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INTRODUCTION |
Vacuolar-type H+-translocating inorganic
pyrophosphatases (V-PPases)1
are primary electrogenic proton pumps that derive their energy from the
hydrolysis of inorganic pyrophosphate (PPi) (1). Long considered to be restricted to plants and certain photosynthetic bacteria, V-PPases have recently been identified in a wide range of
organisms, including prokaryotic extremophiles and the kinetoplastid protists Trypanosoma and Leishmania, the
causative agents of Chagas' disease and leishmaniasis, and the
apicomplexan protists Plasmodium and Toxoplasma,
the causative agents of malaria and toxoplasmosis (reviewed in Ref. 2).
The discovery of V-PPases in these parasitic protists has attracted
much attention. The seemingly complete absence of V-PPases from their
animal hosts has given rise to the exciting possibility that this
enzyme might serve as an effective drug target for a number of the
diseases caused by these pathogens.
All characterized V-PPases are constituted of a single 75-82-kDa
intrinsic membrane protein species that is now known to fall into two
structurally and functionally distinct types, I and II. Type I
V-PPases, as exemplified by the molecular prototype, AVP1 from
Arabidopsis (3), exhibit a near obligate requirement for millimolar K+ for activity. Type II V-PPases, as
exemplified by Arabidopsis AVP2 (4), by contrast, share only
~36% sequence identity with their type I counterparts and are
insensitive to K+.
A property of all V-PPases characterized to date, regardless of whether
they are type I or type II enzymes, which distinguishes them from
soluble PPases, is their high sensitivity to competitive inhibition by
the 1,1-diphosphonate, aminomethylenediphosphonate (AMDP), and their
relative insensitivity to irreversible inhibition by fluoride (5, 6).
Originally identified in screens for PPi analogs capable of
functionally distinguishing V-PPases from soluble PPases and other
phosphohydrolases, diphosphonates such as AMDP that contain a
heteroatom (NH2 or OH) on the bridge carbon are exquisitely
potent V-PPase inhibitors (5). Taking their cue from the finding that
protozoal V-PPases are as sensitive to inhibition by AMDP as their
plant and photosynthetic bacterial counterparts, several investigators
have shown that this compound inhibits the growth of Plasmodium
falciparum (7), Toxoplasma gondii (8), and
Trypanosoma cruzi (9). Although in all cases the apparent
efficacy of AMDP in vivo is markedly lower than its efficacy
in vitro, the potential for the development of derivatives of this compound or alternative V-PPase-specific agents for drug purposes is nevertheless evident.
Those plant V-PPases that have been characterized in detail are found
primarily in vacuolar and Golgi membranes, where their activity
contributes to the transmembrane H+ gradient that drives
H+- and/or electrically coupled secondary transport
processes (1). By analogy, parallel biochemical and immunological
investigations of the V-PPases of trypanosomatid and apicomplexan
protists indicate that they are most closely associated with a
vacuole-like organelle, the acidocalcisome (8, 10-13). Acidocalcisomes
are small, electron-dense vacuolysosome-like acidic compartments,
replete with polyphosphates complexed with Ca2+,
Mg2+, and other mineral ions, that are suspected to play a
dominant role in Ca2+ storage and signaling (reviewed in
Ref. 14). The recent demonstration of V-PPases in the membranes
bounding the contractile vacuoles of Chlamydomonas (15) and
Dictyostelium (16) and the remarkable equivalence of the
composition of these organelles with acidocalcisomes may also be
pertinent to these considerations. Having made this point, it should be
stressed that the association of V-PPases with acidocalcisome-like
membranes does not preclude their association with other membranes. As
indicated by the results of a number of studies of trypanosomatids and
apicomplexans, V-PPase-like immunoreactivity is also to be found in the
plasma membrane (7, 8, 12, 13, 17) and Golgi system (17), at least
under some circumstances.
Several publications have described the preliminary in vitro
biochemical characterization of PPase activities associated with membranes prepared from trypanosomatid and apicomplexan protists (reviewed in Ref. 14). However, the molecular basis for these activities has been less well defined. Only a single type I V-PPase gene has been isolated from T. cruzi (18), and in the
apicomplexa, although genes for both type I and type II enzymes have
been cloned from P. falciparum, PfVP1 and
PfVP2, respectively, all attempts to elucidate the
functional properties of these gene products by heterologous expression
have been unsuccessful (7).
In the following we report the isolation and characterization of a type
I V-PPase, TgVP1, from the apicomplexan protist, T. gondii, and rigorous analyses of the subcellular localization of
the enzyme and of the effects of the V-PPase inhibitor AMDP on parasite
morphology both during and after host cell invasion. We demonstrate
that, upon heterologous expression in yeast, TgVP1 encodes
an intrinsic membrane protein competent in
PPi-dependent H+ transport that is
unique among V-PPases in containing an N-terminal signal sequence
sufficient for targeting proteins to the secretory pathway in T. gondii. Furthermore, using affinity-purified V-PPase-specific antibodies, we demonstrate a dynamic pattern of distribution of the
V-PPase in invading parasites. Under most conditions,
immunofluorescence microscopy of the V-PPase reveals a punctate apical
distribution. However, during invasion of the host cell, this
immunofluorescence undergoes a dramatic redistribution to assume a
collar-like structure at the periphery of the parasite that migrates in
synchrony with the penetration furrow as the parasite enters the host
cell. Given this association of the V-PPase with the invasion
apparatus, it is perhaps surprising that application of even high doses
of AMDP to invading parasites has no significant effect on their
establishment in the host cell, despite the facility with which lower
doses impair intracellular parasite division. These results demonstrate the effects of AMDP at doses much lower than those reported previously and suggest a function for acidocalcisomes in host cell invasion in
T. gondii.
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MATERIALS AND METHODS |
Host Cells and Parasites--
T. gondii RH strain
tachyzoites were maintained by serial passage in primary human foreskin
fibroblast (HFF) cultures grown at 5% CO2 in
bicarbonate-buffered Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% heat-inactivated newborn bovine serum (HyClone)
and an antibiotic mixture of penicillin, streptomycin, and gentamycin
as described (19). The culture medium was replaced with modified
Eagle's medium containing 1% dialyzed fetal bovine serum (Invitrogen)
immediately before infecting the fibroblasts with parasites.
Cloning Reagents--
The PCR primers used for cloning, plasmid
construction, RT-PCR, and 5'-RACE are listed in Table
I. The T. gondii cDNA
pools were prepared from and the 5'-RACE reactions were performed on total RNA and poly(A) mRNA purified from freshly lysed tachyzoites using a SMART RACE Kit (Clontech). DNA sequencing
was by dye terminator chemistry using nested oligonucleotide
primers.
Isolation of Genomic and cDNA Clones of TgVP1--
The
genomic and cDNA clones of TgVP1 were isolated by a
combination of PCR and standard oligonucleotide hybridization screens of genomic and cDNA libraries. Initially, degenerate primers
corresponding to the conserved "universal" V-PPase sequences
DNAGGIAE and WDNAKKYI (primers "Universal1" and "Universal2" in
Table I) were used in PCRs in which RH genomic DNA or tachyzoite
cDNA pools were used as templates. The resulting unique 1028-bp
genomic and 593-bp cDNA PCR products were cloned into pGEM-T vector
(Promega), sequenced, and used as probes for subsequent parallel
screens of an RH genomic library constructed in DASH-II (Stratagene, La
Jolla, CA) and a tachyzoite cDNA library (from the AIDS Reference
and Reagent Repository, National Institutes of Health, Bethesda).
The longest cDNA clone isolated in the hybridization screens was
1989 bp in length and, by comparison with the published sequences of
V-PPases from other sources, appeared to lack the coding sequence for
the first ~240 amino acid residues of the mature polypeptide. The
largest of three overlapping clones from the hybridization screens of
the genomic library yielded two restriction fragments after
EcoRI-HindIII digestion, one of 6.2 and another
of 3.4 kb, that hybridized at high stringency in Southern analyses with
the 1989-bp partial cDNA sequence. These restriction fragments were cloned into the EcoRI-HindIII sites of Bluescript
plasmid pKS+ (Stratagene) and sequenced. In order to deduce
the translation start site of TgVP1 from its genomic
sequence, several 5'-RACE and nested RT-PCRs were performed using the
tachyzoite cDNA pools as template, and the primer combinations
listed in Table I.
Plasmid Construction--
The full-length TgVP1
cDNA (GenBankTM accession number AF320281) was cloned
by RT-PCR of T. gondii tachyzoite cDNA pools using
Pfu DNA polymerase (Stratagene) and sense and antisense primers TgVP.1 and TgVP.3'Y (Table I) corresponding to positions 1-20
and 2430-2451, respectively, of the ORF predicted from the TgVP1 genomic sequence (GenBankTM accession
number AF320282). After digestion with XbaI the resulting
2451-bp PCR product was ligated into the multicloning site of
PvuII-XbaI double-digested yeast expression
vector pYES2 (Invitrogen) to generate pYMD23. Yeast expression vectors
encoding truncated versions of TpVP1 were generated by PCR
amplification of TgVP1 from pYMD23 using Pfu DNA
polymerase and sense primers corresponding to the sequence immediately
downstream of the predicted signal peptide (residues 121-138 of the
TgVP1 cDNA; primer TgVP1.2, Table I) to generate pYMD24,
and with sense primers corresponding to the sequence further downstream
of this (residues 223-240; primer TgVP1.3, Table I) to generate pYMD25.
The T. gondii fluorescent protein and overexpression
plasmids used in this study were constructed in
ptubP30-YFP/sag-chloramphenicol acetyltransferase vector
(20), in which a BglII site separates the 5'-untranslated
region of
-tubulin (21) from the P30 signal sequence (22), and an
AvrII site separates the P30 sequence from the yellow
fluorescent protein-coding sequence (YFP, a derivative of
Aequoria victoria green fluorescent protein, GFP). TgVP1
coding sequences were cloned as BamHI-XbaI
fragments into BglII
(BamHI-compatible)-AvrII (XbaI-compatible) double-digested vector. For the
construction of plasmids pYMD26-27, which contained coding sequences
corresponding to the first 84 and 232 amino acid residues of TgVP1
in-frame with the YFP sequence, the TgVP1 coding sequences
were amplified by PCR from YMD23 using the TgVP5'T sense primer in
combination with the N-term.1 and N-term.2 antisense primers (Table I).
Plasmid pYMD28 containing the full-length TgVP1 cDNA
fused to YFP was similarly generated by PCR using primers TgVP5'T and
TgVP3'T (Table I). Plasmids pYMD31 and pYMD32, encoding
D550N-substituted TgVP1, were constructed from T. gondii and
yeast expression plasmids pYMD28 and pYMD25, respectively, using the
QuickChange Site-directed Mutagenesis Kit (Stratagene) and the
mutagenic primer "D550N" (Table I).
T. gondii expression plasmids pYMD29-30 and pYMD33-34 were
engineered to contain a stop codon upstream of YFP/GFP in the
expression vector. Plasmids pYMD29 and -33 were generated by PCR from
pYMD23 and pYMD31, respectively, using primers TgVP5'T and TgVP.3'Y
(Table I). Plasmids pYMD30 and -34 were generated by ligation of the BamHI-XbaI fragments from pYMD25 and pYMD32,
respectively, to BglII-AvrII double-digested vector.
Plasmid pYES2-AVP1, containing the coding sequence of AVP1,
was constructed as described (23).
Heterologous Expression in Yeast--
Vacuolar
protease-deficient Saccharomyces cerevisiae strain BJ5459
(Mat
, ura3-52, trp1, lys2-801, leu2
1, his3-
200,
pep4::HIS3, prb
1.6R, can1, GAL)
transformants containing pYMD23-25, pYMD32, or pYES2-AVP1 were
generated by the LiOAc/PEG method, selected for uracil prototrophy, and
subjected to membrane fractionation as described (23, 24).
Antibody Purification--
Polyclonal V-PPase antisera
PABTK (324), PABHK (326), and their respective
preimmune sera were affinity-purified against the AVP1 polypeptide of
vacuolar membrane-enriched vesicles purified from
pYES2-AVP1-transformed yeast BJ5459 cells. Membrane protein (100 µg)
was separated and electroblotted as described below, and the band
corresponding to the PABTK-reactive 81-kDa AVP1 polypeptide was cut from the blot and blocked overnight in PBS + 3% BSA. After a
brief wash in PBS containing 0.1% BSA, the excised strips of nitrocellulose filter were placed in a 1:4 dilution of serum in PBS + 3% BSA and incubated for 16 h at 4 °C with shaking. Upon completion of the incubations, the blots were washed three times with
TBS + 0.1% BSA, twice with TBS + 0.1% BSA + 0.1% Nonidet P-40, and 3 more times with TBS + 0.1% BSA, before elution of the antibodies into
150 µl of 0.2 M glycine HCl buffer (pH 2.5) and immediate
neutralization of the eluate with 75 µl ice-cold 1 M
K4PO4 (pH 9.0) containing 3% BSA. Elution and
neutralization were repeated three more times, and the eluates were
pooled and desalted with PBS in a Centricon-30 filtration unit
(Millipore Corp.). The final antibody preparations were stored in PBS
containing 0.05% (w/v) NaN3 at 4 °C.
Measurement of PPi Hydrolysis and H+
Translocation--
PPi hydrolytic activity was assayed as
described (24) except that imidazole-based, rather than Tris- or
BisTris-propane-based, buffers were used throughout to preclude
competition with K+ and other monovalent cations (4). PPase
activities are expressed as µmol of PPi hydrolyzed/mg of
membrane protein/min (= µmol/mg/min). PPi-dependent intravesicular acidification was
monitored fluorimetrically using acridine orange (2.5 µM)
as indicator as described (4).
Parasite Transfection--
The parasites were transfected by
electroporation as described (19). Briefly, 107 purified
parasites were resuspended in Cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM
K2HPO4/KH2PO4, 25 mM Hepes, 2 mM EDTA, 5 mM
MgCl2, 2 mM ATP, and 5 mM
glutathione (pH 7.6)) containing 50 µg of sterilized plasmid DNA.
Electroporation was performed in a 2-mm gap cuvette with a 1.5 kEV
pulse at a resistance setting of 24 ohms using a BTX 600 electroporation system. The electroporated parasites were used to
infect HFF cells grown on coverslips.
Western Analyses--
For Western analysis, vacuolar
membrane-enriched vesicles purified from the yeast transformants or
different membrane fractions from T. gondii were subjected
to denaturation, SDS-PAGE on 10% (w/v) acrylamide gels,
electrotransfer, and immunoreaction with antibodies PABHK
or PABTK, as described (24). Immunoreactive bands were
visualized by ECL (Amersham Biosciences).
Protein Assays--
Protein was estimated by the method of
Bradford (25).
Light Microscopy--
For light microscopy, HFF cells were grown
to confluence on sterilized coverslips in 6-well plates. The confluent
cultures were infected with 5 × 105 parasites and
examined at the times indicated. For the visualization of native
fluorescent proteins, the coverslips were mounted in PBS. For the
immunofluorescence analyses, parasite-infected cells were fixed in 3%
paraformaldehyde and permeabilized with 0.25% Triton X-100 in PBS. The
purified anti-V-PPase sera were used at dilutions of 1:200, and
immunoreaction was detected using FITC-conjugated anti-rabbit
immunoglobulin (1:1000) (Molecular Probes, Inc.). The anti-MIC3
antibodies (26) in the double-immunolabeling experiments were used at a
dilution of 1:1000 and detected using Texas Red-conjugated anti-mouse
immunoglobulin (1:1000) (Molecular Probes, Inc.).
Cells expressing YFP fusion proteins were examined using an Axiovert
microscope (Carl Zeiss, Inc.), equipped with a single emission filter
(505-555 nm; Chroma) and a specific YFP filter (480-495 nm). Cells
that had been reacted with antibodies raised against the V-PPase and/or
MIC3 were examined using FITC (450-480 nm excitation/515-565-nm
emission) and Texas Red (563-585-nm excitation/615-nm-long pass
emission) filter sets, respectively. The images were collected using an
interline chip cooled Orca 9545 CCD camera (Hamamatsu).
Electron Microscopy--
For electron microscopy, infected cells
were fixed in situ with a freshly prepared mixture
containing 1% glutaraldehyde (made from an 8% stock; Electron
Microscopy Sciences, Fort Washington, PA) and 1% osmium tetroxide in
50 mM phosphate buffer (pH 6.2). After adding the fixative
at room temperature, the specimens were incubated at 4 °C for 45 min. The samples were rinsed with distilled water to remove excess
phosphate before being gently scraped off the Petri dishes with a
beveled scraper. Staining was with 0.5% aqueous uranyl acetate for
6-16 h at 4 °C. The samples were dehydrated with acetone and
embedded in an Epon-Araldite resin mixture. Ultrathin (50-70-nm thick)
sections were cut and stained with uranyl acetate and lead citrate and
examined using a Philips 200 electron microscope.
Assays of Parasite AMDP Sensitivity--
The effects of AMDP on
parasite growth were examined in HFF cells grown on coverslips and
infected with tachyzoites at a concentration of 105
parasites per ml. The parasites were allowed to invade for 5 min at
37 °C before aspirating the medium and replacing it with fresh
medium to remove all free parasites. AMDP was added to the medium at
the concentrations indicated after allowing 20-30 min at 37 °C for
the parasites to become established in the host cells. The cultures
were incubated in a humidified atmosphere containing 5%
CO2 for 24 h at 37 °C, after which time the
infected cells were fixed on coverslips in methanol at
20 °C. The
infected cells remaining in the dishes were processed for electron
microscopy as described above.
Parasite replication was assessed by counting the numbers of parasites
per parasitophorous vacuole by direct visualization using a Zeiss
microscope with phase objectives. To ensure random counting, fields
from all regions of the coverslip were counted without prior
microscopic examination, and all vacuoles within each field were
counted. In all experiments the numbers of parasites per vacuole were
determined for between 500 and 700 vacuoles from at least five separate
experiments. To examine the effects of AMDP on host cell invasion,
freshly emerged tachyzoites were incubated with AMDP for 5-10 min
prior to infection. Infected cells were incubated and counted as
described above.
Computer Programs--
For measurements of the susceptibility of
the V-PPase to inhibition by Ca2+, the concentration of
free Ca2+ ([Ca2+]free) was
estimated by substitution of the appropriate stability constants into
the SOLCON program (a kind gift from Dr. Yale Goldman, Department
of Physiology, University of Pennsylvania). The stability constants
were obtained from Martell and Smith (27) and Smith and Martell (28)
and deployed as described (29). Sequences were aligned using ClustalW
1.7 (30). The putative membrane topology of TgVP1 was modeled using
TopPred II, version 1.3 (31), as described for AVP1 (24), PVP (32), and
AVP2 (4).
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RESULTS |
Isolation and Sequence Characteristics of TgVP1--
To screen for
genes encoding V-PPases in T. gondii, degenerate primers
corresponding to the sequences, DNAGGIAE and WDNAKKYI (positions
511-518 and 694-701 in AVP1) (32), conserved among all known V-PPases
(2), were used as primers for PCR amplification of strain RH genomic
DNA and tachyzoite cDNA pools. In so doing a unique 1028-bp genomic
product and unique 593-bp cDNA product were isolated, each of which
was cloned, sequenced, and determined to be capable of encoding a
V-PPase. Both isolates were used as probes for hybridization screens of
the RH genomic and tachyzoite cDNA libraries. Because the cDNA
library screens yielded only truncated and incompletely spliced
cDNAs but the screens of the RH genomic library yielded three
distinct but overlapping clones, the complete sequence of this gene
(GenBankTM accession number AF320281) was derived
ultimately from detailed analyses of a 6985-bp portion of the longest
of the three genomic clones isolated (Fig.
1).

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Fig. 1.
Genomic organization and cDNA sequence of
TgVP1 and deduced amino acid sequence of TgVP1.
The relative positions of introns and exons in the genomic sequence are
indicated by black and shaded boxes,
respectively. Also shown is the Kozak translation initiation sequence
(TCCRTCATGG) (33). The positions of putative transmembrane spans
identified using the TopPred II program (31) are indicated by
lines above the peptide sequence. The N-terminal extension
is indicated as white text on a black background
and the likely signal cleavage site, predicted by SignalP (37), by an
arrow. Conserved residues and motifs of mechanistic
significance that are referred to in the main body of the text are
indicated by black text on a gray
background.
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Given that successive rounds of 5'-RACE and RT-PCR consistently failed
to indicate an in-frame initiator codon 5' of the ATG at position 1350 of the genomic sequence (a site encompassed by a sequence similar to
the consensus Kozak translation initiation sequence (TCCRTCATGG) (33)),
this site was inferred to serve as the start site for the expression of
this gene in tachyzoites (Fig. 1). Accordingly, RT-PCR of T. gondii total RNA and poly(A) mRNA using primers designed to
span the entire predicted ORF yielded a 2451-bp product encoding an
816-amino acid (85 kDa) polypeptide (Fig. 1). Designated TgVP1,
this deduced polypeptide is 50% sequence-identical (65% similar) to
the prototypical type I V-PPase from AVP1 (3) and 57% identical (72%
similar) to the type I V-PPase from P. falciparum (7)
but only 37 and 33% identical (53 and 50% similar) to the type II
enzymes from these organisms, respectively (4, 7).
All of the sequence motifs and residues known to be characteristic of
type I V-PPases are conserved in TgVP1 (Fig. 1). These include the
following: (i) the putative "universal PPase catalytic" motif
DX7KXE found in both soluble and
membrane-associated PPases (34); (ii) Cys677, corresponding
to Cys634 of AVP1, whose substrate-protectable alkylation
by maleimides irreversibly abolishes catalytic activity (35, 36); (iii) residues Glu345 and Asp550, corresponding to
Glu305 and Asp504 in AVP1, which contribute to
the susceptibility of the enzyme to inhibition by the hydrophobic
carbodiimide N,N'-dicyclohexylcarbodiimide (24); and (iv)
Glu473, corresponding to Glu427 of AVP1, which
has been inferred to participate directly in H+
translocation (24) but which is substituted by K in type II V-PPases
(2, 4). Of strategic significance is the conservation of the sequences
TKAADVGADL(VS)GK(IN)E and HK(AN)AV(IT)GDT(IV)GDPLK in TgVP1. Because
these are the sequences recognized by peptide-specific antibodies
PABTK and PABHK (23) deployed in all previous
investigations of V-PPases in protists, confirmation of their
conservation substantiates previous reports (8, 10) of
V-PPase-associated immunoreactivity in T. gondii membranes.
The overall membrane topology predicted for TgVP1 using the TopPred II
program (31) is similar to those predicted for AVP1 (24) and PfVP1 (7)
with the exception of an additional putative N-terminal transmembrane
span encompassing residues 14-34 (Fig. 1). Sequence alignments between
TgVP1, other type I V-PPases from higher plants, and PfVP1 indicate
that the additional N-terminal transmembrane span of TgVP1 resides
within a unique 74-residue N-terminal extension. Analyses of this
N-terminal extension using SignalP (37) and PSORTII (38-40) reveal a
putative signal cleavage site between residues 39 and 40 (Fig. 1).
The results of Southern analyses (data not shown) indicate that the
gene encoding TgVP1 exists as a single copy in the genome of T. gondii RH.
Heterologous Expression of TgVP1 in S. cerevisiae--
To
determine whether it encodes a V-PPase with properties similar to those
of AVP1, TgVP1 was heterologously expressed in yeast. For this purpose,
S. cerevisiae strain BJ5459 was transformed with vector
pYES2 containing the entire coding sequence of TgVP1 (pYMD23) or truncated forms of TgVP1 lacking the coding
sequences for either the 40-residue signal sequence (pYMD24) or the
entire 74-residue N-terminal extension (pYMD25). For comparative
purposes, yeast were also transformed with pYES2-AVP1 vector.
Expression of TgVP1, measured as the presence of a polypeptide of the
appropriate size and immunoreactivity in the vacuolar membrane-enriched
fraction from the yeast transformants, is contingent on deletion of the
coding sequence for the entire 74-residue N-terminal extension.
Vacuolar membrane-enriched vesicles purified from pYMD25-transformed (BJ5459/pYMD25) cells contain an intense PABTK (and
PABHK)-reactive Mr 75,000 band that
reacts with antibody with similar intensity to the corresponding band
in the equivalent membrane fraction from pYES-AVP1-transformed BJ5459
cells but which is absent from the corresponding fractions from pYMD23-
and pYMD24-transformed BJ5459 cells (Fig.
2).

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Fig. 2.
Western analysis of antibody
PABTK-reactive polypeptides in vacuolar membrane-enriched
vesicles purified from pYMD23-, pYMD24-, pYMD25-, or pYES2-AVP1
("AVP1")-transformed S. cerevisiae
BJ5459 cells. Plasmid pYMD23 contains the entire coding
sequence of TgVP1, inclusive of the N-terminal signal peptide; pYMD24
contains the coding sequence of TgVP1 minus the N-terminal
signal peptide (residues 1-41); and pYMD25 contains the coding
sequence of TgVP1 minus both the N-terminal signal peptide
and the remaining 33 residues of the N-terminal extension (residues
1-75). All lanes were loaded with 5 µg of membrane protein, and the
immunoreactive bands were visualized by ECL ("Materials and
Methods"). The immunoreactive bands shown were the only ones
detected.
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TgVP1-mediated PPi Hydrolysis and
PPi-dependent H+
Translocation--
The PABTK-reactive polypeptide detected
in the vacuolar membrane-enriched fraction from BJ5459/pYMD25 cells
catalyzes both PPi hydrolysis and
PPi-dependent H+-translocation.
When assayed in reaction buffer containing 250 µM NaF to
abolish any contribution from contaminating yeast soluble PPase (23),
the kinetics of TgVP1-catalyzed PPi hydrolysis are indistinguishable from those of AVP1. The Km values, PPi concentrations required for maximal activity, and
maximal activities of TgVP1 and AVP1 are 34 and 38 µM
total PPi, 0.3 mM total PPi, and
0.5-0.6 and 0.6-0.7 µmol/mg/min, respectively, when PPi
hydrolysis is assayed in reaction media containing 1.3 mM
Mg2+ (data not shown). As would be predicted for a type I
V-PPase, the hydrolytic activity of heterologously expressed TgVP1,
like AVP1, is K+-activated (Fig.
3). TgVP1-mediated PPi
hydrolysis is measurable in media containing choline chloride, albeit
at a low level, but replacement of this salt with KCl or potassium
gluconate increases activity by 2-3-fold. This behavior is similar to
that seen with heterologously expressed AVP1 except that in the latter
case K+ increases activity by about 8-fold
versus choline (Fig. 3). A similar pattern is seen with
PPi-dependent H+-translocation
except that the nature of the counter-anion is also important.
Substitution of the permeant anion Cl
with the less
permeant anion gluconate decreases the extent of both TgVP1- and
AVP1-mediated intravesicular acidification by at least 60%, whereas
substitution of K+ with choline decreases the extent of
intravesicular acidification by at least 90% (Fig. 3). Previous
investigations of both endogenous and heterologously expressed type I
V-PPases have established that the permeant anion Cl
maximizes PPi-dependent intravesicular
acidification by diminishing the magnitude and therefore the stalling
action of the inside-positive membrane potential that would otherwise
be generated by electrogenic H+ translocation (41).

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Fig. 3.
PPi-dependent
H+ translocation and PPi hydrolysis by vacuole
membrane-enriched fractions purified from pYES2-AVP1- and
pYMD25-transformed S. cerevisiae BJ5459 (AVP1 and
TgVP1, respectively) cells. Intravesicular acidification was
monitored with acridine orange in media containing membrane vesicles
(80 µg of membrane protein), MgSO4 (1.3 mM),
and 100 mM KCl, potassium gluconate (KGlu), or
choline chloride (ChCl). At the times indicated,
H+ translocation was initiated by the addition of
imidazole-PPi (0.3 mM), and the decrease in
fluorescence was measured against time. F% = percentage
change in fluorescence (F). PPi hydrolysis by
vacuolar membrane-enriched vesicles (3-5 µg of membrane protein) was
measured in reaction media (300 µl) containing 100 mM
concentrations of the monovalent cations indicated plus 1.3 mM MgSO4 ("Materials and Methods"). Values
shown are means + S.E. (n = 4).
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Sensitivity of TgVP1 to AMDP and Ca2+--
TgVP1 and
AVP1 are similarly sensitive to inhibition by AMDP (5) but
differentially sensitive to inhibition by Ca2+ (Fig.
4). Although the concentration
dependences for inhibition of TgVP1- and AVP1-mediated PPi
hydrolysis by AMDP superimpose to yield I50 values of 0.9 and 3.0 µM, respectively (Fig. 4A), TgVP1 is
more than 8-fold more sensitive to inhibition by free Ca2+
than AVP1 (Fig. 4B). A total concentration of 1.4 µM, equivalent to a free Ca2+ concentration
of 0.15 µM, is sufficient to inhibit TgVP1-mediated PPi hydrolysis by 50%, whereas concentrations of greater
than 70 and 1.2 µM, respectively, are required to inhibit
AVP1 to the same extent (Fig. 4B).

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Fig. 4.
Sensitivities of TgVP1- and AVP1-mediated
PPi hydrolysis to inhibition by AMDP (A)
or Ca2+ (B). PPi
hydrolysis was measured as described in Fig. 3 except that AMDP or
Ca2+ (CaCl2) were added at the concentrations
indicated. Free Ca2+ ([Ca2+]free)
was estimated using the SOLCON program as described (29). Values shown
are means ± S.E. (n = 3).
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TgVP1 N-terminal Signal Sequence--
To examine whether the
putative signal sequence encompassed by the N-terminal extension of
TgVP1 is capable of targeting peptides to the secretory pathway in
T. gondii, YFP fusion plasmids containing the coding
sequences for either the first N-terminal 84 amino acid residues
(plasmid pYMD26), which encompass the entire N-terminal signal sequence
and cleavage site, or the first 232 residues (pYMD27), which encompass
the N terminus, cleavage site, and first predicted transmembrane span
of the mature protein, or the entire coding region of TgVP1 inclusive
of the N-terminal extension (pYMD28) were constructed and transfected
into tachyzoites (Fig. 5).

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Fig. 5.
T. gondii
tachyzoites expressing TgVP1-YFP protein fusions. T. gondii tachyzoites were transiently transfected with expression
constructs containing the following: coding sequence for the first
N-terminal 84 amino acid residues of TgVP1 (plasmid pYMD26), which
encompass the entire N-terminal signal sequence and cleavage site; the
first 232 residues, encompassing the N terminus, cleavage site and
first predicted transmembrane span of the mature protein (plasmid
pYMD27); or the entire TgVP1 ORF (plasmid pYMD28). In all
cases the sequences encoded were C-terminally fused with YFP. The cells
were examined under an Axiovert microscope equipped with a single
emission filter and specific YFP filter ("Materials and
Methods").
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In all tachyzoites transfected with pYMD26, YFP fluorescence is
observed within dense granules and within the lumen of the parasitophorous vacuole (Fig. 5). In tachyzoites transfected with pYMD27, ~75% of the transfectants exhibit sequestration of the YFP
fluorescence in the endoplasmic reticulum, whereas the remaining 25%
exhibit a punctate fluorescence distribution indicative of the
incorporation of YFP into inclusion bodies (Fig. 5). In tachyzoites transfected with pYMD28, the cells divide only rarely and undergo extensive inflation of the vacuole (Fig. 5).
To determine whether the vacuolate, non-dividing phenotype of pYMD28
transfectants might be attributable to the YFP fusion, two constructs,
pYMD29 and pYMD30, that either contained or lacked the N-terminal
extension but contained a stop codon between the TgVP1 and YFP coding
sequences were engineered (Fig. 6). To
determine whether the YMD28 transfectant phenotype might be because of
PPase activity associated with the expression product, two of the
overexpression constructs, pYMD33 and pYMD34, were engineered to
contain a D550N substitution (Fig. 6). It has been established that the
same substitution at the equivalent position, residue 504, in AVP1
abolishes catalytic activity whether it is measured as PPi
hydrolysis or PPi-dependent H+
translocation (24). Analogous assays of heterologously expressed TgVP1
D550N (construct pYMD32) yield the same result (data not shown).
Expression of the TgVP1 sequences in the tachyzoite transfectants was
monitored in these experiments by indirect immunofluorescence microscopy using purified peptide-specific antibody PABTK
(Fig. 6).

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Fig. 6.
Immunofluorescence localization of TgVP1 in
transiently transfected T. gondii tachyzoites.
The tachyzoites were transiently transfected with expression constructs
containing the TgVP1 cDNA either with (plasmids pYMD29
or -33) or without the coding sequence for the N-terminal extension
(plasmids pYMD30 or -34) or containing the coding sequence for
D550N-mutated TgVP1 (plasmids pYMD33 or -34). The D550N substitution
yielded catalytically inactive enzyme. The transfected tachyzoites were
fixed, permeabilized, and reacted with purified anti-V-PPase serum
PABTK before reaction with the FITC-linked secondary
antibody and epifluorescence microscopy.
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Whereas removal of the YFP tag yields transfectants capable of division
at higher frequency, the extents and rates of division of these cells
are nevertheless markedly decreased. Moreover, whereas expression of
both of the full-length constructs (pYMD29 and -33) results in intense
immunostaining that appears to be membrane-associated (Fig. 6),
expression of the constructs lacking the N-terminal extension (pYMD30
and -34) results in diffuse immunostaining throughout the cell (Fig.
6). Similar experiments were performed using T. gondii
expression plasmid pDHFR-P30-GFP/sag-chloramphenicol acetyltransferase vector (42, 48), which contains the dihydrofolate reductase promoter sequence instead of tubulin, but these
yielded no observable expression. In no case, did expression of
non-functional, D550N-substituted TgVP1 (constructs pYMD33
and pYMD34) abrogate the highly vacuolated, low division
frequency phenotype of the transfectants (Fig. 6). Evidently the
N-terminal extension of TgVP1 contains a signal sequence sufficient for
directing transport to the secretory pathway of T. gondii, but overexpression of the coding sequence of TgVP1,
regardless of whether or not the translation product is catalytically
active, interferes with cell division and elicits increased vacuolation.
Subcellular Localization of Endogenous V-PPase--
By having
determined the disruptive effects of protein fusion and overexpression
techniques in this context, the subcellular localization of
TgVP1-related translation products was assessed by using the same
purified peptide-specific antibody, PABTK, as used in the
experiments presented in Fig. 6. However, as is apparent from the
immunofluorescence micrographs shown in Fig.
7, purification of this antibody before
use is crucial if the results are to be intelligible.
Immunofluorescence microscopy of both free and intracellular tachyzoites using crude preimmune and immune sera and FITC-conjugated secondary antibodies yields remarkably similar results, high intensity fluorescence throughout the tachyzoites (Fig. 7). In striking contrast,
when both antisera are affinity-purified against heterologously expressed AVP1 and the specificities of the purification products verified by Western analyses of membranes isolated from S. cerevisiae transformed with either pYES2-AVP1 or empty pYES2
vector, the results are very different. Whereas microscopy using
immunopurified preimmune serum discloses little or no
immunofluorescence with either intracellular or free tachyzoites,
incubation of the same preparations with immunopurified
PABTK clearly demonstrates a punctate anterior apical
distribution of the antigen (Fig. 7).

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Fig. 7.
Immunofluorescence detection of V-PPase in
intracellular tachyzoites. A, after reaction with
crude preimmune and PABTK sera. B, after
reaction with purified preimmune and PABTK sera. The
infected monolayers were fixed, permeabilized, and reacted with crude
or purified preimmune serum or V-PPase antiserum PABTK
before reaction with FITC-linked secondary antibody.
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Phase-dependent Changes in Subcellular
Distribution--
A striking property of the membrane system with
which the V-PPase is associated in the intact parasite is the degree to
which it is subject to dynamic phase-dependent changes in
organization. Deployment of the same immunopurified antibodies as those
used for free and intracellular tachyzoites for studies of trophozoites during host cell invasion reveals that most of the V-PPase-specific staining assumes a transverse radial distribution soon after the parasite has made contact with the host cell. A collar-like structure is generated that migrates along the length of the parasite in synchrony with and immediately anterior to the apicobasally propagating penetration furrow (Fig. 8). Upon
completion of infection, the V-PPase-associated fluorescence disperses
before reappearing again at the anterior apex of the intracellular
tachyzoite (Figs. 8 and 9). Although this
pattern is reminiscent of the dynamics of microneme protein
redistribution during invasion (43), colocalization experiments on
these and non-invading parasites using the microneme-specific antibody
raised against the secreted microneme protein MIC3 (26) in parallel
with reaction with the V-PPase-specific antibody, demonstrate
segregation, sometimes diametric segregation, of the two antigens from
each other (Fig. 9). MIC3 also propagates as a collar-like structure
along the length of the trophozoite during infection, but its
distribution is not coincident with that of the V-PPase immediately
before and after host cell invasion (Fig. 9).

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Fig. 8.
Immunofluorescence detection of V-PPase in
tachyzoites during their attachment to and infection of HFF cells.
Tachyzoites were added to HFF cells and incubated together for 1-10
min, before washing and removal of the extracellular parasites. The
infected monolayers were fixed, permeabilized, and reacted with
purified V-PPase antiserum PABTK before reaction with
FITC-linked secondary antibody. Shown are tachyzoites at different
stages (A-F) in host cell invasion. Throughout host cell
invasion the immunofluorescence associated with the V-PPase coincides
with the position of the penetration furrow (A-E); on
completion of host cell invasion (F) this immunofluorescence
assumes a punctate apical distribution.
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Fig. 9.
Immunofluorescence detection of V-PPase and
microneme antigen, MIC3, in tachyzoites during their attachment to and
invasion of HFF cells. A, after reaction with
purified PABTK antiserum and anti-rabbit FITC-conjugated
secondary antibody. B, after reaction with MIC3
antiserum and anti-mouse rhodamine-conjugated secondary antibody. HFF
cell invasion was initiated and examined as described in Fig. 8. Shown
are tachyzoites at various stages (a-d) in host cell
invasion. Throughout host cell invasion the immunofluorescence
associated with the V-PPase (A) and MIC3 (B)
coincides with the position of the invasion furrow (a-c);
on completion of host cell invasion (d) the V-PPase and MIC3
immunofluorescences segregate anteriorally and posteriorally,
respectively, in the tachyzoite.
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Effects of AMDP on Cell Invasion and Replication--
Knowing that
TgVP1 and AVP1, when heterologously expressed in yeast, are similarly
sensitive to inhibition by AMDP (Fig. 4) and that reaction of
V-PPase-specific antibody with trophozoites during host cell invasion
is with a collar-like structure that appears to be associated with the
penetration furrow (Fig. 8), we were interested to determine the
efficacy of this 1,1-diphosphonate as an inhibitor of cell invasion
and/or intracellular parasite replication.
The results of these screens were surprising in that AMDP impairs
intracellular parasite replication but exerts little or no effect on
host cell invasion. On the one hand, treatment of extracellular
trophozoites with AMDP at concentrations as high as 100 µM exerts no discernible effect on either the efficiency of host cell invasion or integration (data not shown). On the other
hand, AMDP concentrations as low as 5 µM are sufficient to interfere with intracellular parasite division. Treatment of infected cells with AMDP at concentrations of 5 µM or
greater decreases the number of parasites per vacuole after 24 h
concomitant with the appearance of irregular parasite masses (Fig.
10). Examination of parasites treated
with 10 µM AMDP for 24 h indicates that in many
cases daughter cell budding is stalled, leading to the appearance of
large irregularly shaped parasites (Fig.
11A). No obvious swelling of
the endoplasmic reticulum, nuclear envelope, or Golgi complex occurs,
and no large vacuolar spaces are seen in the parasites. Indeed, these
parasites contain a full array of normal secretory organelles
indicating little or no perturbation of the secretory pathway.
Furthermore, the parasite mitochondrion and apicoplast look similar to
control parasites. However, in a small number of the parasites
(~5%), certain vesicular structures reminiscent of the
acidocalcisomes described in trypanosomes (structures typified by the
loss of most of their luminal electron density upon double-fixation (14)) lose most of their electron density and undergo swelling and
disruption after exposure of the cells to AMDP (Fig.
11B).

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Fig. 10.
Effects of AMDP on the growth and
replication of intracellular T. gondii
tachyzoites. AMDP was added to the culture media after
allowing the parasites 20-30 min to become established in the host
cells. The number of parasites per parasitophorous vacuole was
determined by light microscopy after a further 24 h.
Abnormal parasites are defined as those that do not have a
normal elongated appearance and frequently consist of rounded or
irregularly shaped masses. Shown are the means ± S.E. from a
minimum of five separate experiments in each case.
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Fig. 11.
Effects of treating newly established
cultures with 10 µM AMDP for
24 h at 37 °C as determined by transmission electron
microscopy. A, AMDP stalled daughter cell budding
in some of the parasites, so eliciting the formation of larger
irregularly shaped parasites containing inflated nuclei (N).
B, the acidocalcisomes (A), identified by
dense material associated with their bounding membranes
(arrows), ruptured in some cases. Note that ribosomes and
cytosolic materials seep into the ruptured organelles resulting in
areas of low density cytoplasm. C and G denote
conoid and Golgi structures, respectively.
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DISCUSSION |
The findings reported here constitute the first molecular and
functional characterization of TgVP1, a type I V-PPase from the
apicomplexan protist T. gondii. It is established that
TgVP1 encodes a functional K+-activated,
PPi-dependent H+ pump that bears a
close resemblance to the canonical type I V-PPases from plants. In
addition, it is shown that the V-PPase has a punctate apical
distribution in both free and intracellular tachyzoites at steady state
but a dynamic distribution during host cell invasion. Upon initiation
of infection of the host cell by T. gondii, a collar-like
structure with which most of the immunodetectable V-PPase is associated
is formed. This structure, or the V-PPase that is associated with it,
moves from the anterior to the posterior of the parasite coincident
with propagation of the penetration furrow.
Of the V-PPases that have been defined molecularly to date, TgVP1 is
unusual in its possession of an N-terminal signal peptide. With the
exception of the pump from T. cruzi which like TgVP1 does
have one (18), a survey of all available V-PPase sequences from
archaea, eubacteria, and plants reveals none, not even the type I
V-PPase from P. falciparum (7), that contain a putative N-terminal signal peptide. The precise role of this sequence and why
protists like T. gondii and T. cruzi have it and
others not is not known, although in the case of TgVP1 it has been
shown by the fusion experiments that the N-terminal extension is
sufficient to direct polypeptides to the secretory pathway of T. gondii.
More sequences are needed to decide this issue, but these findings are
consistent with the notion that different parasitic protists have
different intracellular V-PPase distributions and that these are
determined by the 5'-coding sequences of their genes, namely whether
they do or do not specify an N-terminal extension. In the kinetoplastid
protist T. cruzi, low to intermediate resolution
immunological methods localize the V-PPase to acidocalcisomes (11, 44),
whereas the results of more recent immunogold electron microscopic
analyses of T. gondii have been interpreted in terms of
localization of the V-PPase to both acidocalcisomes and the plasma
membrane (10). In Plasmodium, by contrast, in which neither of the V-PPase genes, PfVP1 nor PfVP2 (7), encode
a polypeptide containing an N-terminal extension, V-PPase
immunostaining is predominantly associated with the plasma membrane, an
observation supported by the finding that PfVP1 fusions preferentially
target to the plasma membrane in trophozoites (7). On the basis of these findings, the sufficiency of the N-terminal extension of TgVP1
for entry of this polypeptide into the secretory pathway, and the
presence of this extension not only in TgVP1 but also in its cognate
from T. cruzi, there is a possibility that targeting of the
V-PPase to the acidocalcisome is contingent on initial entry of the
translation product into the secretory pathway.
The pronounced punctate apical distribution of the V-PPase described
here clearly indicates localization of the V-PPase of T. gondii to the membranes bounding acidocalcisomes or similar vesicular structures. Moreover, from what we have determined to be
necessary to maximize monospecificity, the differences between the
results presented here and those published by other investigators are
explicable in terms of the different cross-reactivities of the antibody
preparations employed, namely that unless the antisera used (antisera
generated by our laboratory (23) in all cases) are not first carefully
immunopurified, they react relatively indiscriminately with parasitic
protists. We, like Rodrigues et al. (8), observe plasma
membrane immunostaining as well as a punctate and diffuse staining
throughout the cell when crude antiserum is employed. However, if the
monospecificity of the antiserum is maximized by immunoaffinity
purifying it against heterologously expressed AVP1, only the
structure-specific focused staining we describe here is observed. The
implication is clear, the antisera raised against peptide sequences
TKAADVGADLVGKIE and HKAAVIGDTIGDPLK from AVP1, which have since proven
to contain universal V-PPase antibodies (2), also contain antibodies
that react with other parasite antigens. Because these antibodies were originally intended to be used in plants and yeast, they were raised in
New Zealand White rabbits whose preimmune and immune sera were
prescreened against these organisms, not parasitic protists.
What cannot be determined from the investigations we describe here is
whether the immunofluorescence seen is solely attributable to TgVP1 or
is inclusive of other V-PPases. The antisera used in these studies were
raised against peptides that are conserved among all V-PPases
identified to date and, as such, do not distinguish type I from type II
V-PPases (2). It was disappointing but none of the strategies applied
in an attempt to circumvent this limitation were fruitful.
Overexpression of TgVP1 fusions, for instance YFP fusions, consistently
yielded aberrant nondividing cells, and all of our attempts at
isolating a type II sequence from T. gondii were negative.
There is a possibility that T. gondii does not contain a
type II equivalent, in which case the immunolocalizations obtained are
indeed exclusively attributable to TgVP1, but it is more likely, as is
the case for P. falciparum (7), that the levels of the type
II V-PPase transcript are low, perhaps undetectable in the tachyzoite
and bradyzoite stages that were screened. Because of this it is not
known if the dynamic distribution of immunostaining is specific to a
particular V-PPase or is a property of acidocalcisomes in general.
Confirmation or refutation of these two alternatives will have to await
the results of further screens, completion of the Toxoplasma
genome sequencing project, and/or expansion of the corresponding EST
data base.
The functional significance of the collar-like structure to which the
V-PPase immunolocalizes during host cell invasion is intriguing.
Despite the seeming similarity of the distribution of the V-PPase
antigen with that of micronemes, the lack of colocalization of this
enzyme with the microneme MIC3 antigen dispels the notion of a
micronemal localization. Analogously, equivalent experiments using
antibodies raised against rhoptry and dense granule antigens show
little or no colocalization with the V-PPase (data not shown). The most
logical explanation of these findings is therefore that the V-PPase
immunolocalization detected is primarily to acidocalcisomal membranes
and that in T. gondii these structures redistribute to
assume a collar-like configuration during host cell infection.
A speculation that warrants further investigation is that
acidocalcisomes associate at least transitorily with the cytoskeleton and micronemes at the anterior end of tachyzoites and redistribute with
the penetration furrow during parasite host cell invasion. Previous
studies (45) demonstrating an aligned organization of electron-dense,
acidocalcisome-like organelles at the cell periphery of amastigotes and
in close proximity to the flagellum in trypomastigotes of
Trypanosoma imply acidocalcisome-cytoskeleton interactions.
Moreover, as would be expected if this were the case, subcellular
fractionation of these electron-dense vacuoles often results in their
adherence to and copurification with cell ghosts from
Trypanosoma, Leishmania, Toxoplasma,
and Plasmodium (reviewed in Ref. 14). It is therefore
noteworthy that recent immunofluorescence studies of the
calmodulin-actomyosin complex in intracellular T. gondii
disclose an apically focused pattern very similar to that found for the
V-PPase in this study (46) and that earlier studies demonstrated an
anteriorally displaced concentration of intracellular Ca2+
within unidentified compartments, possibly acidocalcisomes, in the same
organism (47). Knowing that microneme discharge, one of the first
events in host cell invasion, requires the mobilization of
intracellular Ca2+ stores (48), it is conceivable that
acidocalcisomes serve as a source of this Ca2+.
Acidocalcisomes may thereby provide the Ca2+ required for
activating the calmodulin-dependent myosin light chain kinases that regulate the actomyosin motor that governs parasite
motility and host cell invasion (46). If this were the case, the
V-PPase would provide the motive force for acidocalcisomal Ca2+ accumulation by H+/Ca2+ antiport.
The finding that even high levels of AMDP do not block host cell
invasion by T. gondii despite the dynamic pattern of V-PPase immunofluorescence during this process was unexpected. In retrospect, however, it might be expected in that biochemical investigations of the
acidocalcisome-like intracellular structures in trypanosomatids and
P. falciparum, which react with the same antisera as used in
this study, have shown these structures to release Ca2+
upon treatment with V-ATPase inhibitors and/or AMDP (12, 49). Thus, the
inactivity, not the activity, of acidocalcisome-associated H+-pumps like the V-PPase may promote Ca2+
release from these organelles, in which case AMDP would be expected not
to interfere with host cell invasion. It may be instructive to
consider, given how sensitive heterologously expressed TgVP1 is to
inhibition by Ca2+, that Ca2+ release itself
may abolish V-PPase activity, so diminishing or eliminating futile
PPi consumption and H+
gradient-dependent organellar Ca2+ uptake
during host cell invasion.
As an extension of our studies of the in vitro sensitivity
of heterologously expressed TgVP1 to inhibition by AMDP and the studies
made by ourselves and others (7-9) of the in vivo
sensitivity of parasitic protists to this and other 1,1-diphosphonates,
we have explored the morphological consequences to T. gondii of exposure to this agent. As a result, we have determined
that the toxic action of AMDP is evident at concentrations lower than
reported previously. Rodrigues et al. (8), for instance, by
employing a global drug assay based on measurements of
[3H]hypoxanthine incorporation, reported that
concentrations of AMDP as high as 50 µM did not inhibit
tachyzoite proliferation; indeed, 200 µM was not
sufficient for 50% inhibition. We, in contrast, by counting the number
of parasites per vacuole and examining their ultrastructure, have
determined that AMDP concentrations as low as 5 µM
(concentrations commensurate with the I50 for the in
vitro inhibition of TgVP1) interfere with parasite replication. AMDP concentrations of 5-10 µM and above block or stall
cell replication but not cell growth such that the formation of highly
enlarged tachyzoites containing multiple nascent daughter cells
is apparent. The effects of AMDP are accompanied by extensive
enlargement of the parasite nucleus which becomes highly lobed and the
inflation and/or disruption of acidocalcisome-like vesicular structures.
The apparent absence of V-PPases from vertebrates and their likely
involvement in energy conservation and membrane transport make these
enzymes potential targets for the development of antiprotozoal agents.
Notwithstanding these hopes and needs, it should be appreciated that
care is required when interpreting the antiparasitic effects of AMDP.
Two considerations are crucial. First, the efficacy AMDP in
vivo is usually less than its efficacy in vitro, which
likely reflects its bulky anionic character and slow permeation of
membranes. Second, the greater intrinsic sensitivity of V-PPases
versus soluble PPases and other phosphohydrolases to AMDP is
in part attributable to the Km values of V-PPases,
which are at least an order of magnitude greater those of soluble
PPases (5, 41). Therefore, the specificity of AMDP and other
PPi analogs as competitive inhibitors will be critically
dependent on the PPi concentration prevailing in the
compartment in which the inhibitor exerts its effects.