Alterations of concentrations of calcium and arachidonic acid and agglutinations of microfilaments in host cells during Toxoplasma gondii invasion

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Veterinary Parasitology 157 (2008) 21–33 www.elsevier.com/locate/vetpar

Alterations of concentrations of calcium and arachidonic acid and agglutinations of microfilaments in host cells during Toxoplasma gondii invasion Liwei Li a, Xunde Li b, Jie Yan a,* a

b

Department of Medical Microbiology and Parasitology, Medical School of Zhejiang University, Hangzhou, 310058, China Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California, Davis 18830 Road 112, Tulare, CA 93274, USA Received 21 April 2008; received in revised form 4 July 2008; accepted 7 July 2008

Abstract Toxoplasma gondii (T. gondii) invasion of host cells is a complicated process of interaction between parasites and host cells. In the present study we investigated the alterations of free Ca2+ concentration ([Ca2+]i) and cytoskeletons in phagocytic and nonphagocytic host cells and arachidonic acid (AA) concentration in cells supernatant during T. gondii invasion. T. gondii invasion induced significant elevation of intracellular [Ca2+]i in phagocytic cells (J774A.1) but not in non-phagocytic cells (L929). Pretreatment of J774A.1 cells with Phospholipase C (PLC) inhibitor (U73122), or Ca2+ chelators (EGTA, BAPTA/AM) did not block elevations of [Ca2+]i but the elevations were lower and of shorter duration than that in untreated cells. Pre-treatment of tachyzoites with Phospholipases A (PLA) inhibitors (4-BPB and AACOCF3) resulted in a similar pattern of increasing of [Ca2+]i as that in Ca2+ chelators treated cells. Agglutinations of microfilaments were observed in J774A.1 cells but not in L929 cells. No changes of microtubules were observed in either cell. Treatment of cells with cytoskeleton inhibitors (colchicines, cytochalasin-D) resulted in reduced cell infection ratios. AA concentration in J774A.1 cells supernatant reached 8.44-fold of basal concentration after T. gondii infection and those in 4-BPB or AACOCF3 pre-treated cells reached 7.70-fold and 8.09-fold of basal concentration, respectively. However, elevation of AA concentrations induced by 4-BPB or AACOCF3 treated tachyzoites were 3.02-fold and 2.65-fold of basal AA concentration. AA concentration in L929 cells supernatant reached 5.02-fold of basal concentration after T. gondii infection and those in 4-BPB or AACOCF3 pre-treated cells reached 4.75-fold and 4.78-fold of basal concentration, respectively. However, elevation of AA concentrations induced by 4-BPB or AACOCF3 treated tachyzoites were 2.06-fold and 2.43-fold of basal AA concentration. Results indicated that elevations of [Ca2+]i and AA induced by T. gondii invasion were from both host cells and parasites. T. gondii invasion activated host cell PLC and triggered the PLC-PKC signal pathway, which resulted in the flowing of extracellular Ca2+ and the releasing of intracellular Ca2+ pool. Elevated [Ca2+]i induced reorganization of host cell microfilaments. The invasion also activated secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) of the parasite to release AA, which increased the permeability of cell membrane. # 2008 Elsevier B.V. All rights reserved. Keywords: Toxoplasma gondii; Invasion; Host cell; Signal; Calcium; Arachidonic acid; Microfilament; Microtubule

1. Introduction * Corresponding author. Tel.: +86 571 88208297; fax: +86 571 88208294. E-mail address: [email protected] (J. Yan). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.07.007

Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that infects most species of

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mammals including humans (Fayer, 1981). Food-borne infection of T. gondii in humans has been well recognized (Dubey, 1991) and a great potential for waterborne transmission exists (Dubey, 2004). A significant percentage (25–50%) of the world’s human population is chronically infected with T. gondii (Dannemann et al., 1991) although infection is mostly asymptomatic. Toxoplasmosis, the disease caused by T. gondii infection can be severe in infants born to women who were acutely infected during pregnancy (Desmonts et al., 1990). Furthermore, T. gondii appears to be an opportunistic parasite that causes severe disease in immunocompromised individuals (Araujo and Remington, 1987). T. gondii is capable of infecting multiple systems and the most serious disease involves infection in the central nervous system and is known as Toxoplasma encephalitis. This disease affects 30% of AIDS patients (Wong and Remington, 1993). T. gondii infects a wide range of phagocytic and non-phagocytic cells by active invasion rather than entering by conventional phagocytosis (Norrby, 1970; Werk, 1985). Like other apicomplexans, invasion of cells and migration in tissues by T. gondii are accomplished by a gliding motility (Dobrowolski and Sibley, 1996). Once a tachyzoite, the invasive stage of T. gondii, enters a host cell, an intracellular compartment called parasitophorous vacuole (PV) develops. T. gondii invasion of host cells is a complicated process that involves intracellular divalent ions and cytoskeletons. Calcium is a ubiquitous signaling molecule involved in a large number of cellular processes in eukaryotic cells. It is essential for protozoan parasite differentiation (Billker et al., 2004), cytoskeleton dynamics (Sahoo et al., 2004), motility (Arrizabalaga et al., 2004) and cell growth (Luo et al., 2004). Calcium has also been demonstrated as a dependent factor for interactions between protozoan parasites and host cells. One study showed that a rise in calcium concentration was associated with morphological changes of the T. gondii tachyzoites and rapid egress from the host cells (Arrizabalaga and Boothroyd, 2004). Furthermore, mobilization of intracellular Ca2+ plays a role in facilitating parasite invasion. Previously it has been demonstrated that the infection of Trypanosoma cruzi caused elevation of intracellular [Ca2+]i in host cell, and followed by transient reorganization of actin microfilaments in host cell, which resulted in T. cruzi invasion (Rodriguez et al., 1995; Dytoc et al., 1994). In addition to calcium, arachidonic acid (AA) is increasingly recognized as a second messenger, which can mediate multiple cellular activities such as cell apoptosis and inflammation (Rizzo et al., 1999; Levick

et al., 2007; Bonta and Parnham, 1978). AA is produced by Phospholipase A (PLA) and mediates decomposition of the phospholipids on cell membrane (Ronco et al., 2002). It has been documented that PLA is closely related to protozoan parasite pathogencity in Entamoeba sp. (Ravdin et al., 1985) and Trypanosome sp. (Connelly and Kierszenbaum, 1984). After adhesion to host cells, the PLA of Entamoeba histolytica can induce the intracellular Ca2+ flow from targeted host cells and subsequently induce decomposition of the host cell (Ravdin et al., 1985). T. gondii can produce secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) and the production is closely related to virulence of T. gondii (Buitrago-Rey et al., 2002). PLA2 inhibitors can significantly decrease the ability of T. gondii to invade cells (Dubey et al., 1998). Also, T. gondii can trigger AA cascade in mouse resident peritoneal macrophages (Thardin et al., 1993). In our previous study, infection cycle of Leptospira interrogans was compared in fibroblasts and macrophages. The results showed that macrophages always had more adherent leptospires than the fibroblasts, and leptospires adhered more strongly to macrophages than fibroblasts (Li et al., 2007). Although previous works have indicated the changes of calcium, AA and cytoskeletons during the infection of T. gondii, studies have not been done to study the differences of these changes between non-phagocytic and phagocytic cells. In this context, the present study aims to investigate the alterations of free Ca2+ concentration ([Ca2+]i) and AA and changes of microfilaments and microtubules together in non-phagocytic and phagocytic cells during T. gondii invasion. 2. Materials and methods 2.1. Main instruments and reagents Main instruments used in this study include fluorescence microscope (Nikon, TE2000, Japan), optical microscope (Olympus, CX-21, Japan), laser scanning confocal microscope (Zeiss LSM 510, Germany), isotopic liquid scintillation counter (Beckman, LS9800, USA), and CO2 incubator (Forma3111S/N, USA). Reagents used included anti-bovine a-tubulin mouse monoclonal antibodies, fluo-3/AM (C51H50Cl2N2O23, a fluorescence marker for Ca2+), and TRITC (tetramethyl rhodamine isothiocyanate)-conjugated phalloidin from Molecular Probes (Eugene, OR, USA); pluronic F127 (difunctional block copolymer surfactant), U73122 (Aminosteroid, Phospholipase C inhibitor), colchicines (microtubules inhibitor), cytochalasin-D (microfilaments

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inhibitor), EGTA (ethylene glycol tetraacetic acid, an extracellular calcium chelator), BAPTA/AM (C34H40N2O18, an intracellular calcium chelator), AACOCF3 (trifluomethylketone analog of arachidonic acid, cPLA2 inhibitor), and BSA (bovine serum albumin) from Sigma (St. Louis, MO, USA); FITC (fluorescein isothiocyanate)-conjugated affinipure goat anti-mouse IgG (H + L) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); RPMI 1640 from Gibco Laboratories (Grand Island, NY, USA); [5,6, 8, 9, 11, 12, 14, 15-3H(N)]-AA from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA); 4-BPB (4-bromophenacyl Bromide, sPLA2 inhibitor) from Fluka (Buchs, Switzerland); fetal calf serum (FCS) from Sijiqing Biological Products Co. (Hangzhou, China). All other reagents were analytical grade. 2.2. Cell lines and cell culture The phagocytic cell line, murine monocyte-macrophage-like cells (J774A.1) and the non-phagocytic cell line, murine fibroblast cell (L929) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Science Shanghai Branch. For all experiments, both J774A.1 and L929 cells were cultured at 37 8C in a 5% CO2 incubator in RPMI 1640 media supplemented with 10% FCS, 100 U/mL of penicillin and 100 U/mL of streptomycin. 2.3. Parasites T. gondii RH strain was propagated in J774A.1 cells using the method described by Chamberland and Current (1991). Tachyzoites harvested were suspended in RPMI 1640 media and a solution of 105/mL was prepared. Three to four week old female specific pathogen free (SPF) Kunming mouse (15–18 g) (Laboratory Animal Center, Zhejiang Institute for Drug Control, Hangzhou, China) was given 0.5 mL tachyzoites solution by intra-peritoneal injection. Mice were housed individually in sterilized cages fitted with air filters and provided with feed and water ad libitum. At 48 h post injection, mice were euthanized by CO2 asphyxiation and tachyzoites were immediately collected from peritoneal cavity. Tachyzoites were filtered through 5 mm membrane filter, washed 2 in PBS by centrifugation at 1600  g for 5 min, and resuspended in RPMI 1640 media (without antibiotics) containing 2% FCS. A stock solution of tachyzoites at 1.0  107/mL was prepared and stored at 4 8C for less than 24 h before used for experiments.

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2.4. Examination of microfilaments and microtubules Two milliliters of each cell suspension (5  104/mL J774A.1 cell and 2.5  104/mL L929 cell) were inoculated per well in 12-well tissue culture plates (Corning, NY, USA) containing glass coverslips. The plates were incubated in 5% CO2 at 37 8C for 24 h, after which the medium was removed and replaced with 2 mL of antibiotic-free medium per well for an additional 2–3 h. One milliliter of tachyzoites stock solution was inoculated into each well and cultured at 37 8C in 5% CO2 incubator. After 40 min of culture, coverslips were removed from wells, washed 3 with PBS and fixed in methanol at 20 8C for 2 min. Coverslips were submerged in 0.1% Triton X-100 for 5 min, washed 3 with PBS and blocked with 1% BSA/ PBS for 30 min at room temperature. Each coverslip was labeled with 30 mL Phalloidin-TRITC (1:1000 of 1% BSA/PBS) to visualize microfilaments. To visualize microtubules, each coverslip was treated with 1:200 diluted anti-bovine a-tubulin mouse monoclonal antibodies, then labeled with 1:100 diluted FITC-conjugated affinipure goat anti-mouse IgG (H + L). Coverslips were incubated in a wet box in the dark for 40 min at room temperature followed by 3 wash with PBS. Coverslips were dried at room temperature, placed on a slide upside down and mounted with glycerol. Each experiment was performed in triplicate. Slides were examined with fluorescent microscope for microfilaments (excitation at 568 nm, emission at 585 nm) and for microtubules (excitation at 488 nm, emission at 568 nm). 2.5. Experiments of effects of cytoskeleton inhibitors, PLC inhibitor and Ca2+chelators on T. gondii infection J774A.1 and L929 cells were cultured on coverslips as described as above. Monolayers were treated with colchicines (30 mg/L), cytochalasin-D (3 mg/L), EGTA (2 mmol/L), BAPTA/AM (100 mmol/L) or U73122 (10 mmol/L), respectively, for 30 min at 37 8C. Then each well was washed 3 with PBS and inoculated with 1.0 mL of tachyzoites stock solution. Coverslips were incubated at 37 8C for 10, 30, 60 and 120 min, respectively. After infection, coverslips were recovered and fixed the same way as above, stained with Gimsa stain, placed on a slide upside down and mounted and sealed with Canada balsam (neutral). Slides were examined with an optical microscope for parasites infection in cells (magnification 1000). The infection

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ratio was defined as [(no. of infected cells)/(no. of cells examined)]  100% (at least 100 cells examined). 2.6. Measurement of [Ca2+]i Cells were grown on coverslips (0.8 cm  0.8 cm) in 3.5 cm diameter Petri dishes. Each Petri dish (containing four coverslips) were inoculated with 2 mL of J774A.1 (1.0  105/mL) or L929 (5.0  104/mL) cells and incubated at 37 8C for 24 h. Coverslips were removed from Petri dishes, washed 3 with D-Hanks solution (NaCl 8.00 g/L, KCl 0.40 g/L, Na2HPO4
12H2O 0.12 g/L, KH2PO4 0.06 g/L, D-glucose 1.00 g/L, NaHCO3 0.35 g/L), and stained with 100 mL stain solution (10 mmol/L fluo-3/AM, 0.04% (w/w) Pluronic F-127, and 0.2% BSA in RPMI 1640 media) at 37 8C for 30 min. Stain solution was removed and coverslips were placed onto the bottom of a specially designed small glass chamber with the side contains monolayer upwards. The glass chamber can hold 0.8 cm  0.8 cm coverslips and be fitted under the lens of microscope. It is capable of holding 200 mL solution and the bottom of it is as thin and transparent as coverslips. Coverslips were washed 3 with D-Hanks solution followed by adding 200 mL RPMI 1640 media. The small chamber was placed under the lens of laser scanning confocal microscope for measuring [Ca2+]i in cells. The images of free Ca2+ complex were analyzed with Zeiss software associated with the laser scanning confocal microscope and [Ca2+]i was expressed as % of fluorescence intensity (excitation at 488 nm, emission at 530 nm). After measuring [Ca2+]i, the culture media was removed by aspiration and 200 mL tachyzoites stock solutions were added to each chamber. Chambers were incubated at 37 8C and [Ca2+]i was measured every 5 min until 50 min post inoculation of tachyzoites. 2.7. Experiments of effects of Ca2+chelators, and PLA and PLC inhibitors on [Ca2+]i in phagocytic cells J774A.1 cells were cultured on coverslips in Petri dish, stained and washed the same way as above. RPMI 1640 media with supplement of 2% FCS were added EGTA (2 mmol/L), BAPTA/AM (100 mmol/L) or U73122 (10 mmol/L), respectively. Two hundred microliters of each of these media were respectively added to chambers and incubated at 37 8C for 30 min. After removing media and washing 3  with PBS, 200 mL of tachyzoites stock solutions were added to each chamber and [Ca2+]i were measured in the same manner as above.

Additionally, experiments were conducted to treating parasites and to both treating cells and parasites. Tachyzoites were treated with both 20 mmol 4-BPB (for 20 min) and 20 mmol AACOCF3 (for 10 min) at room temperature and washed 3 with PBS by centrifugation at 1600  g for 5 min. Treated tachyzoites were inoculated into monolayers of cells without treatment and [Ca2+]i was measured in the same manner as above. Both host cells and parasites were treated with EGTA (2 mmol/L, 30 min) and BAPTA/AM (100 mmol/L, 30 min), or 4-BPB(20 mmol, 20 min) and AACOCF3 (20 mmol, 10 min), respectively. Then treated cells and tachyzoites were washed 3 with PBS and incubated together. [Ca2+]i was measured in the same manner as above. 2.8. Measurement of AA concentrations in supernatant of J774A.1 and L929 cells J774A.1 and L929 cells were cultured on coverslips the same way as described in Section 2.4. Cell monolayers were labeled with 0.5 mCi [5, 6, 8, 9, 11, 12, 14, 15-3H (N)]-AA in RPMI 1640 media at 37 8C for 24 h and washed 3 with PBS. Two hundred microliters of tachyzoite stock solution were inoculated into each well and incubated at 37 8C for 10, 20, 40, 60, 80 or 100 min. Plates were centrifuged at 1000  g for 10 min, then supernatants were collected by aspiration and examined using the isotopic liquid scintillation counter. The concentrations of AA are expressed as counts per minute (CPM). 2.9. Experiments of effects of PLA inhibitors on AA concentration in supernatant of J774A.1 and L929 cells Tachyzoites were treated with 20 mmol 4-BPB for 15 min or with 20 mmol AACOCF3 for 10 min at room temperature and washed 3  with PBS by centrifugation at 1600  g for 5 min. Two hundred microliters of treated tachyzoites solution were inoculated to 0.5 mCi [5, 6, 8, 9, 11, 12, 14, 15-3H (N)]-AA labeled J774A.1 and L929 cell monolayers on coverslips cultured the same way as above. After incubation at 37 8C for 10, 20, 40, 60, 80, and 100 min, supernatants were collected by aspiration and AA was measured the same way as above. In addition to the experiments of treating tachyzoites, experiments were conducted on treated cells and treated cells and tachyzoites with PLA inhibitors.

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J774A.1 and L929 cells were cultured on coverslips and cell monolayers were labeled with 0.5 mCi [5, 6, 8, 9, 11, 12, 14, 15-3H (N)]-AA and washed the same way as above. Monolayers were treated with 20 mmol 4-BPB for 15 min or 20 mmol AACOCF3 for 10 min, respectively, at 37 8C. After 3 washing with PBS, 200 mL of treated tachyzoites (20 mmol 4-BPB, 15 min; 20 mmol AACOCF3, 10 min) or untreated tachyzoites stock solutions were inoculated into each well and incubated at 37 8C for 10, 20, 40, 60, 80, or 100 min. Supernatants were collected by aspiration and AA was measured the same way as above.

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3. Results 3.1. Effects of T. gondii infection on structure of microfilaments and microtubules of host cells Agglutination of microfilaments was observed in J774A.1 cells after T. gondii infection (Fig. 1b). However, no agglutination of microfilaments was observed in L929 cells after T. gondii infection (Fig. 1d). Apparent changes in structures of microtubules were neither observed between uninfected J774A.1 cells (Fig. 2a) and infected J774A.1 cells (Fig. 2b), nor between uninfected L929 cells (Fig. 2c) and infected L929 cells (Fig. 2d).

2.10. Statistical analysis Data were analyzed statistically by using ANOVA. Results were expressed as arithmetic means  standard deviation. Statistical significance was assumed when P < 0.05.

3.2. Effects of cytoskeleton inhibitors, PLC inhibitor and Ca2+ chelators on T. gondii infection The ratios of T. gondii infected J774A.1 cells and L929 cells were shown in Table 1. A time-dependent

Fig. 1. Fluorescent microscopy for observation of microfilaments in host cells before and after T. gondii invasion. Cell monolayers were labeled with Palloidin-TRITC (1:1000 of 1% BSA/PBS); Slides were examined with fluorescent microscope (Nikon, TE2000) (excitation at 568 nm, emission at 585nm). Arrowheads show host cell microfilaments. (a) J774A.1 cells before T. gondii invasion; (b) J774A.1 cells after T. gondii invasion; (c) L929 cells before T. gondii invasion; (d) L929 after T. gondii invasion. Bars: 20 mm.

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Fig. 2. Fluorescent microscopy for observation of microtubules in host cells before and after T. gondii invasion. Cell monolayers were labeled with 1:100 diluted FITC-conjugated affinipure goat anti-mouse IgG (H + L); slides were examined fluorescent microscope (Nikon, TE2000) (excitation at 488 nm, emission at 568 nm). Arrowheads show host cell microtubules. (a) J774A.1 cells before T. gondii invasion; (b) J774A.1 cells after T. gondii invasion; (c) L929 cells before T. gondii invasion; (d) L929 after T. gondii invasion. Bars: 20 mm.

increase of infection ratios were observed in both cells either treated with inhibitors or untreated. Compared to infection ratios in untreated cells, infection ratios decreased in both J774A.1 and L929 cells treated with cytoskeleton inhibitors (colchicines, cytochalasin-D), PLC inhibitor (U73122) and Ca2+chelators (EGTA and BAPTA/AM). Infection ratios decreased significantly in microfilaments inhibitor (cytochalasin-D), PLC inhibitor and Ca2+chelators treated J774A.1 cell. Infection

ratios in this group of cells were all significantly lower than those in untreated J774A.1 cells at all time points after infection (P < 0.01). 3.3. Intracellular [Ca2+]i in host cells during T. gondii invasion The intracellular [Ca2+]i (expressed as % of fluorescence intensity) was 102.0% in J774A.1 cells

Table 1 T. gondii infection ratios of J774A.1 and L929 cells pre-treated with cytoskeleton inhibitors, PLC inhibitors and Ca2+ chelators Group

Colchicines Cytochalasin-D U73122 EGTA BAPTA/AM Control a a

Infection ratios of J774A.1 cells

Infection ratios of L929 cells

10 min

30 min

60 min

120 min

10 min

30 min

60 min

120 min

8.35  1.06 3.68  0.47 4.32  0.59 6.41  0.82 5.39  0.65 12.2  1.35

35.4  1.98 18.8  2.32 24.2  2.66 30.4  2.48 27.8  2.37 50.3  2.19

52.8  2.60 26.5  3.50 35.4  3.74 40.6  2.99 38.2  3.12 68.4  3.44

60.7  2.34 29.8  3.70 38.7  3.82 44.7  3.02 41.5  2.87 73.8  2.75

8.85  1.15 8.98  1.82 8.76  1.31 8.80  1.27 7.53  1.52 9.03  1.38

44.6  1.85 44.1  1.96 42.9  1.77 45.2  1.83 42.6  1.64 46.2  2.46

46.7  2.38 45.9  2.43 48.2  2.19 46.3  1.97 45.4  2.26 52.9  2.24

56.2  2.58 54.6  2.92 58.5  2.69 58.8  2.73 57.7  2.51 60.8  2.45

Cells without treatment with cytoskeleton inhibitors, PLC inhibitors and Ca2+ chelators.

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Fig. 3. Comparison of changes of [Ca2+]i in J774A.1 cells and L929 cells during T. gondii invasion. [Ca2+]i were expressed as fluorescence intensity which was measured using laser scanning confocal microscope (Zeiss LSM 510) (excitation at 488 nm, emission at 530 nm).

before T. gondii infection (basal calcium levels). Increasing of [Ca2+]i was observed after T. gondii infection of J774A.1 cells. A peak of [Ca2+]i was observed at 35 min after infection, which was 1219.7% and significantly higher (P < 0.01) than the basal calcium levels. After 50 min of infection, [Ca2+]i still remained at a high level (883.64%) (Fig. 3). In L929 cells, the [Ca2+]i before T. gondii infection was 103.7%. After T. gondii infection, [Ca2+]i remained almost the same level as that before infection (P > 0.05) (Fig. 3).

induced elevation of [Ca2+]i but the peak concentration was only 305.6%, which was lower than that in untreated J774A.1 cells. [Ca2+]i remained almost the same level as that before infection in cells treated with Ca2+ chelators infected with tachyzoites treated with Ca2+ chelators (P > 0.05) (Fig. 4).

3.4. Effects of Ca2+ chelators, PLA and PLC inhibitors on [Ca2+]i in J774A.1 cells

As shown in Fig. 5, the AA concentration (CPM) measured in J774A.1 cells supernatant before T. gondii infection was 10,317 (basal AA level). T. gondii invasion induced elevation of AA concentration in supernatant of J774A.1 cells. At 40 min after infection, the AA concentration peaked to 87,042, which was 8.44-fold of the basal AA level (P < 0.001). At 80 min after infection, the AA concentration decreased to the level before infection and remained stable until 140 min after infection. Pre-treatment of J774A.1 cells with 4-BPB or AACOCF3 resulted in similar increasing of AA concentration as that in untreated cells (Fig. 5). Concentrations also peaked at 40 min after infection, and the peaks were 78,968 (7.70-fold of basal AA level) and 80,782 (8.09-fold of basal AA level), respectively, in 4-BPB and AACOCF3 treated cells. Both concentrations decreased at 80–100 min after infection and remained stable until 140 min after infection. Tachyzoites treated with 4-BPB or AACOCF3 could still induce the elevation of AA concentration in supernatant of untreated J774A.1 cells (Fig. 5). However, peak

In J774A.1 cells treated with PLC inhibitor U73122, T. gondii invasion induced elevated [Ca2+]i but the peak concentration was only 458.3%, and this was lower than that in untreated J774A.1 cells (1219.7%). In addition to that, after 50 min of infection, the [Ca2+]i decreased to a level close to basal calcium levels (168.6%). In J774A.1 cells infected with tachyzoites treated with PLA inhibitors (4-BPB and AACOCF3), the concentration peaked to 688.9% at 35 min and decreased to 217.8% after 50 min of infection. Data were similar in cells treated with PLA inhibitors infected with tachyzoites treated with PLA inhibitors. The patterns of effects of Ca2+ chelators on [Ca2+]i in J774A.1 cells were similar, at 35 min after infection, [Ca2+]i peaked to 667.24% in BAPTA/AM treated cells and peaked to 798.25% in EGTA treated cells. After 50 min of infection, in both treatment types concentrations decreased to levels close to basal calcium levels. In J774A.1 cells treated with both EGTA and BAPTA/AM, T. gondii invasion still

3.5. AA concentrations in J774A.1 cells supernatant and effects of PLA inhibitors on it during T. gondii invasion

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Fig. 4. Effects of Ca2+ chelators and Phospholipases inhibitors on [Ca2+]i in J774A.1 cells during T. gondii invasion. [Ca2+]i were expressed as fluorescence intensity which was measured using laser scanning confocal microscope (Zeiss LSM 510) (excitation at 488 nm, emission at 530 nm).

concentrations were 44,560 (3.02-fold of basal AA level) and 38,517 (2.65-fold of basal AA level), respectively, in cells infected with 4-BPB and AACOCF3 treated tachyzoites (Fig. 5). Both data were significantly lower than peak AA concentration in supernatant of untreated J774A.1 cells infected with untreated tachyzoites (P < 0.01). 3.6. AA concentrations in supernatant of L929 cells and effects of PLA inhibitors on it during T. gondii invasion As shown in Fig. 6, the AA concentration (CPM) measured in supernatant of L929 cells before T. gondii infection was 9657 (basal AA level). T. gondii invasion induced elevation of AA concentration in supernatant of L929 cells. At 40 min after infection, the AA concentration peaked to 48,478, which was 5.02-fold of the basal AA level (P < 0.01). At 80 min after infection, the AA concentration decreased to the level before infection and remained stable until 140 min after infection. Pre-treatment of L929 cells with 4-BPB or AACOCF3 resulted in similar increasing of AA concentration as that

in untreated cells (Fig. 6). Concentrations also peaked at 40 min after infection, and the peaks were 45,877 (4.75fold of basal AA level) and 46,207 (4.78-fold of basal AA level), respectively, in 4-BPB and AACOCF3 treated cells. Both concentrations decreased at 80–100 min after infection and remained stable until 140 min after infection. Tachyzoites treated with 4-BPB or AACOCF3 could still induce the elevation of AA concentration in supernatant of untreated L929 cells (Fig. 6). However, peak concentrations were 19,921 (2.06-fold of basal AA level) and 23,436 (2.43-fold of basal AA level), respectively, in cells infected with 4-BPB and AACOCF3 treated tachyzoites (Fig. 6). Both data were significantly lower than peak AA concentration in supernatant of untreated L929 cells infected with untreated tachyzoites (P < 0.01). 4. Discussion Adhesion to host cells by some bacteria (pathogenic Escherichia coli and Salmonella) (Dytoc et al., 1994; Philpott et al., 1995; Clark et al., 1998; Wadsworth and Goldfine, 1999) and protozoa (Trypanosoma brucei)

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Fig. 5. Changes of AA concentrations in supernatants of J774A.1 cells during T. gondii invasion. Cell monolayers were labeled with 0.5 mCi [5, 6, 8, 9, 11, 12, 14, 15-3H (N)]-AA; culture supernatants were collected and examined with multi-purpose scintillation counter. AA concentrations were presented by the radio intensity which is expressed as counts per minute (CPM).

(Rodriguez et al., 1995; Schenkman et al., 1988; Tardieux et al., 1992) can trigger reorganization of Factin and invaginations of host cell membrane, which facilities pathogen to enter host cells. Few reports are available on the roles of microfilaments during T. gondii

invasion. Dobrowolski and Sibley (1996) reported that T. gondii invasion was critically dependent on actin filaments in the parasite and the invasion was actively powered by an actin-based contractile system in the parasite. Morisaki et al. (1995) demonstrated that

Fig. 6. Changes of AA concentrations in supernatants of L929 cells during T. gondii invasion. Cell monolayers were labeled with 0.5 mCi [5, 6, 8, 9, 11, 12, 14, 15-3H (N)]-AA; culture supernatants were collected and examined with multi-purpose scintillation counter. AA concentrations were presented by the radio intensity which is expressed as Counts per minute (CPM).

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Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. In the present study, we observed apparent agglutinations of microfilament in J774A.1 cells after T. gondii infection (Fig. 1b) but not in L929 cells. Interestingly, agglutinations of microfilament were not observed in J774A.1 cells pre-treated with cytochalasin-D (microfilaments inhibitor). In addition to that, ratios of infected cells in such treated cells decreased significantly (Table 1). The results presented above thus suggested that microfilament functioned differently in phagocytic and non-phagocytic host cells during T. gondii invasion and the functions were critical for successful infection in phagocytic cells by T. gondii. Rodriguez et al. (1995) proved that invasion of nonphagocytic cells by T. cruzi was independent of pseudopodia formation and actin polymerization, indicating that the mechanism of invasion of nonphagocytic cell was distinct from that of phagocytic cell, which might also be the case for T. gondii. In this study we did not observe any apparent changes in microtubules in either J774A.1 or L929 cells after T. gondii infection. It was in accordance with the previous report that invasion of T. gondii required the actin cytoskeleton of the parasite but not that of the host cells (Dobrowolski and Sibley, 1996). However, another studies showed that host cell microtubules were associated with the development of parasitophorous vacuole (Andrade et al., 2001) and colchicines (microtubules inhibitor) treatment altered the shape of the parasitophorous vacuole containing tachyzoites (Melo et al., 2001). These apparent conflicting observations may be due to differences in experimental conditions and facilities. Fluorescent microscopy could fail in imaging or capturing weak fluorescent structures. Some researchers use transmission electron microscopy to observe microtubules in cells infected with T. gondii (Melo et al., 2001). It was interesting that we observed decreases of T. gondii infection ratios in both J774A.1 cells and L929 cells pre-treated with colchicines or cytochalasin-D, the mechanism of the decreases could be due to the suppression of effects of microtubules which can form a barrier for the phagolysosomal fusion during T. gondii infection (Andrade et al., 2001). Ca2+ is one of the most common second messengers that is involved in multiple processes of cell metabolisms including cell growth, proliferation and death. Changes of intracellular [Ca2+]i is one of the main factors that leads to the interaction between protozoan parasites and host cells (Burleigh and Andrews, 1995; Sikha et al., 2002). In the present study, the intracellular

[Ca2+]i in non-phagocytic cells (L929) did not change significantly before and after tachyzoite invasion. This finding was in accordance with the previous reports. Some researchers proved that invasion of non-phagocytic cells, such as KB cells (human epidermoid carcinoma epithelial cell line) and HF cells (human foreskin fibroblast cells), was parasites’ initiative activity and the [Ca2+]i of host cells did not change much or only increased slightly (Pingret et al., 1996; Lovett and Sibley, 2003). In contrast, present study showed that [Ca2+]i elevated significantly in phagocytic cells (J774A.1) after T. gondii infection. At 35 min after infection, the concentration was as much as 12-fold of the basal calcium level. The present results plus previous findings indicated that [Ca2+]i of host cell changed differently and thus Ca2+ related signal transduction might vary in phagocytic and nonphagocytic host cells after T. gondii invasion. The mechanisms of elevation of host cell [Ca2+]i are partly due to T. gondii activation of PLC in the host cell membrane. The PLC functions by catalyzing the PIP2 (phosphatidylinositol bisphosphate) to produce IP3 (inositol triphosphate) and 2-DG (diacylglycerol). The 2-DG can induce the release of Ca2+ which is normally attached to the endoplasmic reticulum (Rodriguez et al., 1995; Salter and Hicks, 1995). The IP3 acts as a second messenger and can also mediate intracellular calcium release (Lovett et al., 2002). This may explain why in our results the [Ca2+]i in PLC inhibited J774A.1 cells was significantly lower than that in untreated cells. In the present study, pre-treatment of J774A.1 cells with Phospholipase C (PLC) inhibitor (U73122), or Ca2+ chelators (EGTA, BAPTA/AM) did not block elevations of [Ca2+]i but the elevations were lower and of shorter duration than that in untreated cells. Results revealed that elevation of [Ca2+]i induced by T. gondii invasion had two sources, one was the extracellular Ca2+ flowing and the other one was intracellular calcium releasing. Our data paralleled with previous findings that mobilization of intracellular calcium upon attachment of T. gondii tachyzoites to host cells was required for invasion (Vieira and Moreno, 2000) and that extra- and intracellular Ca2+ mobilization was required for T. gondii to enter host cells (Bonhomme et al., 1999). More and more studies proved that Ca2+ from parasites, rather than host cell, was more important for T. gondii invasion. Stommel et al. (1997) demonstrated that chelating of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA did not prevent the activation of parasites. This result suggested that Ca2+ that activated parasite motility might reside near or

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within the parasite itself. Pezzella et al. (1997) proved that depletion of the Ca2+ in the external medium with EGTA reduced tachyzoite invasion, and depletion of Ca2+ from intratachyzoite pools with A23187 + EGTA inhibited invasion. Furthermore, recent findings indicated that calcium released from the parasite’s intracellular pools was necessary and sufficient to induce some of the events critical for invasion and egress (Bouchot et al., 1999; Arrizabalaga and Boothroyd, 2004). In the present study, chelation of host cell calcium with EGTA or BAPTA/AM did not block the elevation of [Ca2+]i in J774A.1 cells after T. gondii invasion (Fig. 4), though the elevations were lower than that in untreated cells. This result indicated that not only Ca2+ from host cells plays an important role in T. gondii invasion, but also Ca2+ from parasites itself was critical for T. gondii invasion. The interactions between T. gondii and host cell were Ca2+ dependent and the sources of Ca2+ were not only from host cell but also from parasite itself. AA is produced by Phospholipase A (PLA) decomposing the phospholipids on the cell membrane (Ronco et al., 2002) and is related to protozoan parasite pathogencity (Ravdin et al., 1985;Connelly and Kierszenbaum, 1984). A previous research showed PLA2 inhibitors could significantly decrease the ability of T. gondii to invade cells (Dubey et al., 1998). In our study, AA concentrations increased as much as 8.44fold in supernatant of J774A.1 cell infected with tachyzoites. AA concentrations in 4-BPB or AACOCF3 (PLA inhibitors) treated cells infected with untreated tachyzoites were around the same level as that in untreated cells. However, pre-treatment of tachyzoites with 4-BPB or AACOCF3 resulted in much lower elevation of AA concentrations in supernatant of untreated cells. Results revealed that sources of AA during T. gondii invasion were from both host cells and parasites. As a consequence of interaction between parasite and host cells, T. gondii invasion triggered self PLA to release AA to increase permeability and fluidity of host cell membrane which facilitated parasites entry. Previous works found that AA could alter permeability and fluidity of cell membrane and induce the intracellular Ca2+ store activation. Beck et al. (1998) proved that AA could lead hyperpolarization of the cell membrane and increased electrochemical gradient for Ca2+ in endothelial cells. Fleming and Mellow (1995) demonstrated that AA caused Ca2+ efflux from endoplasmic reticulum. Collectively, our data plus these findings indicated the process of T. gondii invasion of host cells was not solely or simply a phagocytic event, but parasites played an active role. Alterations of

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concentrations of Ca2+ and AA were closely correlated and elevations of AA were at least beneficial to the increasing of [Ca2+]i. These changes worked together to complete the process of T. gondii invasion of host cells. Hu et al. (2005) showed that Ca2+ efflux from endoplasmic reticulum mediated apoptosis of nonphagocytic cells. Interestingly, in the previous study we demostrated that T. gondii triggered almost exclusively necrosis in phagocytic cells and apoptosis in nonphagocytic cells (Li et al., 2006). Necrosis is now believed to stimulate inflammation, while apoptosis is anti-inflammatory (Savill and Fadok, 2000; Fadok et al., 1998). Our results suggested that T. gondii may contribute to disease through their ability to trigger inflammation in different host cell. In summary, T. gondii invasion of host cells is a complicated process of interaction between parasite and host cells which requires series changes in host cells and parasites. Calcium plays an important role for tachyzoite invasion and the sources of calcium are from both host cells and parasites. T. gondii infection also induces elevations of AA concentration and agglutination of microfilaments in phagocytic host cells. Further investigations are needed to discover the relations among these changes and how these changes function together during the T. gondii invasion in different types of host cells. Acknowledgements This study was supported by the National Natural Science Foundation of China (no. 30300294). We thank Professor Da-gang Xu (The Department of Microbiology and Parasitology, Shanghai Second Medical University, Shanghai, China) for his generous gift of the Toxoplasma gondii RH strain. References Andrade, E.F., Stumbo, A.C., Monteiro-Leal, L.H., Carvalho, L., Barbosa, H.S., 2001. Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion? J. Submicrosc. Cytol. Pathol. 33, 337–341. Araujo, F.G., Remington, J.S., 1987. Toxoplasmosis in immunocompromised patients. Eur. J. Clin. Microbiol. 6, 1–2. Arrizabalaga, G., Boothroyd, J.C., 2004. Role of calcium during Toxoplasma gondii invasion and egress. Int. J. Parasitol. 34, 361–366. Arrizabalaga, G., Ruiz, F., Moreno, S., Boothroyd, J.C., 2004. Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation. J. Cell Biol. 165, 653–662.

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