Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St, New Haven, CT, 06520-8022, USA
Author for correspondence (e-mail:
keith.joiner{at}yale.edu)
Accepted 21 January 2003
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Summary |
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Key words: Rhoptry, Secretion, Toxoplasma, Pleiotropic, Antisense RNA
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Introduction |
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T. gondii enters the host cell by an active, orientation-dependent
penetration mechanism that is powered by the actin cytoskeleton of the
parasite (Dobrowolski and Sibley,
1996). Rhoptries discharge at the time of host cell invasion
(Aikawa et al., 1977
;
Carruthers and Sibley, 1997
)
and the released proteins include those encoded by the rhoptry gene family
(ROP1, 2, 3, 4, 6, 7 and 8) that range in size from 42-68
kDa (Ossorio et al., 1992
;
Joiner and Dubremetz, 1993
;
Beckers et al., 1994
;
Beckers et al., 1996
). The
function of these rhoptry proteins remains unknown, although they are thought
to have roles in host cell attachment and invasion, establishment and
maintenance of the parasitophorous vacuole membrane (PVM) and replication in
the PV. It has been suggested that rhoptries secrete a lytic product or
penetration-enhancer factor (PEF) to facilitate T. gondii invasion
into the host cell (Lycke et al.,
1975
; Schwartzman,
1986
). ROP1 is associated with PEF, but disruption of the
ROP1 gene showed no effect on invasion
(Kim et al., 1993
;
Soldati et al., 1995
).
Similarly, a deletion of RAP1 rhoptry gene in Plasmodium
falciparum did not affect parasite invasion or growth
(Baldi et al., 2000
).
We have previously shown that the T. gondii ROP2 protein is
localized to the PVM with its N-terminal domain exposed to the host cell
cytosol (Beckers et al., 1994).
In vitro import assays of ROP2 deleted in the putative mitochondrial transit
sequence in the N-terminus (residues 98-12) support the hypothesis that ROP2
interacts with the mitochondrial import machinery and mediates the tight
association of host mitochondria to the PVM (Sinai et al., 2001). However,
this model has not been tested in vivo.
Because rhoptries are assumed to be essential for parasite invasion,
studies of rhoptry/gene function are especially complicated by the fact that
T. gondii cannot survive outside the host cell, thus precluding a
gene knockout strategy that ablates parasite invasion. This may potentially
explain our inability in previous attempts to generate a ROP2 gene
knockout (C. J. M. Beckers and K.A.J., unpublished). In addition, rhoptry
genes are often part of multigene families, and/or are tandemly repeated
(Beckers et al., 1996), further
complicating a gene knockout approach. To circumvent these problems, we
applied a recently developed ribozyme-modified antisense RNA strategy
(Nakaar et al., 1999
;
Nakaar et al., 2000
) to lower
the expression of ROP2 gene. In this review we show that targeted
depletion of ROP2 results in: (1) disruption of rhoptry biogenesis and an
impairment of cytokinesis; (2) reduction in the association of host cell
mitochondria with PVM of the parasite, and a reduced sterol uptake from the
host cell; (3) reduced capacity of parasites to invade and replicate in human
fibroblasts, and attenuation of virulence in mice. These data suggest that
rhoptry discharge, and in particular the release of ROP2, is critical for
parasite invasion, replication and parasite-host cell interaction.
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Materials and Methods |
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A fragment including the chloramphenicol acetyl transferase (CAT) gene was
amplified by PCR with T3 (5'-GACTAGTAATTAACCCTCACTAAAGGG-3') and
T7 (5'-GAGCTCCAATTCGCCCGATC-3') primers using the 220NTP3 cassette
as DNA template (Nakaar et al.,
1998). This sequence was inserted in a cohesive end ligation to
the SpeI site of pASRP2 generating pASRP2-CAT. These constructs were
verified by sequencing at the W. M. Keck Sequencing Center, Yale University
School of Medicine. Other constructs, pminCAT and pAS-HXCAT, have previously
been described (Nakaar et al.,
2000
).
Parasite cultures and transfection
RH strain of T. gondii was cultivated in Vero or human foreskin
fibroblasts (HFF) cells. These cells were routinely grown in MEM and
-MEM, respectively, supplemented with antibiotics, 1 mM L-glutamine and
10% FBS (Gemini Bio-Products, Calabasas, CA). Typically, 10-20 µg of
pASRP-CAT purified by a commercially available kit (Qiagen) was used to
electroporate 107 tachyzoites that were purified from host cells by
syringe passage (21G needle) as previously described
(Roos et al., 1994
). After 24
hours, 20 µM chloramphenicol was added to the medium
(Soldati and Boothroyd, 1993
)
and the medium containing the selection drug was changed every 3-4 days. When
the monolayer of cells neared lysis, the parasites were sub-cultured onto
fresh cells. After two passages, the parasites were purified and plated by
limiting dilution into 96-well plates. Stable transformants were cloned and
amplified into mass cultures.
Uracil incorporation and invasion assays
Parasite replication was assayed by measuring the incorporation of
[3H]-uracil (Amersham) into parasites as previously described
(Nakaar et al., 1999).
Invasion assays were performed using confluent cultures of HFF in 6-well
plates that were inoculated with 106 freshly lysed-out parasites.
After 30 minutes, most of the parasites that had not invaded were washed off
and fresh medium was added. The cultures were incubated overnight at 37°C
and parasitophorous vacuoles were counted in 50-100 random light microscopy
fields.
Immunoblots
Between 105 and 106 parasites were separated on
SDS-PAGE and the material was transferred onto nitrocellulose filters. The
filters were probed with T34A7 (Sadak et
al., 1988), anti-ROP2, 3, 4 antibody and with goat anti-mouse
horseradish peroxidase polyclonal antibody as secondary antibody or with
anti-NTPase antibody as previously described
(Nakaar et al., 1999
). Both
primary and secondary antibodies were generally used in 1:1000 dilution.
Detection was by ECL kit (Amersham Pharmaceuticals Biotech).
Immunofluorescence assay, mitotracker labeling, transmission electron
microscopy and morphometric analysis
Immunofluorescent microscopy was done as previously described
(Hoppe et al., 2000). For
electron microscopy, monolayers of Vero cells were infected with RH or
recently isolated stable clones expressing ROP2 antisense (ROP2AS)
for 2-3 days until parasite vacuoles were detectable by light microscopy.
Infected cultures are fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate
(pH 7.4) for 30 minutes and post-fixed in 1% osmium tetroxide in the same
buffer. Fixed samples were processed for epon plastic embedding and thin
sectioned for transmission electron microscopy using standard protocols.
Morphometric analysis and mitotracker labeling of parasites were completed
using a procedure similar to that previously described
(Sinai et al., 1997
).
Immunoelectron microscopy
A monolayer of HFF cells were infected with ROP2AS-1 and
ROP2AS-10 parasites for 24-48 hours and fixed in 8% PFA in 0.25 M
HEPES, pH 7.4 for 2 days at 4°C. Infected monolayers were scraped and
pelleted in 10% fish skin gelatin, infiltrated overnight with 2.3 M glucose at
4°C, and frozen in liquid nitrogen. Ultrathin cryosections were obtained
and incubated with T43A7 mAb (1:100), washed, and then incubated with 5 nm
protein A-gold Conjugate (1:70). The washed sections were postfixed in 1%
glutaraldehyde and contrasted with 1.8% methyl cellulose and 0.5% uranyl
acetate and examined by transmission electron microscope.
Preparation of LDL, lipoprotein-deficient serum (LPDS) or LDL labeled
with NBD-C ([NBD-C]-LDL)
The protocols of isolation of human LDL (density 1.019 to 1.063 g/ml) from
fresh plasma by zonal density gradient ultracentrifugation, preparation of
LPDS by ultracentrifugation of fetal bovine serum (FBS) and incorporation of
the fluorescent lipid NBD-C into LDL have been described
(Coppens et al., 2000).
Incubation of infected cells with [NBD-C]-LDL and filipin and
fluorescence microscopy
To visualize fluorescent cholesterol acquired by T. gondii,
synchronized infected cells were incubated in culture medium containing 10%
LPDS. After 24 hours, cells were labeled with [NBC-C]-LDL and treated as
previously described (Coppens et al.,
2000). For cytochemical staining of ß-hydroxysterols with
filipin, infected cells were incubated in culture medium containing 10% FBS,
fixed in paraformaldehyde before incubation with filipin and viewed by
fluorescence microscope as previously described
(Coppens et al., 2000
).
In vivo assay
All animals were treated and handled according to institutional established
guidelines. A set of 10 Swiss-Webster mice (8-10 weeks old) in each group was
injected intraperitoneally with 105 parasites of ROP2AS-1,
ROP2AS-7, ROP2AS-20 and RH parasites. Survival of mice was monitored
twice daily.
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Results |
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|
|
Light microscopy indicated that the morphology of ROP2-deficient parasites
is atypical in comparison to the characteristic banana shape of their
wild-type (WT) counterparts. When host cells were infected, a significant
number (>60%) of aborted parasite vacuoles were detected that deviated from
the typical rosettes of WT parasites (data not shown). A portion (up to 30%)
of the vacuoles appeared morphologically normal at the light microscopic
level. Transmission electron microscopy (TEM) confirmed that aborted parasite
vacuoles contained grossly enlarged parasites bearing multiple nuclei (2-7)
that were arrested in the late stages of cell division
(Fig. 2A,B)
(Hager et al., 1999). The
daughter cells were formed internally and released from the mother cell, but
the assembly of the cytokinetic furrow to segregate the maturing cells
appeared to be inhibited, as indicated by a large number of empty vesicles
aligning at the base of the cytokinetic plane
(Fig. 2A,B).
|
Immunofluorescence microscopy of extracellular (Fig. 2K-R) and intracellular (data not shown) parasites demonstrated both an alteration in the pattern (Fig. 2N-R) and a decrease in intensity (Fig. 2M,O-R) of ROP2 fluorescent labeling in the majority (>80%) of parasites. In some cases, however, the pattern and intensity of ROP2 staining in the antisense clone was not altered in comparison to WT RH (compare panels K and L). These data indicate that ROP2 expression can be specifically abrogated by an antisense RNA strategy, although there is phenotypic heterogeneity in the antisense clones.
|
Toxoplasma has differential mechanisms of sorting and secreting
secretory proteins from the three regulated secretory organelles, i.e.
micronemes, rhoptries and dense granules
(Ngô et al., 2000).
Whereas ROP2-deficient parasites exhibited no morphological defects in the
biogenesis of dense granules and micronemes as assessed by qualitative
immunofluorescence microscopy and electron microscopy (data not shown), the
formation of mature rhoptries was specifically abrogated in greater than 80%
of the parasites. In WT parasites, flask-shaped rhoptries were assembled and
typically docked in lateral alignment near the apical pole of the cell (data
not shown). Depleting the constitutive expression of the putative
transmembrane protein ROP2 generated several notable rhoptry phenotypes
(Fig.
2,Fig. 2). The
extremity of phenotypes correlates with the level of ROP2 depletion, hence
ROP2AS-7 and ROP2AS-20 exhibiting the more striking changes
than ROP2AS-1 and ROP2AS-10.
In all ROP2AS clones examined, most rhoptries were no longer
aligned in linear fashion and were not restricted to the apical half of the
parasite (Fig.
2A,K-N).
There was a qualitative reduction in rhoptry cellular density, albeit the
quantification of organelle number per cell was hindered by the incomplete
segregation of parasites. The categorical club shape of mature rhoptries was
not formed, or maintained (Fig.
2C-F,H-J
and legends). In parasites with the higher level of ROP2 depletion
(ROPAS-7 and ROPAS-20), rhoptries were infrequently observed
and they exhibited an irregular shaped basal portion, and the distal neck was
aberrant from the typical homogenous electron dense lumen. Parasites
exhibiting a lower level of ROP2 reduction (ROPAS-1 and
ROPAS-20) contained more rhoptry-like tubules but they were
incompletely segregated and appeared as connected clusters
(Fig. 2H-I). Immunolabelling by
cryoelectron microscopy confirmed that these clusters contained ROP2/3/4 as
detected by mAb T34A7 (Fig.
2H-J) and cathepsin B (data not shown). Cathepsin B (TgCP1) is a
cysteine protease that is specifically localized to the lumenal core of the
rhoptry basal portion (Que et al.,
2002). All of these morphologic abnormalities in rhoptry shape
were interspersed, even within parasites in the same vacuole, with rhoptries
which were morphologically normal (Fig.
2G). Two major conclusions derive from these observations. First,
ROP2 appears to be an important determinant in the normal formation of mature
rhoptries. Second, parasites tolerate reduction of ROP2 expression levels only
to a point, which is on the cusp between formation of morphologically normal
and abnormal rhoptries.
To evaluate the hypothesis that secreted ROP2 mediates the tight
association between host mitochondria and the PVM
(Sinai et al., 1997; Sinai et
al., 2001), we determined the association with host cell mitochondria
qualitatively by mitotracker staining (Fig.
3A,B) and quantitatively by morphometric TEM
(Fig. 3C-E). The association
between host cell mitochondria and the PVM was significantly reduced in
ROP2-deficient parasites in both analyses; most notable was a 90% reduction in
the linear density of PVM membrane that was tethered to host mitochondria
(Fig. 3E). The association of
vacuolar membrane with endoplasmic reticulum appeared qualitatively
unaffected. These results suggest that ROP2 is necessary and may be sufficient
for mitochondrial association. Such association could potentially facilitate
the salvage from the host cells of lipids for which the parasite is
auxotrophic (Foussard et al.,
1991
).
|
Cholesterol is concentrated in parasite rhoptries, as determined by both
biochemical and morphological data
(Foussard et al., 1991;
Coppens et al., 2000
). We
tested the hypothesis that the rhoptries may be involved in the trafficking or
storage of lipids from the host cell. Cholesterol content, as detected by
filipin, was lower in the ROP2-deficient parasites as compared with WT
parasites (Fig. 4, compare
panels A-A' with B-B').
|
We have recently demonstrated that the parasite can efficiently access
cholesterol from host lysosomal compartments by an active mechanism, which is
independent of vesicular fusion, and requires parasite viability
(Coppens et al., 2000).
Incubation of infected fibroblasts with fluorescent cholesterol
(NBD-C,22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)23,24-bisnor-5-cholen-3ß)-ol)
incorporated into low density lipoproteins (LDL) led to a lower uptake of
NBD-C into ROP2AS parasites (Fig.
4, compare panels C-C' with D-D'). Hence, lowering ROP2 levels
results in aberrant incorporation of cholesterol into rhoptries.
Antisense ROP2 clones were markedly impaired in cell invasion. To monitor invasion, the number of vacuoles established after infection was determined in three ROP2-deficient clones (ROP2AS-1, ROP2AS-7 and ROP2AS-20). Compared to control parasites, the ROP2-deficient clones produced 6-13-fold fewer vacuoles (Fig. 5A), indicating that antisense clones are not able to efficiently invade human cells. Once intracellular, all the ROP2 antisense clones, in comparison to control parasites, exhibited between 50-90% reduction in their ability to take up [3H] uracil after 24 hours postinfection (Fig. 5B). Although uracil incorporation taken over time is a more accurate measurement of parasite replication, the limited quantities of ROP2-depleted parasites (see below) coupled with the problem of host cell lysis by parasites precluded these experiments. To discriminate between reduced proliferation of intracellular parasites and diminished ability to invade host cell, we monitored the number and size of the vacuoles. In the ROP2AS-7 and ROP2AS-20 clones, the majority of the vacuoles had between 4-8 parasites compared to 16 parasites in the WT (Fig. 5C). These values correspond to 14-21 hours doubling times for the antisense clones, in comparison to approximately 7 hours for WT parasites. Hence, ROP2 deficiency not only compromised invasion of host cells by T. gondii but also its intracellular replication.
|
To test the effect of the ROP2-deficiency in vivo, naïve mice were challenged with ROP2AS parasites and survival was monitored for two weeks. These parasites are derived from the highly virulent parental RH strain (LD50=1). All the mice in the control died by the sixth day after challenge, whereas in ROP2AS-1 and ROP2AS-20 groups there were no survivors at 8 days (Fig. 5D). In contrast, a third of mice survived a challenge with ROP2AS-7 by day 15 after infection. These data indicate that ROP2 deficiency attenuates the virulence of T. gondii in mice.
ROP2 expression gradually recovered in the ROP2AS clones, generally within 3-4 weeks (Fig. 6). Normalization of ROP2 levels correlated with restoration of the normal rhoptry morphology, organelle association, cholesterol uptake and virulence in mice. These data further confirm the association between low ROP2 levels and the phenotypes illustrated in Figs 2, 2, 3, 4, 5. This observation is also the probable explanation for variability in mouse virulence, and the capacity of even the ROP2AS-7 clone to cause a lethal in vivo infection (Fig. 5D).
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Discussion |
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We have recently demonstrated that a tyrosine-based motif (YXX) in the
cytoplasmic tail of ROP2 mediates its faithful targeting to mature rhoptries
most probably by interacting with the µ1 chain of the AP-1 adaptor complex
(Hoppe et al., 2000
). Dominant
negative interference with the tyrosine-binding pocket of µ1 adaptin
abolished its binding to transmembrane rhoptry proteins ROP2 and ROP4, and
disrupted the steady state formation of mature rhoptries
(Ngô, et al., 2003
).
Ablation of µ1 adaptin expression by antisense RNA altered rhoptry
biogenesis and was detrimental to parasite survival. In contrast, deletion of
the gene for the soluble protein ROP1 altered the honeycombed architecture of
the rhoptry lumen in the basal portion, but not the defined flask shape and
formation of rhoptries (Soldati et al.,
1995
). In Plasmodium, gene knockout of the rhoptry
protein RAP1, which forms a hetero-complex with soluble RAP2 and RAP3,
inhibits the delivery of the latter two cargo proteins to rhoptries
(Baldi et al., 2000
).
Similarly, the transmembrane microneme protein MIC6 in Toxoplasma is
proposed to complex with soluble MIC2 and MIC4 and is required for the
delivery of these soluble proteins to micronemes
(Reiss et al., 2001
). Although
we have no evidence to implicate ROP2 in hetero-complex formation, it is
conceivable that other rhoptry proteins can associate with the transmembrane
ROP2 to mediate the formation of mature rhoptries. Whether ROP2 functions
independently or in conjunction with other proteins, it is intriguing that
disruption of a transmembrane cargo interferes with the maturation of a
regulated secretory organelle. These data indicate that ROP2 is an essential
determinant of rhoptry shape and biogenesis.
Antisense RNA-mediated depletion of either ROP2 transmembrane cargo (this
study) or µ1 adaptin (Ngô et al.,
2003) disrupts normal rhoptry formation and consequently causes
defects in cell division and growth. In contrast to parasites expressing ROP2
antisense RNA, the TGN and endosomal vacuoles are drastically distorted in
AP-1-depleted parasites, but the rhoptries maintain their club shape. Although
both manipulations generated aborted parasite vacuoles, the specific block in
parasite division is distinct. AP-1-depleted parasites complete the formation
of cortical membrane complex along the cytokinetic furrow, but the daughter
cells are unable to segregate. Toxoplasma acquires cholesterols from
the host cell by an endocytosis pathway mediated in part by the endosomal
GTPase rab5 (Robibaro et al.,
2002
) and cholesterols are eventually trafficked to the rhoptries
for secretion. Deficiency of cholesterol uptake in ROP2-depleted parasites may
explain the lack of lipid materials needed for the synthesis of plasma
membrane and cortical cisternae to complete cytokinesis. It remains to be
determined whether a critical rhoptry component (e.g. protease) that is
required for unzipping the daughter cell surfaces is missorted in
AP-1-depleted parasites. Nevertheless, the antisense effects appear to be
specific to functions of ROP2 and AP-1 adaptin.
It is important to note that in our experiments, ROP2AS clones in
early passage were used. At this point, the diminution of ROP2 expression is
very pronounced with the accrual of multiple phenotypes. Although the
phenotype is variable, with only approximately 60% of the parasites displaying
pronounced morphologic defects at the light microscopic level, all
abnormalities observed are probably ascribable to an alteration in ROP2
expression. Supporting this assertion, when parasites were maintained in
continuous passage for 3-4 weeks, ROP2 levels were restored leading to the
recovery of defects in growth, morphology, association of host mitochondria
with the PVM, and mouse virulence (Fig.
6). Hence, it is highly probable that recovery of ROP2 expression
occurred after ROP2AS clones were inoculated into mice, explaining
the variable and modest alteration in parasite virulence. Because the
antisense strategy inherently does not usually generate complete inhibition or
complete loss of function of a gene, it is not suitable for the creation of
null mutants (Stuart and Wold,
1985; van der Krol et al.,
1988
; Liu et al.,
1994
; Shi et al.,
2000
). In the case of ROP2AS, a clonal population
expressing a basal level of ROP2 is probably favored for survival. Whether the
eventual recovery of ROP2 expression involves an RNA suppressor mechanism or
degradation of antisense RNA is not known, but loss of antisense RNA
inhibitory effects is in concordance with the data in Leishmania and
trypanosomes (Zhang and Matlashewski,
1997
; Ngô et al.,
1998
). These data further underscore the utility of an antisense
RNA approach to study essential gene function, as deletion of a critical gene
produces a lethal phenotype.
ROP2 is expressed at all invasive stages of the parasite (tachyzoite,
bradyzoite and sporozoite) potentially reflecting its essential role in
parasite biology. In addition, ROP2 and its family are potent humoral and
T-cell antigens (Saavedra et al.,
1996; Jacquet et al.,
1999
). It also carries B-cell epitopes because antibodies against
the ROP2 antigen are present in more than 85% of T.
gondii-seropositive individuals (Van
Gelder et al., 1993
). These findings, coupled with the essential
nature of ROP2 in mediating pathogen-host cell interaction, infectivity and
virulence validate this protein as a potential vaccine candidate. It is
probable that rhoptry function is conserved in all related coccidian parasites
raising the intriguing prospects that these organelles may serve as novel drug
targets. The data presented herein may thus open up new avenues for
chemotherapeutic and immunologic intervention against these devastating
parasitic diseases.
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Acknowledgments |
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Footnotes |
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