LPS, then infected with 1 x 105
tachyzoites, using the Griess colorimetric reaction (24).
Briefly, supernatant (100 µl) was reacted in a mixture of 50 µl 1%
sulfanilamide (Sigma-Aldrich) and 50 µl 0.1%
N-(-1-napthyl)ethylene diamine dihydrochloride
(Sigma-Aldrich) in 3% phosphoric acid at room temperature. The
absorbance was read after 5 min in a microtiter plate reader at 540 nm
in reference to a standard nitrite quantitative curve.
NF-B analysis
Murine splenocytes were plated into 2-well Labtek chamber slides
(Nalge Nunc International) at 1 x 107/well,
incubated for 5 h, then incubated with samples (10 host cells: 1
parasite) for 2 h. Medium was then removed from the chamber slides
and the cells washed in PBS before being fixed in as described above.
Cells were stained with NF-B anti-p65 polyclonal rabbit Ab
(Santa Cruz Biotechnology, Santa Cruz, CA) at 4 µg/ml in PBS with
2.5% goat serum for 45 min, followed by FITC-labeled anti-rabbit
IgG (Sigma-Aldrich) at a dilution of 1/50. Nuclear localization of
NF-B was confirmed by 4',6'-diamidino-2-phenylindole dihydrochloride
hydrate (Sigma-Aldrich) staining. Slides were mounted in
FluorSave reagent (Calbiochem, La Jolla, CA) and viewed with a
fluorescent microscope (Olympus, Melville, NY). The percentage of cells
expressing NF-B p65 was calculated from averages obtained from
duplicated conditions where four different fields of 20–40 cells for
each condition were counted. This experiment was repeated twice. Means
and SE were calculated from the total dataset.
Statistical analysis
Statistical significance was assessed by the Student’s
t test and/or the one-way ANOVA with Tukey’s multiple
comparison test, where indicated, using GraphPad PRISM version 3.0 for
Windows software (GraphPad, San Diego, CA). Comparisons were considered
significant at p 
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Optimal HSP70 inhibition was achieved using a combination of
antisense and quercetin treatment. Parasites treated with quercetin
alone reduced HSP70 expression by 50% compared with untreated
parasites, while treatment with the active antisense oligonucleotide
AntiA alone reduced stress protein expression by 55% (one-way ANOVA
with Tukey’s multiple comparison test; Fig. 1
). Transfection of T. gondii
tachyzoites with AntiA antisense oligonucleotides and subsequent
treatment with quercetin reduced HSP70 expression by 77% (significant
at p < 0.05; one-way ANOVA with Tukey’s multiple
comparison test) after samples were stressed in vitro, compared with
values generated in control samples, and by 69% compared with the
SenseA oligonucleotides (Fig. 1); the effect was
concentration-dependent (data not shown). Parasites were assessed for
cell viability (typically >90%) and counted, then collected and
assayed for expression of HSP70 over a 96-h period as described
previously (22, 23). HSP70 expression was reduced in
parasites for up to 72 h posttreatment both in vitro and in vivo
(by 77 and 87%, respectively), but expression of the stress protein
returned to control levels after this period. Control oligonucleotides
showed increasing antisense effect with increases in sequence
homogeneity corresponding to the complementary region of HSP70 targeted
by the active antisense oligonucleotide. For example, the SenseA
oligonucleotides inhibited HSP70 expression by 27% compared with
untreated tachyzoites, but this was not statistically significant.
Likewise, the 16% reduction in HSP70 expression seen in the presence
of the AntiB antisense oligonucleotides (four sequence mismatches) was
not significant, but treatment with AntiC (two sequence mismatches)
achieved a 50% reduction in parasite stress protein expression, which
was a significant reduction (p < 0.05; one-way
ANOVA with Tukey’s Multiple Comparison test).
|
). In parasite samples pooled from
four mice per condition, expression of HSP70 by the virulent RH and ENT
strains was reduced by 87 and 78%, respectively, whereas expression of
HSP70 in the avirulent strains, ME49 and C, was reduced by 50% in both
cases.
|
Tachyzoites were rarely detected in the liver and lungs of
infected mice. However, spleens from mice infected with virulent RH
strain parasites contained significantly more
(p < 0.001; Student’s t test)
parasites than did mice infected with virulent parasites treated to
reduce HSP70 expression. In fact, for virulent infections, a 45% (RH
strain) and 25% (ENT strain) reduction in splenic parasite burden was
observed after HSP70 inhibition in these parasites (Fig. 3). Avirulent strains, which were
detected in much lower numbers in the spleen compared with virulent
strains, showed no significant difference between normal and
HSP70-treated parasites (Fig. 3).
|
Parasites cultivated in the presence of activated macrophage host
cells were positive for BAG1 by 48 h postinoculation; however,
detection of bradyzoite-specific Ags varied between strains and
treatment groups. In virulent strains, detection of bradyzoite-specific
Ags was always a low percentage of the total population (<5%)
compared with expression of the tachyzoite-specific SAG1 molecule;
however, virulent strains with reduced HSP70 expression showed
significantly increased expression of the BAG1 bradyzoite marker
compared with untreated virulent parasites (p
< 0.05; one-way ANOVA with Tukey’s multiple comparison test). BAG1
expression in RH strain was increased by 13–62% when parasites were
treated for HSP70 expression, while in the other virulent strain
examined, ENT, BAG1 expression rose by 18–75% upon HSP70 inhibition
(Fig. 4).
|
). Effect of T. gondii HSP70 on iNOS activity
The combination of IFN- and LPS stimulated expression of iNOS
in RAW 264.7 cells (Fig. 5). Infection of
these cells with tachyzoites of an avirulent strain (ME49) of T.
gondii caused a 45% increase in expression of this protein
(significant at p < 0.001; one-way ANOVA with Tukey’s
multiple comparison test). Treatment with the AntiA antisense
oligonucleotides did not affect the ability of the avirulent
tachyzoites to induce iNOS expression. In contrast, infection of
IFN-/LPS-primed RAW 264.7 cells with virulent tachyzoites (RH
strain) dramatically reduced expression of iNOS (by 95%,
p < 0.001; one-way ANOVA with Tukey’s multiple
comparison test). Treatment of RH tachyzoites with the AntiA antisense
oligonucleotides significantly (p < 0.01;
one-way ANOVA with Tukey’s multiple comparison test) reversed this
effect (expression of iNOS was 4-fold higher in the presence of
transfected parasites), but the expression of this enzyme in the
presence of the transfected parasites was still substantially (79%)
less than that of the uninfected RAW 264.7 cells
(p < 0.001; one-way ANOVA with Tukey’s
multiple comparison test).
|
and LPS, confirmed the affect of
the AntiA antisense oligonucleotides on iNOS expression. Thus, IFN-
and LPS stimulated NO production in RAW 264.7 cells and
this was further enhanced (by 45%, significant at p <
0.01; one-way ANOVA with Tukey’s multiple comparison test) by
infection with avirulent ME49 tachyzoites (Fig. 6). NO production was similarly enhanced
by ME49 tachyzoites that had been treated with AntiA antisense
oligonucleotides. In contrast, NO production by RAW 264.7 cells
infected with virulent RH strain tachyzoites was >90% lower than in
uninfected cells (significantly reduced, p < 0.001;
one-way ANOVA with Tukey’s multiple comparison test), but treatment
with AntiA antisense oligonucleotides abrogated this effect; NO
production by RAW 264.7 cells infected with transfected RH strain
tachyzoites was four to five times higher (significant at
p < 0.05; one-way ANOVA with Tukey’s multiple
comparison test) than in the cells infected with normal parasites and
only 50% reduced compared with the uninfected IFN-/LPS-primed
cells.
|
B
In the absence of inducing signals, 92% of uninfected splenocytes
showed only cytoplasmic NF-B expression (Fig. 7). Con A, a known stimulator of NF-B
translocation to the nucleus, induced nuclear uptake of p65 in 40% of
cells after 3 h. Infection with tachyzoites of avirulent strains
of T. gondii also stimulated substantial translocation of
NF-B to the nucleus; >65% of ME49 strain-exposed cells and >50%
of C strain-exposed cells were positive for the presence of nuclear
p65. This translocation was only slightly affected by treatment of the
parasites with AntiA antisense oligonucleotides; translocation of
NF-B to the nucleus occurred in 44% of ME49 strain-exposed cells
and in 40% of C strain-exposed cells. However, nuclear translocation
of NF-B was reduced in cells exposed to RH strain and ENT strain
tachyzoites when compared with avirulent infections; 29% of RH
strain-exposed cells and 34% of ENT strain-exposed cells
were positive for nuclear p65. This effect was
significantly reversed (p < 0.05; one-way
ANOVA with Tukey’s multiple comparison test) by treatment of the
virulent parasites with AntiA antisense oligonucleotides. Thus, >60%
of both transfected RH strain-exposed cells and transfected ENT
strain-exposed cells were positive for nuclear p65.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One of the most dramatic phenotypes evident among strains of T. gondii relates to the variation observed in severity of acute infection in the murine model (25). In mice, virulent strains such as RH commonly produce massive tachyzoite proliferation. Additionally, while both virulent and avirulent strains show rapid dissemination from the point of infection, significant differences occur in terms of the absolute parasite burden achieved in infected tissues between virulent strains such as RH and avirulent strains such as ME49 (25). In this study, virulent parasites expressing reduced levels of HSP70 showed decreased parasite burden in mouse tissues compared with normal virulent strain infections: a 45% (RH strain) and 25% (ENT strain) reduction in parasite numbers was observed in infected mouse spleens after 4 days of infection following HSP70 inhibition in these parasites. This is an impressive result considering the transient nature of inhibition of HSP70 in this system (a maximum of 72 h). Avirulent strains, which were detected in much lower numbers in spleens compared with virulent strains, showed no significant difference between normal and HSP70-treated parasites. Whether this is due to the conversion of the avirulent parasites into the more slowly growing bradyzoite stage or the more effective control of tachyzoite growth during acute infection by avirulent strains is unknown. However, our results with HSP70 inhibition in virulent strains implicate HSP70 expression as a component of the virulence phenotype, somehow facilitating ongoing replication of the proliferative tachyzoite form in the face of cyst formation and reduced replication in avirulent and HSP70-reduced virulent strains. This hypothesis is further supported by our observations of increased differentiation signaling, in terms of BAG1 expression, in virulent parasites treated to reduce HSP70 expression. Thus, inhibition of HSP70 expression in virulent strains of T. gondii (RH and ENT) had profound effects on strain propensity to differentiate following stress. Culture of treated virulent strains in a murine macrophage-like cell line (RAW 264.7) stimulated to produce the parasiticidal molecule, NO, initiated induction of the bradyzoite-specific marker BAG1, which was significantly increased (62% rise in HSP70-reduced RH strain; 75% in HSP70-reduced ENT strain) compared with expression in untreated virulent parasites. No significant effects were observed as a result of HSP70 inhibition in avirulent strains, though the trend was for reduced stage conversion, which would fit with the role of HSP70 in stage conversion of avirulent T. gondii described by Weiss and Kim (26).
Virulent strains of T. gondii used in this study (RH and
ENT) had profound effects on the responsiveness of RAW 264.7 cells and
murine splenocytes, inhibiting both iNOS expression and consequent
production of NO, and the translocation of NF-B from the cytoplasm
to the nucleus. This later observation confirms that of Butcher et al.
(13). These effects were dependent on HSP70. Thus, when
host cells were infected with virulent tachyzoites treated to reduce
HSP70 expression, levels of iNOS message were significantly increased
and HSP70-related inhibition of iNOS transcription translated to
downstream interference with NO production by virulent T.
gondii. Similarly, virulent parasites expressing reduced levels of
HSP70 were unable to inhibit translocation of NF-B from the
cytoplasm to the nucleus. In contrast, infection with avirulent strains
ME49 and C induced high levels of host iNOS expression and NO
production, regardless of the level of HSP70 expression in these
parasites. Likewise, inhibition of HSP70 in avirulent parasites had no
significant effects on translocation of NF-B to the nucleus. These
results provide a molecular basis for the inhibition of iNOS expression
since NF-B plays a crucial role in expression of this enzyme
(11).
An important question raised by our work is, how does HSP70 from
virulent T. gondii tachyzoites interfere with NF-B and
subsequently, iNOS activity? A number of precedents exist for HSP70
modulation of NF-B uptake to the nucleus; however, the mechanism by
which HSP70 could interfere with the activation of NF-B nuclear
uptake is not yet fully elucidated. In cases involving HSP70-mediated
inhibition of NF-B, Feinstein et al. (18) proposed that
stress proteins physically interact with IB. The association of
inhibitory IB with NF-B subunits occurs via interaction with
IB ankyrin domains with nuclear localization sites present in the
p50 and p65 proteins. Nuclear localization site homologs in human HSP70
have been described, thus raising the potential for HSP70 interaction
with ankyrin domains present in IB. This activity could result in
prevention of IB phosphorylation and subsequent dissociation of
NF-B. However, this explanation seems unlikely to account for the
effect of T. gondii HSP70, because it has recently been
shown that although tachyzoites do invade macrophages without
activating NF-B, they trigger normal phosphorylation-dependent
degradation of IB, thus indicating that the block on NF-B
translocation occurs downstream of these events (13).
Other possibilities are that HSP70, which is capable of migrating to
the nucleus upon induction of the stress response, impedes NF-B
nuclear translocation by competing for access to the same nuclear pore
complexes through which NF-B must be transported to the nucleus
(18). HSP70 could potentially inhibit NF-B activation
by direct interaction with one or more of the NF-B constituents.
Upon dissolution of the NF-B/IB complex after signal-induced
activation, NF-B is left in a temporarily unstable state that may
imitate that of a denatured protein. HSP70 could, in a move which
adheres to its functional capacity as a molecular chaperone, bind to
and therefore, prevent the nuclear translocation of NF-B as it
dissociates from IB. Thus, the subcellular localization of the HSP70
protein and the timing of its production may be important to promoting
suppressive effects. Feinstein et al. (18) hypothesized
that following activation of the signals necessary to induce NF-B
nuclear translocation and iNOS induction, a period of time is required
such that HSP70 is present concurrently with the HSP70-sensitive step
in this cascade of events. Thus, HSP70 expression begins within 30 min
and continues to accumulate for the next several hours. During this
time, translocation of host NF-B would commence, resulting in
dissolution of the NF-B/IB complex. Nuclear uptake of the
transcription factor then commences, but at this time, HSP70 levels are
still low, so initial nuclear uptake is probably not impeded. However,
within the next phase of activation, HSP70 levels are sufficient to
reduce NF-B uptake to the nucleus. This timing fits very nicely with
the timings of suppression of immune activities observed by Butcher et
al. (13). Furthermore, our observation that the inhibition
of NF-B translocation was not complete (an observation also made by
Butcher et al., Ref. 13) fits the above sequence of
events. Thus, this is a plausible explanation for the immunomodulatory
effects of T. gondii HSP70, but it begs the question of how
the parasite HSP70 gains access to the host cytoplasm and nucleus. It
will be interesting to see whether HSP70 is released during, or shortly
after, host cell invasion by virulent tachyzoites, along with several
other proteins that could conceivably interfere with host
signaling pathways (13). We do have preliminary
evidence that HSP70 is indeed secreted by T. gondii;
analysis of precipitated proteins separated by two-dimensional
electrophoresis and probed with a parasite specific anti-HSP70 Ab
detected parasite HSP70 in the culture supernatant at 4 h
postinfection (data not shown). Furthermore, a number of examples from
other parasites have described extracellular HSP70; for example, a
72-kDa Plasmodium falciparum HSP related to the 78-kDa
glucose-regulated stress protein of mammals called Pfgrp
(27) was detected in sera of people exposed to this
parasite (28), and is expressed on the surface of infected
hepatocytes (29).
There are several ways by which HSP70 might arrive at the cell surface and from there, move to the extracellular space. For example, in P. falciparum it is hypothesized that before merozoite formation, HSP70 associates with incipient membranes destined to constitute the merozoite surface, and within this molecular complex, locates to the cell surface (30). Additionally, HSP70 may be cytoplasmic during early parasite development but adheres to the surface of parasites after differentiation, or during host cell lysis (30). Alternatively, parasite HSP70 may become extracellular by passive anchoring to the cell surface of proteins released from dead or dying cells; for example, promonocytic cells are able to interact with and internalize HSP70 members (31). The exact nature of HSP70 release from the T. gondii remains to be elucidated; however, detection of this protein in the culture medium of infected cells suggests that T. gondii HSP70 is at least accessible to the host machinery, and therefore, in the position to exert the immunomodulatory effects observed in this study. This mechanism could help to explain why the inhibition of iNOS and NO production by virulent T. gondii in vitro was so efficient (95% reduction in iNOS expression in 2.5 x 107 activated RAW cells exposed to only 105 tachyzoites).
An additional issue raised by our results is why HSP70 from virulent T. gondii tachyzoites has such dramatic effects, whereas the same protein in avirulent strains of the parasite fails to affect host cell responsiveness or parasite differentiation? Molecular genetic studies show that the HSP70 gene is single copy in both virulent and avirulent strains of T. gondii (14). However, sequence analysis has revealed that the genes encoding HSP70 in virulent and avirulent strains are identical at the amino acid level, with the exception of the number of seven residue repeat units (GGMPGGM) at the 3'-end of the gene. In avirulent strains, five copies of this repeat are detected, while virulent parasites have only four (15). As the only difference between strains in terms of genetic structure, the deletion of this unit may have important implications for the synthesis and stability of this protein, which in turn may affect its efficacy in terms of immunomodulation and stage conversion. It is possible that HSP70, a molecule whose three-dimensional configuration is still being investigated, selectively presents its hypervariable C termini to the host immune system, which in turn may result in increased infectivity while concurrently acting as a trigger for components which protect the parasite from host defense mechanisms such as NO. Whether this accounts for the differential HSP70-directed response of virulent and avirulent strains to the host immune response and induction of parasite differentiation remains to be seen.
A potential limitation of the protocols used in this study to inhibit HSP70 expression by T. gondii (i.e., the combination of transient transfection with antisense oligonucleotides and quercetin) is that quercetin and other related flavonoids are known to induce multiple changes in the cells exposed to them (32, 33, 34, 35). The complex nature of the relationship between flavonoid compounds such as quercetin, heat shock proteins, and the immune response obviously requires further characterization, but it should be emphasized that, in this study, only parasites were treated with quercetin; the host cells that were used to examine the effects of parasite HSP70 on various parameters of the immune response were not exposed directly to quercetin at any time. Thus, the effects we have observed are highly likely to be the result of inhibition of parasite HSP70 by the combined action of the antisense oligonucleotides and quercetin. However, it is remotely conceivable that quercetin, independent of its effects on parasite HSP70, may have contributed to the effects on key host immune effector events and the promotion of bradyzoite formation that were observed in this study. Experiments with stable HSP70-knockout parasites, where transcription levels as well as expression can be confirmed, may definitively resolve this issue.
Regardless of the HSP70 sensitive step in the cascade of events leading
to inhibition of NF-B activation and iNOS transcription in virulent
T. gondii, HSP70 does appear to play a role in the ability
of virulent parasites to modulate immune response factors. Our results
implicate HSP70 as a means by which virulent strains of
Toxoplasma evade proinflammatory responses. The fact that
the effects of HSP70 are more apparent in virulent than avirulent
strains is consistent with observations of unrestrained virulent strain
asexual replication compared with the apparently enforced encystations
and subsequent development of quiescent parasitic forms observed in
avirulent strain infection. We hypothesize that HSP70 may have
different functions in avirulent and virulent T. gondii
parasites: in avirulent parasites, which readily undergo cyst
formation, HSP70 may be part of the machinery involved in stage
conversion, facilitating an essential evasion strategy of the parasite.
The role of HSP70 in virulent parasites, which do not readily
differentiate into cyst-dwelling bradyzoites, could represent an
alternative mechanism by which the parasite avoids the host immune
response. This stress protein may be part of an important survival
strategy by which virulent strains down-regulate activation of NO
production, thereby removing one of the principal parasiticidal
mechanisms from the host-parasite equation.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Alan M. Johnson, Department of Cell and Molecular Biology, University of Technology, Westbourne Street, Gore Hill, Sydney, New South Wales, 2065, Australia. E-mail address: alan.johnson{at}uts.edu.au
3 Abbreviations used in this paper: iNOS, inducible NO synthase; HSP70, 70-kDa heat shock protein; PS, penicillin and streptomycin; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication November 27, 2001. Accepted for publication May 14, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and correlates with the down-regulated expression of inducible nitric oxide synthase and other markers of macrophage activation. J. Immunol. 151:3672.[Abstract]
triggers antitoxoplasmal activity of IFN- primed macrophages. J. Immunol. 147:2340.[Abstract]
response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-. J. Immunol. 150:3982.[Abstract]
induced L-arginine dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous TNF-. J. Immunol. 148:568.[Abstract]
B transcription factors. Oncogene 18:6853.[Medline]
B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J. Immunol. 163:1457.B: a specific inhibitor of NF-B transcription factor. Science 242:540.B and Rel proteins: evolutionarily conserved proteins of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4701.B family member RelB is required for innate and adaptive immunity to Toxoplasma gondii. J. Immunol. 163:4453.B. J. Immunol. 167:2193.-induced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem. 272:1402.B activation. J. Biol. Chem. 271:17724.This article has been cited by other articles:
|
|
 |
|
  H. Fang, F. Aosai, H.-S. Mun, K. Norose, A. K. Ahmed, M. Furuya, and A. Yano Anaphylactic reaction induced by Toxoplasma gondii-derived heat shock protein 70 Int. Immunol., October 1, 2006; 18(10): 1487 - 1497. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zimmermann, P. J. Murray, K. Heeg, and A. H. Dalpke Induction of Suppressor of Cytokine Signaling-1 by Toxoplasma gondii Contributes to Immune Evasion in Macrophages by Blocking IFN-{gamma} Signaling J. Immunol., February 1, 2006; 176(3): 1840 - 1847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Cheng, C. Cenciarelli, G. Nelkin, R. Tsan, D. Fan, C. Cheng-Mayer, and I. J. Fidler Molecular Mechanism of hTid-1, the Human Homolog of Drosophila Tumor Suppressor l(2)Tid, in the Regulation of NF-{kappa}B Activity and Suppression of Tumor Growth Mol. Cell. Biol., January 1, 2005; 25(1): 44 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mamani-Matsuda, J. Rambert, D. Malvy, H. Lejoly-Boisseau, S. Daulouede, D. Thiolat, S. Coves, P. Courtois, P. Vincendeau, and M. D. Mossalayi Quercetin Induces Apoptosis of Trypanosoma brucei gambiense and Decreases the Proinflammatory Response of Human Macrophages Antimicrob. Agents Chemother., March 1, 2004; 48(3): 924 - 929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Debierre-Grockiego, N. Azzouz, J. Schmidt, J.-F. Dubremetz, H. Geyer, R. Geyer, R. Weingart, R. R. Schmidt, and R. T. Schwarz Roles of Glycosylphosphatidylinositols of Toxoplasma gondii: INDUCTION OF TUMOR NECROSIS FACTOR-{alpha} PRODUCTION IN MACROPHAGES J. Biol. Chem., August 29, 2003; 278(35): 32987 - 32993. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
