B

FIGURE 10.4 Immunofluorescence microscopy of TgVPl.

(A) The acidocalcisome localization of TgVPl in isolated tachyzoites is shown using polyclonal antibodies against the enzyme (red, 1 : 1000).

(B) The lack of co-localization of the reaction against TgVPl and ROP1, a rhoptry protein, is shown, using polyclonal antibodies against TgVPl (red, 1 : 1000) and monoclonal antibodies against ROP1 (green, 1 : 1000). Bar = 10 pm.

This figure is reproduced in color in the color plate section.

face of the membrane (Figure 10.5B). Tachyzoites show acidocalcisomes with different degrees of preservation of the electron-dense material, from an almost totally empty vacuole (Figure 10.5A, left arrows) to others containing a considerable amount of electron-dense material (Figure 10.5A, right arrows). The membrane of the T. gondii acidocalcisome is about 8 nm thick (Figure 10.5B). In some circumstances the electron-dense material can be found in aggregates within the organelle, apparently surrounded by an internal membrane (Figure 10.5C, arrow), as occurs in some Phytomonas species (Miranda et al., 2004), but no direct evidence for the presence of an internal membrane has been obtained. Occasionally acidocalcisomes of Toxoplasma can be seen fusing with each other, being found in a polymorphic organization (Figures 10.5D, 10.5E).

A large number of acidocalcisomes can be seen in electron spectroscopic images of whole cells directly dried on Formvar-coated grids (Figure 10.6A). The advantage of this type of preparation is the observation of the whole parasite (and whole organelles) without the addition of fixatives and

FIGURE 10.5 Transmission electron microscopy of tachyzoites of T. gondii.

(A) Thin section of a T. gondii tachyzoite showing several acidocalcisomes in different regions of the cell, with different degrees of preservation of the electron-dense material (arrows). Bar = 500 nm.

(B) and (C) High magnification of acidocalcisomes. Note the single membrane and the small amount of electron-dense material (B) and the amount of electron-dense material accumulated in the matrix of the organelle (C, arrow). Scale bars: (B) = 100 nm, (C) = 200 nm.

(D) and (E) Acidocalcisomes fusing with each other (arrows). Bars: (D) = 100 nm, (E) = 200 nm. Cells were washed twice in PBS, fixed in Karnovsky, post-fixed in OsO4 and embedded in Polybed 812 epoxide resin. Sections were stained for 30 minutes in uranyl acetate and for 5 minutes in lead citrate, and observed in a JEOL 1200EX electron microscope operating at 80 kVV

FIGURE 10.5 Transmission electron microscopy of tachyzoites of T. gondii.

(A) Thin section of a T. gondii tachyzoite showing several acidocalcisomes in different regions of the cell, with different degrees of preservation of the electron-dense material (arrows). Bar = 500 nm.

(B) and (C) High magnification of acidocalcisomes. Note the single membrane and the small amount of electron-dense material (B) and the amount of electron-dense material accumulated in the matrix of the organelle (C, arrow). Scale bars: (B) = 100 nm, (C) = 200 nm.

(D) and (E) Acidocalcisomes fusing with each other (arrows). Bars: (D) = 100 nm, (E) = 200 nm. Cells were washed twice in PBS, fixed in Karnovsky, post-fixed in OsO4 and embedded in Polybed 812 epoxide resin. Sections were stained for 30 minutes in uranyl acetate and for 5 minutes in lead citrate, and observed in a JEOL 1200EX electron microscope operating at 80 kVV

FIGURE 10.6 Electron probe X-ray microanalysis of whole cells adhered to formvar-coated grids.

(A) Electron spectroscopic image (ESI) of T. gondii. Bar = 1 pm.

(B) Corresponding X-ray spectrum obtained from the sample region arrowed in Figure 10.6A (arrow). Note the presence of high amounts of oxygen, sodium, magnesium, phosphorus, chlorine, potassium, calcium and zinc within these organelles. Copper signal comes from the electron microscope grid and titanium signal from the specimen holder. Specimens were analyzed in a LEO 912 Omega scanning transmission electron microscope. X ray point measurements were collected for 150 seconds using a Li-drifted Si-detector (front area 30 mm2) equipped with an ATW atmospheric window. The microscope was operated at 80 kV using a tungsten filament, in the scanning transmission (STEM) imaging mode, and spot size was 63 nm.

FIGURE 10.6 Electron probe X-ray microanalysis of whole cells adhered to formvar-coated grids.

(A) Electron spectroscopic image (ESI) of T. gondii. Bar = 1 pm.

(B) Corresponding X-ray spectrum obtained from the sample region arrowed in Figure 10.6A (arrow). Note the presence of high amounts of oxygen, sodium, magnesium, phosphorus, chlorine, potassium, calcium and zinc within these organelles. Copper signal comes from the electron microscope grid and titanium signal from the specimen holder. Specimens were analyzed in a LEO 912 Omega scanning transmission electron microscope. X ray point measurements were collected for 150 seconds using a Li-drifted Si-detector (front area 30 mm2) equipped with an ATW atmospheric window. The microscope was operated at 80 kV using a tungsten filament, in the scanning transmission (STEM) imaging mode, and spot size was 63 nm.

other chemicals used in the routine procedures for transmission electron microscopy. This reduces significantly the extraction of material from the acidocalcisomes, and therefore allows observation of the organelle in its 'native' state (Luo et al., 2001). In these preparations, acidocalcisomes can be seen as spherical electron-dense organelles randomly spread throughout the cell body (Figure 10.6A). Approximately 10 acidocalcisomes, with diameters varying between ~150 and ~400 nm, are observed per cell. X-ray microanalysis (Luo et al., 2001) (Figure 10.6B) reveals considerable amounts of oxygen, sodium, magnesium, phosphorus, chlorine, potassium, calcium, and zinc concentrated in these compartments, similarly to what has been reported previously in the acidocalci-somes of trypanosomatids (Scott et al., 1997; Rodrigues et al., 1999; Miranda et al., 2000, 2004; Lefurgey et al., 2001).

T. gondii acidocalcisomes have been shown to possess a plasma membrane-type Ca2+-ATPase (PMCA), involved in Ca2+ influx, with similarity to vacuolar Ca2+-ATPases of other unicellular eukary-otes (Bouchot et al., 2001; Luo et al., 2001), and two proton pumps - a vacuolar H+-ATPase (V-H+-ATPase) and a vacuolar H+-pyrophosphatase (V-H+-PPase) - involved in their acidification (Moreno et al., 1998; Rodrigues et al., 2000; Luo et al., 2001; Drozdowicz et al., 2003). No second messengers have been demonstrated to be involved in Ca2+ release from acidocalcisomes of T. gondii. However, a gene with similarity to the previously described two-pore channel 1 (TPC1), the Arabidopsis thaliana Ca2+-dependent Ca2+-release channel (Furuichi et al., 2001), has been found in the genome of T. gondii (583.m05406) (Chen et al., 2006). The predicted protein sequence shows 12 transmembrane segments distributed in 2 domains typical of these kinds of channels, which are present in the plant vacuoles, and it is possible that this channel might be present in the acidocalcisomes.

Although the Ca2+ content of acidocalcisomes is very high (probably in the molar range), most of it is bound to poly P and can be released only upon alkalinization (Moreno and Zhong, 1996) or after poly-P hydrolysis (Rodrigues et al., 2002).

A gene encoding the acidocalcisome Ca2+-ATPase (TgA1) (Table 10.1) was identified in T. gondii (Luo et al., 2001). This gene was able to complement yeasts deficient in the vacuolar Ca2+-ATPase gene PMC 1, providing genetic evidence for its function (Luo et al., 2001). The protein product is closely related to the family of plasma membrane calcium ATPases (PMCA). A sequence analysis of conserved core sequences of all PMCA-type Ca2+-ATPases has identified a subclus-ter within these sequences that is formed by the acidocalcisome Ca2+-ATPases of T. gondii, Trypanosoma cruzi, T. brucei, and Dictyostelium discoideum, and the vacuolar Ca2+-ATPases of yeast and Entamoeba histolytica (Luo et al., 2001). A common feature of these pumps is the lack of a calmodulin-binding domain, in contrast to other PMCA-type Ca2+-ATPases. Mutants deficient in TgA1 were shown to have decreased virulence in vitro and in vivo due to their deficient invasion of host cells (Luo et al., 2005). Biochemical analysis revealed that the tachyzoite poly P content was drastically reduced, and that the basal Ca2+ levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions (Luo et al., 2005).

The V-H+-ATPase was first identified in T. gondii by its sensitivity to bafilomycin A1, a specific inhibitor of this proton pump when used at low concentrations (Bowman et al., 1988). In experiments using intact tachyzoites loaded with the fluorescent calcium indicator fura-2, bafilomycin A1 was able to release calcium from an intracellu-lar compartment of T. gondii (Moreno and Zhong, 1996). The V-H+-ATPase was also shown, by immunofluorescence microscopy, to localize in acidocalcisomes and in the plasma membrane, where it has a role in regulating intracellular pH homeostasis (Moreno et al., 1998).

A V-H+-PPase activity was also found in T. gondii (Rodrigues et al., 2000), and this enzyme was also shown to localize in acidocalcisomes of T. gondii

(Rodrigues et al., 2000; Luo et al., 2001; Drozdowicz et al., 2003)(Figure 10.4). The gene encoding the T. gondii enzyme (TgVPl) was cloned and sequenced, and a truncated version of the enzyme (without the N-terminal) could be functionally expressed in yeast (Drozdowicz et al., 2003). Interestingly, the V-H+-PPase-specific staining of T. gondii 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 (Drozdowicz et al., 2003). Upon completion of infection, the V-H+-PPase-associated fluorescence disperses before reappearing again at the anterior apex of the intracellular tachyzoite (Drozdowicz et al., 2003). In recent work a chimera of the T. gondii V-H+-PPase, with or without the N-terminal extension of T. cruzi V-H+-PPase at its N-terminus, has shown improved expression levels, enough to complement yeasts deficient in the soluble pyrophosphatase (Drake et al., 2004). The acidocalcisome enzyme belongs to the K+-stimulated group of V-H+-PPases (type I) (Rodrigues et al., 2000; Drozdowicz et al., 2003), and has been successfully used as a marker for acidocalcisome purification - not because is only localized in these organelles, but because it is heavily concentrated in them (Rodrigues et al., 2002).

A number of genes have been identified in the genome of T. gondii that could potentially encode for additional acidocalcisome transporters. For example, a Ca2+/H+ exchanger similar to those present in the vacuole of yeast and plants has been annotated, probably erroneously, as a Ca2+/Na+ antiporter (see section 10.3.1) (20.m03897); a putative phosphate transporter (49.m03192); two putative chloride channels (57.m01751 and 80.m02270); a neutral and basic amino-acid transporter (583.m05611); a Zn2+ transporter (52.m01632); and Na+/H+ exchangers (129.m00252 and 541.m01159) (Chen et al., 2006). Alternatively, however, some or all of these transporters could be located in the plasma membrane.

All acidocalcisomes described so far have been found to have high levels of phosphorus in the

TABLE 10.2 Pyrophosphate and polyphosphate levels in tachyzoites

Phosphorous compounds

Concentration (mM)

Pyrophosphate

7.950 ± 0.160

Short-chain poly P

24.000 ± 0.500

Long-chain poly P

0.043 ± 0.005

Data from Rodrigues et al., 2002.

Data from Rodrigues et al., 2002.

form of inorganic pyrophosphate (PPi) and polyphosphate (poly P). Quantitative measurements of PPi and different poly Ps in T. gondii are shown in Table 10.2. T. gondii acidocalcisomes are especially rich in short-chain poly Ps such as poly P3 (Rodrigues et al., 2000; Moreno et al., 2001).

PPi is a byproduct of many biosynthetic reactions (synthesis of nucleic acids, coenzymes, and proteins, activation of fatty acids, and isoprenoid synthesis), and its hydrolysis by inorganic pyrophosphatases makes these reactions thermo-dynamically favorable. None of these pathways has been found in T. gondii acidocalcisomes. One possibility is that PPi is there as a byproduct of the hydrolysis of poly P, or as an intermediate for its synthesis. Only three reactions are known to use PPi in T. gondii: one catalyzed by phospho-fructokinase (Peng et al., 1995), another by the V-H+-PPase responsible for acidification of acidocalcisomes (Rodrigues et al., 2000; Drozdowicz et al., 2003), and the third by an inorganic pyrophosphatase (Luo and Moreno, unpublished results). Since PPj is charged and polar, any movement through the acidocalcisome membrane probably involves a transporter. A transmembrane trans -porter that shuttles PPi between intracellular and extracellular compartments has been identified in several mammalian tissues (Ho et al., 2000) and a similar channel in the acidocalcisome membrane would explain PPi accumulation after its synthesis through anabolic reactions occurring in the cytosol or other compartments, or its release into the cytosol to serve as substrate for the V-H+-PPase.

Poly P accumulates in very large amounts in acidocalcisomes (Table 10.2). The storage of phosphate as poly P reduces the osmotic effect of large pools of this important compound. Short- and long-chain poly P levels also rapidly decreased upon exposure of tachyzoites to agents that mobilize Ca2+, such as calcium ionophores (ionomycin), alkalinizing agents (NH4Cl), or inhibitors of the V-H+-ATPase (bafilomycin A^ (Rodrigues et al., 2002). This would suggest a role for poly P in the adaptation of the parasites to environmental stress.

The low sulfur content detected by elemental analysis (Figure 10.6) suggested a low content of proteins within acidocalcisomes. However, in addition to the proton and calcium pumps, another enzymatic activity has been detected. Acidocalcisomes from T. gondii were shown to contain a polyphosphatase activity (Rodrigues et al., 2002).

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