Inositol 1,4,5‐trisphosphate receptor determines intracellular Ca2+ concentration in Trypanosoma cruzi throughout its life cycle

Regulation of intracellular Ca2+ concentration ([Ca2+]i) is vital for eukaryotic organisms. Recently, we identified a Ca2+ channel (TcIP 3R) associated with intracellular Ca2+ stores in Trypanosoma cruzi, the parasitic protist that causes Chagas disease. In this study, we measured [Ca2+]i during the parasite life cycle and determined whether TcIP 3R is involved in the observed variations. Parasites expressing R‐GECO1, a red fluorescent, genetically encoded Ca2+ indicator for optical imaging that fluoresces when bound to Ca2+, were produced. Using these R‐GECO1‐expressing parasites to measure [Ca2+]i, we found that the [Ca2+]i in epimastigotes was significantly higher than that in trypomastigotes and lower than that in amastigotes, and we observed a positive correlation between TcIP 3 R mRNA expression and [Ca2+]i during the parasite life cycle both in vitro and in vivo. We also generated R‐GECO1‐expressing parasites with TcIP 3R expression levels that were approximately 65% of wild‐type (wt) levels (SKO parasites), and [Ca2+]i in the wt and SKO parasites was compared. The [Ca2+]i in SKO parasites was reduced to approximately 50–65% of that in wt parasites. These results show that TcIP 3R is the determinant of [Ca2+]i in T. cruzi. Since Ca2+ signaling is vital for these parasites, TcIP 3R is a promising drug target for Chagas disease.


Regulation of intracellular Ca 2+ concentration ([Ca
] i ) is vital for eukaryotic organisms. Recently, we identified a Ca 2+ channel (TcIP 3 R) associated with intracellular Ca 2+ stores in Trypanosoma cruzi, the parasitic protist that causes Chagas disease. In this study, we measured [Ca 2+ ] i during the parasite life cycle and determined whether TcIP 3 R is involved in the observed variations. Parasites expressing R-GECO1, a red fluorescent, genetically encoded Ca 2+ indicator for optical imaging that fluoresces when bound to Ca 2+ , were produced. Using these R-GECO1-expressing parasites to measure [Ca 2+ ] i , we found that the [Ca 2+ ] i in epimastigotes was significantly higher than that in trypomastigotes and lower than that in amastigotes, and we observed a positive correlation between TcIP 3 R mRNA expression and [Ca 2+ ] i during the parasite life cycle both in vitro and in vivo. We also generated R-GECO1-expressing parasites with TcIP 3 R expression levels that were approximately 65% of wild-type (wt) levels (SKO parasites), and [Ca 2+ ] i in the wt and SKO parasites was compared. The [Ca 2+ ] i in SKO parasites was reduced to approximately 50-65% of that in wt parasites. These results show that TcIP 3 R is the determinant of [Ca 2+ ] i in T. cruzi. Since Ca 2+ signaling is vital for these parasites, TcIP 3 R is a promising drug target for Chagas disease.
Trypanosoma cruzi is the parasitic protist that causes Chagas disease in Latin America. At present, only two drugs are available for Chagas disease (benznidazole and nifurtimox), and these often induce severe side effects and are effective for only the acute phase of the disease. Since no practical drug or vaccine for Chagas disease is available, new treatments are greatly needed [4]. The life cycle of the parasite comprises two phases, the insect and mammalian phases [5]. In the insect vector (the reduviid bug), the epimastigote replicates and transforms into a metacyclic trypomastigote (metacyclogenesis). A nonproliferating metacyclic trypomastigote invades a mammalian host, and is then transformed into an amastigote inside a wide variety of nucleated cells. The intracellular amastigote multiplies by binary fission, and is then transformed back into a trypomastigote, which is released into the circulation after host cell disruption.
[Ca 2+ ] i regulation is vital for T. cruzi, and the molecular mechanisms of [Ca 2+ ] i regulation in the parasite are thought to be quite different from those in mammalian cells [6]. In fact, no homologs of the typical Ca 2+ transporters-ROCs, SOCs, or Na + /Ca 2+ exchangers, have been detected in Trypanosomes. The results of a proteome analysis of T. brucei suggested that a putative VGCC is localized to the flagellum [7]. Two homologs of plasma membrane Ca 2+ ATPase (PMCA) have also been reported; one is localized on the plasma membrane, and the other is localized to the acidocalcisome of T. brucei [8]. In addition, a SERCA has been shown to be localized to the ER of T brucei like mammalian cells [9]. However, no RyR homologs have been reported. Recently, we identified an IP 3 R homolog in T. cruzi (TcIP 3 R), and showed that it is mainly localized to the ER. When the expression level of TcIP 3 R is reduced to less than one-half of that in wild-type (wt) cells, the parasite cannot grow [10]. Therefore, TcIP 3 R may be a promising drug target [11]. We also previously showed that TcIP 3 R regulates parasite growth, transformation, infectivity, and virulence in mammalian hosts, indicating that TcIP 3 R is an important regulator of the parasite life cycle [10,12]. In fact, experiments using classical Ca 2+ indicators, such as Fura-2, showed that Ca 2+ signaling is important for host cell invasion [10,13,14] as well as proliferation and transformation [15].
In this paper, we reported the successful preparation of parasites expressing R-GECO1 (a red fluorescent, genetically encoded Ca 2+ indicator for optical imaging), which is a green fluorescent protein (GFP) variant that fluoresces only upon binding to Ca 2+ [16]. It has recently been reported that other parasites including Plasmodium falciparum and Toxoplasma gondii that express genetically encoded Ca 2+ indicators are useful for investigating Ca 2+ signaling in the parasite [17,18]. Importantly, our findings revealed that analysis of T. cruzi expressing R-GECO1 revealed that the [Ca 2+ ] i in the parasite changes significantly during its life cycle, and that TcIP 3 R is the determinant of [Ca 2+ ] i in T. cruzi.

Cell culture
Epimastigotes of the T. cruzi Tulahuen strain were cultured as previously described [21]. The mammalian stages of the parasites were maintained in HeLa cells or 3T3-Swiss albino cells (Health Science Research Resources Bank, Tokyo, Japan), and trypomastigotes were collected from subcultures of infected 3T3-Swiss albino cells by centrifugation as previously described [22]. Metacyclogenesis was performed as previously described [23]. Quantitative real-time RT-PCR analysis was performed as previously described [10] Expression of R-GECO1 in T. cruzi A total of 1 9 10 7 epimastigotes were resuspended in Amaxa Basic Ò Parasite Nucleofector Kit 2 solution (Lonza, K€ oln, Germany). The resuspended wt or TcIP 3 R SKO parasites [10] were mixed with 10 lg of pTREX (neo R )/R-GECO1 or pTREX (pur R )/R-GECO1, respectively, and then electroporated with an Amaxa Nucleofector Device (Lonza) using program U-033. Stable wt transformants expressing R-GECO1 were selected by incubating the cells for 30 days in LIT medium containing 0.5 mgÁmL À1 G418, and then clonal derivatives were isolated by limiting dilution. The stable SKO transformants expressing R-GECO1 were selected by incubating the cells for 30 days in LIT medium containing 0.25 mgÁmL À1 G418 and 3 lgÁmL À1 puromycin. Amastigotes or trypomastigotes stably expressing R-GECO1 were transformed from the epimastigotes expressing R-GECO1 using the methods described above.

Detection of [Ca 2+ ] i by fluorescence microscopy
Fluorescence images of the parasites were acquired using a fluorescent microscope (Axio Imager M2; Carl Zeiss Co. Ltd., Oberkochen, Germany) or a laser confocal microscope (Nikon A1R, Nikon Co. Ltd., Tokyo, Japan). After acquiring fluorescence images in normal culture medium, the maximal fluorescence signal (F max ) of R-GECO1 in individual parasites was determined by treating them with high Ca 2+ solution (10 mM) and ionomycin (0.26 mM; Nacalai Tesque, Kyoto, Japan), which increase the cytosolic Ca 2+ content of parasites so as to saturate R-GECO1 with Ca 2+ . To convert fluorescence intensity to Ca 2+ concentration, the following formula was used:

Statistical analysis
Statistical analysis was performed with SIGMA PLOT ver. 12 software (Systat Software, Inc., San Jose, CA, USA) using one-way ANOVA or Student's t-test.

Results and Discussion
The [Ca 2+ ] i changes significantly during the life cycle of T. cruzi To investigate the changes in the [Ca 2+ ] i in T. cruzi during its life cycle, parasites expressing R-GECO1 were prepared. The R-GECO1 gene was cloned in the T. cruzi pTREX expression vector [19], in which R-GECO1 is expressed under the ribosomal promoter; therefore, R-GECO1 was constitutively expressed throughout the parasite life cycle. We at first investigated whether the fluorescence signal in the parasites expressing R-GECO1 was reduced after treatment with a cell-permeant Ca 2+ chelator BAPTA-AM. After replacement of the parasite medium with PBS, BAPTA-AM (final concentration 100 lM) was added to the PBS to reduce Ca 2+ , and [Ca 2+ ] i was measured after 3 min (Fig. 1A). Treatment of the parasites with BAPTA-AM significantly reduced the parasite [Ca 2+ ] i , indicating that R-GECO1 works as a Ca 2+ indicator in the parasites. We also found that the parasites were killed by the treatment.
To exclude variations in signal intensity among the parasite clones, a clonal derivative was isolated and used for the experiments. Figure 1B shows the R-GECO1 signal in the epimastigote, trypomastigote, and amastigote. Although the signal was detected throughout the cytoplasm of the parasites at all stages, the signal intensity was quite different among the different life cycle stages, and the maximal fluorescence intensities of R-GECO1 obtained in the presence of 260 lM ionomycin and 10 mM Ca 2+ were similar. The [Ca 2+ ] i was calculated from the R-GECO1 signal intensity and compared among the three stages, as described in the Methods section (Fig. 1C). The [Ca 2+ ] i was clearly higher in amastigotes (579 AE 204 nM) and epimastigotes (327 AE 44 nM) than in trypomastigotes (85 AE 39 nM). These results indicated that the [Ca 2+ ] i changes significantly during the progression of the parasite life cycle. Ca 2+ oscillation was not detected at any stage.

Live cell imaging revealed changes in the [Ca 2+ ] i of T. cruzi parasitizing host cells
We further investigated the changes in the [Ca 2+ ] i of parasites during intracellular growth. 3T3-Swiss cells were infected with R-GECO1-expressing trypomastigotes, and then the growth of one parasite was monitored under a fluorescent microscope ( Fig. 2A). We successfully monitored them until the parasite divided three times within a host cell. The results showed that the [Ca 2+ ] i in amastigotes did not change significantly after division.
Amastigotes divide several times within host cells, transform into trypomastigotes, and then lyse the host cells. We investigated whether the [Ca 2+ ] i in trypomastigotes parasitizing host cells was decreased similar to that observed in tissue culture-derived trypomastigotes (Fig. 2B, Movie S1). The results showed that the [Ca 2+ ] i in trypomastigotes within host cells (Fig. 2B, arrowhead in the right panel) was significantly lower than that in amastigotes (Fig. 2B, arrow in the right  panel).
These results indicate that amastigotes are able to maintain [Ca 2+ ] i even in environments where the Ca 2+ concentration is very low, such as the cytosol of host cells, and that the [Ca 2+ ] i in the trypomastigote when inside host cells is significantly lower than that in the amastigote [Ca 2+ ] i .

TcIP 3 R is the determinant of [Ca 2+ ] i in T. cruzi
Previously, we reported that TcIP 3 R mRNA expression varied significantly among the parasite life cycle stages [10]. Here, we investigated a possible correlation between TcIP 3 R mRNA expression and the [Ca 2+ ] i of the parasite (Fig. 3A). There was a positive correlation between the parasite [Ca 2+ ] i at each life stage and the TcIP 3 R mRNA expression level (R 2 = 0.66). These results suggest that TcIP 3 R is important for the regulation of [Ca 2+ ] i in T. cruzi.
To investigate whether TcIP 3 R is involved in the regulation of [Ca 2+ ] i in T. cruzi, the level of TcIP 3 R was reduced in parasites expressing R-GECO1, and the [Ca 2+ ] i in wt and mutant parasites was measured (Fig. 3B). We previously found three TcIP 3 R genes in the genome of the T. cruzi Tulahuen strain, and we prepared single-knockout (SKO) parasites, in which one of the TcIP 3 R genes was disrupted by homologous recombination [10]. We observed the specific disruption of only one TcIP 3 R gene by Southern blot analysis and an approximately 35% reduction in TcIP 3 R expression levels in the SKO parasites, and these parasites show various phenotypes, such as inhibition of epimastigote growth [10]. Since the knockout cassette used to prepare the SKO parasites contained a neomycin resistance gene, the transformants were selected with G418. Then, the R-GECO1 gene was cloned into an expression plasmid vector for T. cruzi containing a puromycin resistance gene (pTREX(pur R )), and then this plasmid was transfected into the SKO parasites. SKO parasites expressing R-GECO1 were selected in culture medium containing G418 and puromycin. For the control, wt Tulahuen strain parasites were transfected with pTREX(pur R )/R-GECO1 and selected in culture medium containing puromycin. Since the expression level of R-GECO1 among the parasite clones might vary, the fluorescence intensity in the parasites was randomly measured without cloning. The fluorescence intensity in the SKO parasites was significantly lower than that in the wt parasites. Importantly, the TcIP 3 R expression level in the SKO parasites was reduced to approximately 65% of wt levels [10], and the R-GECO1 signal in SKO parasites was reduced to approximately 50% of wt levels.
Next, we investigated whether Ca 2+ influx from the extracellular fluid or efflux to the extracellular fluid is important for maintenance of [Ca 2+ ] i (Fig. 3C). Excessive amounts of CaCl 2 or BAPTA, a noncell-permeable Ca 2+ chelator, was added to the cultivation medium of epimastigotes expressing R-GECO1, and the [Ca 2+ ] i in treated parasites was compared to that in untreated parasites after 2 h. No increase was detected in parasites treated with CaCl 2 compared to that in untreated parasites. When the Ca 2+ in the culture medium was chelated by the addition of BAPTA, we speculated that [Ca 2+ ] i might be reduced by PMCA function. However, the [Ca 2+ ] i in parasites treated with BAPTA was not reduced when compared with that in untreated parasites. These results indicate that T. cruzi do not constitutively import or export Ca 2+ . Therefore, Ca 2+ released from intracellular Ca 2+ store(s) into the cytosol by TcIP 3 R should be effectively returned to the store(s) by SERCA [24,25].
In animal cells, [Ca 2+ ] i are kept at low concentrations (~100 nM) in the absence of extracellular stimuli [26]. Phosphoinositide phospholipase C (PI-PLC) is activated in response to signals from cell surface receptors, and it catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate IP 3 , which activates IP 3 R and transiently increases [Ca 2+ ] i [27]. Our present data indicate that [Ca 2+ ] i in epimastigotes and amastigotes is constitutively high. Recently, it has been reported that Trypanosoma brucei PI-PLC may be constitutively activated [28]. Furthermore, the molecular properties of T. cruzi PI-PLC have been reported to be similar to that of T. brucei PI-PLC [29] (e.g., plasma membrane localization). Together, these findings suggest that constitutive activation of T. cruzi PI-PLC might maintain high [Ca 2+ ] i through constitutive TcIP 3 R activation.
According to the cell boundary theorem, [Ca 2+ ] i is determined by the balance between Ca 2+ influx and efflux, and Ca 2+ release via IP 3 R does not result in The amastigotes (one cell) that divided once (two cells), twice (four cells), and three times (eight cells) are shown. Bar, 5 lm. (B) A bright-field image of cells that are heavily infected with R-GECO1expressing amastigotes (arrow) and trypomastigotes (arrowhead) is shown (left). A representative microscopic image was obtained with an inverted microscope (IX72; Olympus, Tokyo, Japan). A video of the same field is available as Movie S1. Note that the trypomastigotes in the host cell move intensely. A fluorescent image of the same field is also shown (right). Bar, 10 lm.
higher [Ca 2+ ] i [30,31]. In mammalian cells, Ca 2+ influx increases through the SOC mechanism activated by Ca 2+ release from the ER, thereby resulting in an increase of [Ca 2+ ] i [25]. Furthermore, it might be possible that [Ca 2+ ] i in T. cruzi is not always increased through TcIP 3 R directly but the parasites have some unknown mechanism(s) to increase Ca 2+ influx. Interestingly, since amastigotes parasitize the host cell cytoplasm, where the concentration of Ca 2+ is much lower than the [Ca 2+ ] i in amastigotes, the parasites may not receive Ca 2+ from outside through a SOClike mechanism. However, how amastigotes maintain high [Ca 2+ ] i within the host cells remains unknown at present.
In conclusion, our present study revealed that basal [Ca 2+ ] i levels in T. cruzi are determined by the level of TcIP 3 R expression. Since Ca 2+ signaling is essential for the parasite and the primary structure of TcIP 3 R shares low similarity with that of mammalian IP 3 Rs, TcIP 3 R, the key Ca 2+ signaling molecule, is a promising drug target for Chagas disease. The fluorescence intensity of 20 parasites was measured. Statistical analysis between the groups was performed using Student's t-test. ***P < 0.001. (C) R-GECO1expressing epimastigotes were treated with 10 mM CaCl 2 or 10 mM BAPTA for 2 h; fluorescence was measured, and [Ca 2+ ] i was calculated and compared to that in untreated parasites. The fluorescence intensity of 20 parasites was measured.