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Babesia gibsoni: preferential multiplication in reticulocytes is related to the presence of mitochondria and a high concentration of adenosine 5′-triphosphate in the cells
Experimental Parasitology 102 (2002) 164–169 www.elsevier.com/locate/yexpr
Babesia gibsoni: preferential multiplication in reticulocytes is related to the presence of mitochondria and a high concentration of adenosine 50-triphosphate in the cells Masahiro Yamasaki, Osamu Yamato, Mohammad Alamgir Hossain, Ja-Ryong Jeong, Hye-Sook Chang, Hiroyuki Satoh, and Yoshimitsu Maede* Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan Received 9 April 2002; received in revised form 10 March 2003; accepted 28 March 2003
Abstract To determine the cause of the predilection of Babesia gibsoni for reticulocytes, the parasites were cultivated with various types of reconstituted erythrocyte ghosts, which were prepared by resealing erythrocyte ghosts together with variously treated erythrocyte lysate, in vitro. The level of parasitemia in the culture with reconstituted reticulocyte ghosts containing untreated reticulocyte lysate was significantly higher than that in the culture with reconstituted normocyte (mature erythrocyte) ghosts containing untreated normocyte lysate. The removal of mitochondria from reconstituted reticulocyte ghosts by filtration or centrifugation resulted in decreased of parasitemia in those cultures. In contrast, when mitochondria from reticulocytes were loaded into reconstituted normocyte ghosts, the parasitemia in the ghosts loaded mitochondria was increased to the same level as that in reconstituted reticulocyte ghosts. Furthermore, the parasitemia in the culture with reconstituted normocyte ghosts was proportional to the concentration of adenosine 50 -triphosphate in the ghosts. These results suggested that mitochondria of reticulocytes might enhance the multiplication of B. gibsoni through the generation of adenosine 50 -triphosphate within the cells. Ó 2003 Elsevier Science (USA). All rights reserved. Index Descriptors and Abbreviations: Babesia gibsoni; reticulocyte; mitochondria; ATP, adenosine 50 -triphosphate; ATPase, adenosinetriphosphatase; ADP, adenosine 50 -diphosphate; HK erythrocyte, erythrocyte with high concentrations of potassium; GSH, reduced glutathione; RBC, red blood cell; PB, phosphate buffer; PBS, phosphate-buffered saline; PCV, packed cell volume
1. Introduction We have demonstrated that Babesia gibsoni, a blood parasite that causes hemolytic anemia in dogs, preferentially invades and multiplies in reticulocytes compared to normocytes (mature erythrocytes) in vitro (Murase et al., 1993). We also reported that B. gibsoni multiplied well in some canine erythrocytes with inherited high concentrations of potassium (HK), glutamate, aspartate, glutamine, and reduced glutathione (GSH), called HK erythrocytes (Yamasaki et al., 2000b). Because reticulocytes also contain larger amounts of both glutamate and GSH than normocytes, those previous *
Corresponding author. Fax: +81-11-709-7296. E-mail address: [email protected] (Y. Maede).
findings suggested that the higher multiplication of B. gibsoni in reticulocytes was partly due to the high concentrations of glutamate and GSH in the cells (Yamasaki et al., 2000a). On the other hand, previous studies also suggested that the B. gibsoni parasite preferred reticulocytes to HK erythrocytes because the level of parasitemia was much higher in reticulocyte-rich culture than in HK erythrocyte culture (Murase et al., 1993; Yamasaki et al., 2000b). In general, reticulocytes still contain some micro-organelles such as mitochondria in the cytoplasm, but lose them during maturation. Mature erythrocytes, as well as HK erythrocytes, have no more micro-organelles in their cytoplasm. Since mitochondria are energy-converting organelles in all eukaryotic cells, they seem to play a central role to determine the intracellular environment of reticulocytes, such as a higher
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00052-3
M. Yamasaki et al. / Experimental Parasitology 102 (2002) 164–169
concentration of ATP, which may be suitable for multiplication of B. gibsoni. To identify the cause of the predilection of B. gibsoni for reticulocytes, we investigated the effects of mitochondria in reticulocytes on the multiplication of B. gibsoni.
2. Materials and methods 2.1. Preparation of canine normocytes and reticulocytes Canine normocytes and reticulocytes were prepared as reported previously (Maede and Inaba, 1985). Whole blood from normal dogs was washed three times with 10 mM phosphate-buffered saline (PBS, pH 7.4) and filtered through an a-cellulose/micro-crystalline cellulose column to remove leukocytes and platelets (Beutler et al., 1976). The filtered RBCs were washed with PBS. For preparing reticulocytes, five dogs were bled of about 10 ml/kg for three days. On the third day after bleeding, 120 ml of whole blood from each dog with experimental anemia was collected and reticulocytes were obtained by using Percoll discontinuous gradient centrifugation (Yamasaki et al., 2000a). All dogs with the experimental anemia were treated with iron and recovered from the anemia within one week after the bleeding. 2.2. Preparation of canine HK erythrocytes The HK erythrocytes were obtained from dogs with HK erythrocytes, which have been maintained since 1986 in our laboratory. The HK erythrocytes contained high potassium (123:6 5:9 mM), low sodium (54:1 21:6 mM), whereas the normal cells contained low potassium (10:7 3:1 mM), high sodium (153:0 44:9 mM) concentrations. The concentrations of glutamate, aspartate, glutamine, and reduced glutathione (GSH) in HK cells increased to 92, 63, 13, and 5 times the mean value in the normal canine erythrocytes, respectively (Maede et al., 1982).
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ghost loaded with reticulocyte lysate (R[R] ghosts) were prepared as described previously (Yamasaki et al., 2000a). For preparing normocyte and reticulocyte lysates, packed normocytes and reticulocytes were resuspended in PBS to yield a PCV of 70%. Each suspension was mixed with two volumes of lysing buffer (5 mM phosphate buffer (PB), 1 mM Mg-ATP, and pH 7.4), and centrifuged at 5000g for 30 min at 4 °C to remove intact RBCs and destroyed membranes. The supernatants was collected and stored at 4 °C until use. For preparing normocyte and reticulocyte ghosts, normocyte and reticulocyte suspensions with the PCV adjusted to 70% with PBS were dialyzed according to the method of Olson and Kilejian (1982). In the present study, 5 mM PB was used as the lysis buffer. The contents of the dialysis tube were used as ghosts. One volume of the ghosts, eight volumes of lysate, and one volume of 1.2 M NaCl with 1 mM Mg-ATP were mixed and incubated at 37 °C for 1 h. After the incubation, the mixtures were washed twice with PBS as described above and used as reconstituted erythrocyte ghosts. To remove intracellular mitochondria, the reticulocyte lysate was filtered using a 0.45-lm pore-size filter, or centrifuged at 20,000g for 20 min at 4 °C. Seven types of reconstituted erythrocyte ghosts prepared were as follows (Table 1); N[N], R[R], N[fN], R[fR], N[cN], R[cR], and N[pR]. Of these, N[fN] and R[fR] ghosts were reconstituted normocyte (N) and reticulocyte (R) ghosts loaded with filtered normocyte [fN] and reticulocyte [fR] lysates, respectively. N[cN] and R[cR] ghosts were loaded with centrifuged normocyte [cN] and reticulocyte lysate [cR], respectively. A pellet was obtained only when the reticulocyte lysate was centrifuged. The pellet was resuspended in the normocyte lysate [pR] and then loaded into normocyte ghosts (N[pR] ghosts). For preparing the reconstituted normocyte ghosts containing various concentrations of ATP, 1.0, 2.5, 5.0, and 10 mM of Mg-ATP were added to the ghost suspension, and loaded into normocyte ghosts. 2.4. Culture of B. gibsoni
2.3. Preparation of reconstituted erythrocyte ghost Reconstituted normocyte ghost loaded with normocyte lysate (N[N] ghosts) and reconstituted reticulocyte
For preparation of B. gibsoni-infected erythrocytes, blood was collected from an experimentally B. gibsoniinfected dog with a parasitemia rate of that varied from
Table 1 Types of reconstituted erythrocyte ghosts prepared for the present study Abbreviations
Type of reconstituted ghosts
Treatment of lysate loaded into ghost
N[N] R[R] N[fN] R[fR] N[cN] R[cR] N[pR]
Normocyte ghosts Reticulocyte ghosts Normocyte ghosts Reticulocyte ghosts Normocyte ghosts Reticulocyte ghosts Normocyte ghosts
Normocyte lysate Reticulocyte lysate Filtered normocyte lysate Filtered reticulocyte lysate Centrifuged normocyte lysate Centrifuged reticulocyte lysate Normocyte lysate including the pellet from reticulocyte lysate
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4.8 to 6.2% and washed with PBS solution. B. gibsoni was cultivated as described in the previous report (Yamasaki et al., 2000a). 2.5. Measurement of intracellular ATP in reconstituted erythrocyte ghosts The concentration of intracellular ATP in reconstituted erythrocyte ghosts was measured according to the method of Beutler (1984). 2.6. Electron microscopy of reconstituted erythrocyte ghosts Reconstituted erythrocyte ghosts prepared as described above were sedimented at 5000g for 10 min and fixed in 2.5% (v/v) glutaraldehyde, 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 °C. They were rinsed at least three times in ice-cold 0.1 M phosphate buffer, pH 7.4 and then postfixed in 2% OsO4, 0.1 M phosphate buffer, pH 7.4 at 4 °C for 2 h. After rinsed in buffer and H2 O, the ghosts were dehydrated in ethanol, and embedded in epoxy resin. The epoxy resin consisted of 9.1 ml of Quetol-812 (Nissin EM), 6.2 ml of dodecenyl succinic anhydride (Nissin EM), 4.6 ml of methyl nadic anhydride (Nissin EM) and DMP-30 (Nissin EM). Most thin sections were stained with uranyl acetate and lead citrate, and examined with an electron microscope (JEM1210, Nissin EM). 2.7. Statistics Statistical analysis was carried out using StudentÕs t test.
3. Results 3.1. The multiplication of B. gibsoni in intact reticulocytes and HK erythrocytes When B. gibsoni-infected erythrocytes (RBC) were separately cultivated with dog HK erythrocytes, normal dog normocytes, and normal dog reticulocytes, the amount of parasitemia was significantly higher in reticulocyte culture than in the normocyte culture. The parasitemia in the HK erythrocyte culture was intermediate between those in the reticulocyte and the normocyte cultures (Fig. 1). 3.2. The multiplication of B. gibsoni in various reconstituted erythrocyte ghosts Table 1 shows various reconstituted erythrocyte ghosts prepared for this experiment. When B. gibsoniinfected RBCs were cultivated in either N[N] or R[R]
Fig. 1. Babesia gibsoni were cultivated in normocytes (open circles), reticulocytes (closed circles), and HK erythrocytes (open squares). Data are expressed as means SD (n ¼ 3). * Significantly different from the value for the normocyte culture (p < 0:05). y Significantly different from the value for the HK erythrocyte culture (p < 0:05).
ghosts, the numbers of parasitized cells began to increase at cultivation day 1. During cultivation for 6 days, a difference in the multiplication of the parasite was observed between N[N] and R[R] cultures. In R[R] culture, the level of parasitemia increased parabolically and reached 3:5 0:7% at day 5, whereas the highest parasitemia in N[N] culture was 2:1 0:6% at day 5 (Figs. 2a and b). When B. gibsoni-infected RBC were cultivated in either R[fR] or R[cR] ghosts, the level of parasitemia in each culture was lower than that in R[R] culture, and almost the same level as that in N[N] culture (Fig. 2b). On the other hand, when N[pR] ghosts were used for the cultivation of B. gibsoni, the level of parasitemia in N[pR] culture was higher than that in R[cR] culture, and the level of parasitemia in N[pR] culture was almost the same as that in R[R] culture during the cultivation period (Fig. 2a). Next, the concentrations of intracellular ATP in N[N], R[R], N[cN], R[cR], and N[pR] ghosts were measured. As shown in Table 2, the concentrations of intracellular ATP in R[R] and N[pR] ghosts were 4 and 1.8 times that in N[N] ghosts, respectively, whereas those in N[cN] and R[cR] ghosts were at almost the same level as that in N[N] ghosts. As shown in Fig. 3a, the electron microscopic observation of the pellet from reticulocyte lysate revealed many mitochondria. Several mitochondria were also
M. Yamasaki et al. / Experimental Parasitology 102 (2002) 164–169
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Fig. 2. Babesia gibsoni was cultivated in reconstituted erythrocyte ghosts. (a) N[N] (open circles), N[fN] (open squares), N[cN] (open triangles), and N[pR] (closed circles) ghost. (b) R[R] (closed circles), R[fR] (closed squares), and R[cR] (closed triangles) ghosts. Data are expressed as means SD (n ¼ 3). * Significantly different from the value for the N[N] culture (p < 0:05).
Table 2 The concentrations of intracellular ATP in N[N], R[R], N[cN], R[cR], and N[pR] ghosts Ghost type N[N] ATP concentration (lmol=109 ghosts) *
1:7 0:58
R[R]
6:9 1:1
N[cN]
R[cR]
N[pR]
1:6 0:79
1:6 1:2
3:1 0:50
Data are expressed as means SD (n ¼ 3). Values are significantly (p < 0:05) higher than those of N[N] ghosts.
observed within N[pR] ghosts (Fig. 3b), whereas no organelles were observed in the all observed N[N] ghosts (data not shown). Reconstituted normocyte ghosts containing various concentrations of ATP were prepared by incubating the ghosts with 1, 2.5, 5, or 10 mM ATP. The concentrations of intracellular ATP in those reconstituted normocyte ghosts are shown in Table 3. When the reconstituted normocyte ghosts were used as host cells for the cultivation of B. gibsoni, proportional increases of parasitemia were observed at the concentrations of 0.29, 1.7, and 2.8 mM ATP, while the level of parasitemia at 5.4 mM ATP was almost the same as that at 2.8 mM ATP (Table 3).
4. Discussion As described elsewhere, previous studies suggested that high concentrations of glutamate and GSH in reticulocytes might be a cause of the preferential multiplication of B. gibsoni in reticulocytes (Yamasaki et al.,
2000a). In the present study, however, B. gibsoni parasites multiplied well in reticulocytes even compared with HK dog erythrocytes possessing higher concentrations of glutamate and GSH than reticulocytes (Yamasaki et al., 2000a,b). This suggested that reticulocytes might contain other essential substances for multiplication of B. gibsoni. Reticulocytes are immature erythrocytes and differ from normocyte in their biochemical and morphologic characteristics. Reticulocytes have higher concentrations of ATP, GSH, amino acids and nucleic acids than do normocytes (Mons, 1990). The most prominent difference between reticulocytes and normocytes, as well as HK erythrocytes, is that reticulocytes still contain some micro-organelles, such as mitochondria in their cytoplasm, while both normocyte and HK erythrocytes have no micro-organelles. Thus, it was hypothesized that the preferential multiplication of B. gibsoni in reticulocytes might depend on those characteristics of reticulocytes, especially the presence of mitochondria in the cells, because mitochondria are an energy-converting organelles involved in the energy metabolism of the cell.
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Fig. 3. Electron microscopic observation of N[pR]. (a) The pellet, which was obtained from reticulocyte lysate by centrifugation, included many mitochondria 25,000. (b) N[pR] ghost. Mitochondria are observed in an N[pR] ghost (arrowheads) 20,000. Scale bar ¼ 500 nm.
Table 3 Concentrations of intracellular ATP and the level of parasitemia of B. gibsoni in reconstituted normocyte ghosts loaded with various concentrations of ATP Concentration of added ATP 1 mM (N[N]) 9
ATP concentration (lmol=10 ghosts) Parasitemia (%) with each ghostsa
0:59 0:38 2:15 0:54
2.5 mM 1:7 0:60 3:05 0:22
5 mM
10 mM
2:8 0:74 3:55 0:56
5:4 0:58 3:5 0:22
Data are expressed as means SD (n ¼ 3). Values are significantly (p < 0:05) higher than those of reconstituted normocyte ghosts with 1 mM Mg-ATP. a Babesia gibsoni was cultivated in reconstituted normocyte ghosts with various concentrations of Mg-ATP. *
The present study clearly showed that the mitochondria play a central role in the preferential multiplication of B. gibsoni parasites in reticulocytes. That is, the level of parasitemia in the culture using reconstituted normocyte ghosts loaded with mitochondria from reticulocytes (N[pR]) was increased to the same level as in the culture using the reconstituted reticulocyte ghosts (R[R]). In contrast, when mitochondria were removed from the R[R] ghosts, the level of parasitemia in the culture of those ghosts was decreased to that in the culture using the reconstituted normocyte ghosts (N[N]). In general, reticulocytes generate a considerable amount of ATP via the tricarboxylic acid cycle in mitochondria in addition to glycolysis, whereas ATP is chiefly generated by glycolysis in normocytes. As a result, reticulocytes have a higher concentration of intracellular ATP than normocytes. In the present study, the higher
concentration of intracellular ATP observed in N[pR] ghosts seemed to be generated by mitochondria embedded in the ghosts. Furthermore, the present study also showed that the level of parasitemia in the cultures with reconstituted normocyte ghosts was proportional to the intracellular ATP concentration of each reconstituted normocyte ghost. From these results, we concluded that the predominant cause of the predilection of B. gibsoni parasites for reticulocytes was chiefly the high concentration of ATP generated by mitochondria in the cells. In a previous study, we postulated that a high concentration of intracellular GSH, a tripeptide, which protects intracellular components from oxidative attack, might be an important factor in promoting the high multiplication of B. gibsoni in HK cells as well as reticulocytes. A recent study of de novo synthesis of GSH in malaria-infected erythrocytes has shown that the
M. Yamasaki et al. / Experimental Parasitology 102 (2002) 164–169
infection with Plasmodium falciparum causes drastic changes in the GSH metabolism of infected erythrocytes (Luersen et al., 2000). That is, infected erythrocytes lose GSH at a rate 40-fold higher than non-infected cells. It has also been reported that the formation of reactive oxygen species in P. falciparum-infected erythrocytes is increased as a result of hemoglobin digestion by the parasite, resulting in an enhanced requirement for GSH (Hunt and Stocker, 1990). Our recent study also showed that superoxide anions were produced in B. gibsoni-infected erythrocytes (Otsuka et al., 2001). These results indicated that the de novo synthesis of the tripeptide in erythrocytes was also essential for the parasiteÕs survival. In erythrocytes, even mature ones, GSH is synthesized from glutamate and 2 amino acids via 2 enzymatic reactions accompanied by conversion of ATP to ADP and inorganic phosphate. Thus, increased GSH synthesis may result in increased utilization of ATP in infected erythrocytes, indicating that a high concentration of ATP is of great advantage for the multiplication of B. gibsoni in erythrocytes. On the other hand, the penetration of host erythrocytes by the B. gibsoni parasite may be linked to intracellular ATP as reported for malaria parasites (Elford et al., 1995). It has been shown that the entry of the malaria parasite into erythrocytes requires the presence of ATP in the host cell cytoplasm (Dluzewski et al., 1983; Olson and Kilejian, 1982; Rangachari et al., 1986). The role of intracellular ATP in the multiplication of B. gibsoni, however, has not been elucidated. If it is clarified, it will undoubtedly be possible to unravel some of the more complex mechanisms concerning the invasion and development of the parasite in host erythrocytes. Acknowledgments This work was supported in part by a grant from the Science Research Fund of the Ministry of Education, Science, and Culture of Japan.
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References Beutler, E., West, C., Blume, K.G., 1976. The removal of leukocytes and platelets from whole blood. J. Lab. Clin. Med. 88, 328–333. Beutler, E., 1984. Adenosine triphosphate (ATP). In: Red Cell Metabolism. Grune & Stratton Inc., Orlando, pp. 122–123. Dluzewski, A.R., Rangachari, K., Wilson, R.J.M., Gratzer, W.B., 1983. A cytoplasmic requirement of red cells for invasion by malarial parasites. Mol. Biochem. Parasitol. 9, 145–160. Elford, B.C., Cowan, G.M., Ferguson, D.J.P., 1995. Parasite-regulated membrane transport processes and metabolism control in malariainfected erythrocytes. Biochem. J. 308, 361–374. Hunt, N.H., Stocker, R., 1990. Oxidative stress and the redox status of malaria-infected erythrocytes. Blood Cells 16, 499–526. Luersen, K., Walter, R.D., Muller, S., 2000. Plasmodium falciparuminfected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione. Biochem. J. 346, 545–552. Maede, Y., Kasai, N., Taniguchi, N., 1982. Hereditary high concentration of glutathione in canine erythrocytes associated with high accumulation of glutamate, glutamine, and aspartate. Blood 59, 883–889. Maede, Y., Inaba, M., 1985. (Na, K)-ATPase and ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation. J. Biol. Chem. 260, 3337–3343. Mons, B., 1990. Preferential invasion of malarial merozoits into young red blood cells. Blood Cells 16, 299–312. Murase, T., Iwai, M., Maede, Y., 1993. Direct evidence for preferential multiplication of Babesia gibsoni in young erythrocytes. Parasitol. Res. 79, 269–271. Olson, J.A., Kilejian, A., 1982. Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum. J. Cell Biol. 95, 757–762. Otsuka, Y., Yamasaki, M., Yamato, O., Maede, Y., 2001. Increased generation of superoxide in erythrocytes infected with Babesia gibsoni. J. Vet. Med. Sci. 63, 1077–1081. Rangachari, K., Dluzewski, A., Wilson, R.J.M., Gratzer, W.B., 1986. Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton. Nature 324, 364–365. Yamasaki, M., Otsuka, Y., Yamato, O., Tajima, M., Maede, Y., 2000a. The cause of the predilection of Babesia gibsoni for reticulocytes. J. Vet. Med. Sci. 62, 737–741. Yamasaki, M., Asano, H., Otsuka, Y., Yamato, O., Tajima, M., Maede, Y., 2000b. Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni. Am. J. Vet. Res. 61, 1520–1524.
Babesia gibsoni: preferential multiplication in reticulocytes is related to the presence of mitochondria and a high concentration of adenosine 50-triphosphate in the cells Masahiro Yamasaki, Osamu Yamato, Mohammad Alamgir Hossain, Ja-Ryong Jeong, Hye-Sook Chang, Hiroyuki Satoh, and Yoshimitsu Maede* Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan Received 9 April 2002; received in revised form 10 March 2003; accepted 28 March 2003
Abstract To determine the cause of the predilection of Babesia gibsoni for reticulocytes, the parasites were cultivated with various types of reconstituted erythrocyte ghosts, which were prepared by resealing erythrocyte ghosts together with variously treated erythrocyte lysate, in vitro. The level of parasitemia in the culture with reconstituted reticulocyte ghosts containing untreated reticulocyte lysate was significantly higher than that in the culture with reconstituted normocyte (mature erythrocyte) ghosts containing untreated normocyte lysate. The removal of mitochondria from reconstituted reticulocyte ghosts by filtration or centrifugation resulted in decreased of parasitemia in those cultures. In contrast, when mitochondria from reticulocytes were loaded into reconstituted normocyte ghosts, the parasitemia in the ghosts loaded mitochondria was increased to the same level as that in reconstituted reticulocyte ghosts. Furthermore, the parasitemia in the culture with reconstituted normocyte ghosts was proportional to the concentration of adenosine 50 -triphosphate in the ghosts. These results suggested that mitochondria of reticulocytes might enhance the multiplication of B. gibsoni through the generation of adenosine 50 -triphosphate within the cells. Ó 2003 Elsevier Science (USA). All rights reserved. Index Descriptors and Abbreviations: Babesia gibsoni; reticulocyte; mitochondria; ATP, adenosine 50 -triphosphate; ATPase, adenosinetriphosphatase; ADP, adenosine 50 -diphosphate; HK erythrocyte, erythrocyte with high concentrations of potassium; GSH, reduced glutathione; RBC, red blood cell; PB, phosphate buffer; PBS, phosphate-buffered saline; PCV, packed cell volume
1. Introduction We have demonstrated that Babesia gibsoni, a blood parasite that causes hemolytic anemia in dogs, preferentially invades and multiplies in reticulocytes compared to normocytes (mature erythrocytes) in vitro (Murase et al., 1993). We also reported that B. gibsoni multiplied well in some canine erythrocytes with inherited high concentrations of potassium (HK), glutamate, aspartate, glutamine, and reduced glutathione (GSH), called HK erythrocytes (Yamasaki et al., 2000b). Because reticulocytes also contain larger amounts of both glutamate and GSH than normocytes, those previous *
Corresponding author. Fax: +81-11-709-7296. E-mail address: [email protected] (Y. Maede).
findings suggested that the higher multiplication of B. gibsoni in reticulocytes was partly due to the high concentrations of glutamate and GSH in the cells (Yamasaki et al., 2000a). On the other hand, previous studies also suggested that the B. gibsoni parasite preferred reticulocytes to HK erythrocytes because the level of parasitemia was much higher in reticulocyte-rich culture than in HK erythrocyte culture (Murase et al., 1993; Yamasaki et al., 2000b). In general, reticulocytes still contain some micro-organelles such as mitochondria in the cytoplasm, but lose them during maturation. Mature erythrocytes, as well as HK erythrocytes, have no more micro-organelles in their cytoplasm. Since mitochondria are energy-converting organelles in all eukaryotic cells, they seem to play a central role to determine the intracellular environment of reticulocytes, such as a higher
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00052-3
M. Yamasaki et al. / Experimental Parasitology 102 (2002) 164–169
concentration of ATP, which may be suitable for multiplication of B. gibsoni. To identify the cause of the predilection of B. gibsoni for reticulocytes, we investigated the effects of mitochondria in reticulocytes on the multiplication of B. gibsoni.
2. Materials and methods 2.1. Preparation of canine normocytes and reticulocytes Canine normocytes and reticulocytes were prepared as reported previously (Maede and Inaba, 1985). Whole blood from normal dogs was washed three times with 10 mM phosphate-buffered saline (PBS, pH 7.4) and filtered through an a-cellulose/micro-crystalline cellulose column to remove leukocytes and platelets (Beutler et al., 1976). The filtered RBCs were washed with PBS. For preparing reticulocytes, five dogs were bled of about 10 ml/kg for three days. On the third day after bleeding, 120 ml of whole blood from each dog with experimental anemia was collected and reticulocytes were obtained by using Percoll discontinuous gradient centrifugation (Yamasaki et al., 2000a). All dogs with the experimental anemia were treated with iron and recovered from the anemia within one week after the bleeding. 2.2. Preparation of canine HK erythrocytes The HK erythrocytes were obtained from dogs with HK erythrocytes, which have been maintained since 1986 in our laboratory. The HK erythrocytes contained high potassium (123:6 5:9 mM), low sodium (54:1 21:6 mM), whereas the normal cells contained low potassium (10:7 3:1 mM), high sodium (153:0 44:9 mM) concentrations. The concentrations of glutamate, aspartate, glutamine, and reduced glutathione (GSH) in HK cells increased to 92, 63, 13, and 5 times the mean value in the normal canine erythrocytes, respectively (Maede et al., 1982).
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ghost loaded with reticulocyte lysate (R[R] ghosts) were prepared as described previously (Yamasaki et al., 2000a). For preparing normocyte and reticulocyte lysates, packed normocytes and reticulocytes were resuspended in PBS to yield a PCV of 70%. Each suspension was mixed with two volumes of lysing buffer (5 mM phosphate buffer (PB), 1 mM Mg-ATP, and pH 7.4), and centrifuged at 5000g for 30 min at 4 °C to remove intact RBCs and destroyed membranes. The supernatants was collected and stored at 4 °C until use. For preparing normocyte and reticulocyte ghosts, normocyte and reticulocyte suspensions with the PCV adjusted to 70% with PBS were dialyzed according to the method of Olson and Kilejian (1982). In the present study, 5 mM PB was used as the lysis buffer. The contents of the dialysis tube were used as ghosts. One volume of the ghosts, eight volumes of lysate, and one volume of 1.2 M NaCl with 1 mM Mg-ATP were mixed and incubated at 37 °C for 1 h. After the incubation, the mixtures were washed twice with PBS as described above and used as reconstituted erythrocyte ghosts. To remove intracellular mitochondria, the reticulocyte lysate was filtered using a 0.45-lm pore-size filter, or centrifuged at 20,000g for 20 min at 4 °C. Seven types of reconstituted erythrocyte ghosts prepared were as follows (Table 1); N[N], R[R], N[fN], R[fR], N[cN], R[cR], and N[pR]. Of these, N[fN] and R[fR] ghosts were reconstituted normocyte (N) and reticulocyte (R) ghosts loaded with filtered normocyte [fN] and reticulocyte [fR] lysates, respectively. N[cN] and R[cR] ghosts were loaded with centrifuged normocyte [cN] and reticulocyte lysate [cR], respectively. A pellet was obtained only when the reticulocyte lysate was centrifuged. The pellet was resuspended in the normocyte lysate [pR] and then loaded into normocyte ghosts (N[pR] ghosts). For preparing the reconstituted normocyte ghosts containing various concentrations of ATP, 1.0, 2.5, 5.0, and 10 mM of Mg-ATP were added to the ghost suspension, and loaded into normocyte ghosts. 2.4. Culture of B. gibsoni
2.3. Preparation of reconstituted erythrocyte ghost Reconstituted normocyte ghost loaded with normocyte lysate (N[N] ghosts) and reconstituted reticulocyte
For preparation of B. gibsoni-infected erythrocytes, blood was collected from an experimentally B. gibsoniinfected dog with a parasitemia rate of that varied from
Table 1 Types of reconstituted erythrocyte ghosts prepared for the present study Abbreviations
Type of reconstituted ghosts
Treatment of lysate loaded into ghost
N[N] R[R] N[fN] R[fR] N[cN] R[cR] N[pR]
Normocyte ghosts Reticulocyte ghosts Normocyte ghosts Reticulocyte ghosts Normocyte ghosts Reticulocyte ghosts Normocyte ghosts
Normocyte lysate Reticulocyte lysate Filtered normocyte lysate Filtered reticulocyte lysate Centrifuged normocyte lysate Centrifuged reticulocyte lysate Normocyte lysate including the pellet from reticulocyte lysate
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4.8 to 6.2% and washed with PBS solution. B. gibsoni was cultivated as described in the previous report (Yamasaki et al., 2000a). 2.5. Measurement of intracellular ATP in reconstituted erythrocyte ghosts The concentration of intracellular ATP in reconstituted erythrocyte ghosts was measured according to the method of Beutler (1984). 2.6. Electron microscopy of reconstituted erythrocyte ghosts Reconstituted erythrocyte ghosts prepared as described above were sedimented at 5000g for 10 min and fixed in 2.5% (v/v) glutaraldehyde, 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 °C. They were rinsed at least three times in ice-cold 0.1 M phosphate buffer, pH 7.4 and then postfixed in 2% OsO4, 0.1 M phosphate buffer, pH 7.4 at 4 °C for 2 h. After rinsed in buffer and H2 O, the ghosts were dehydrated in ethanol, and embedded in epoxy resin. The epoxy resin consisted of 9.1 ml of Quetol-812 (Nissin EM), 6.2 ml of dodecenyl succinic anhydride (Nissin EM), 4.6 ml of methyl nadic anhydride (Nissin EM) and DMP-30 (Nissin EM). Most thin sections were stained with uranyl acetate and lead citrate, and examined with an electron microscope (JEM1210, Nissin EM). 2.7. Statistics Statistical analysis was carried out using StudentÕs t test.
3. Results 3.1. The multiplication of B. gibsoni in intact reticulocytes and HK erythrocytes When B. gibsoni-infected erythrocytes (RBC) were separately cultivated with dog HK erythrocytes, normal dog normocytes, and normal dog reticulocytes, the amount of parasitemia was significantly higher in reticulocyte culture than in the normocyte culture. The parasitemia in the HK erythrocyte culture was intermediate between those in the reticulocyte and the normocyte cultures (Fig. 1). 3.2. The multiplication of B. gibsoni in various reconstituted erythrocyte ghosts Table 1 shows various reconstituted erythrocyte ghosts prepared for this experiment. When B. gibsoniinfected RBCs were cultivated in either N[N] or R[R]
Fig. 1. Babesia gibsoni were cultivated in normocytes (open circles), reticulocytes (closed circles), and HK erythrocytes (open squares). Data are expressed as means SD (n ¼ 3). * Significantly different from the value for the normocyte culture (p < 0:05). y Significantly different from the value for the HK erythrocyte culture (p < 0:05).
ghosts, the numbers of parasitized cells began to increase at cultivation day 1. During cultivation for 6 days, a difference in the multiplication of the parasite was observed between N[N] and R[R] cultures. In R[R] culture, the level of parasitemia increased parabolically and reached 3:5 0:7% at day 5, whereas the highest parasitemia in N[N] culture was 2:1 0:6% at day 5 (Figs. 2a and b). When B. gibsoni-infected RBC were cultivated in either R[fR] or R[cR] ghosts, the level of parasitemia in each culture was lower than that in R[R] culture, and almost the same level as that in N[N] culture (Fig. 2b). On the other hand, when N[pR] ghosts were used for the cultivation of B. gibsoni, the level of parasitemia in N[pR] culture was higher than that in R[cR] culture, and the level of parasitemia in N[pR] culture was almost the same as that in R[R] culture during the cultivation period (Fig. 2a). Next, the concentrations of intracellular ATP in N[N], R[R], N[cN], R[cR], and N[pR] ghosts were measured. As shown in Table 2, the concentrations of intracellular ATP in R[R] and N[pR] ghosts were 4 and 1.8 times that in N[N] ghosts, respectively, whereas those in N[cN] and R[cR] ghosts were at almost the same level as that in N[N] ghosts. As shown in Fig. 3a, the electron microscopic observation of the pellet from reticulocyte lysate revealed many mitochondria. Several mitochondria were also
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Fig. 2. Babesia gibsoni was cultivated in reconstituted erythrocyte ghosts. (a) N[N] (open circles), N[fN] (open squares), N[cN] (open triangles), and N[pR] (closed circles) ghost. (b) R[R] (closed circles), R[fR] (closed squares), and R[cR] (closed triangles) ghosts. Data are expressed as means SD (n ¼ 3). * Significantly different from the value for the N[N] culture (p < 0:05).
Table 2 The concentrations of intracellular ATP in N[N], R[R], N[cN], R[cR], and N[pR] ghosts Ghost type N[N] ATP concentration (lmol=109 ghosts) *
1:7 0:58
R[R]
6:9 1:1
N[cN]
R[cR]
N[pR]
1:6 0:79
1:6 1:2
3:1 0:50
Data are expressed as means SD (n ¼ 3). Values are significantly (p < 0:05) higher than those of N[N] ghosts.
observed within N[pR] ghosts (Fig. 3b), whereas no organelles were observed in the all observed N[N] ghosts (data not shown). Reconstituted normocyte ghosts containing various concentrations of ATP were prepared by incubating the ghosts with 1, 2.5, 5, or 10 mM ATP. The concentrations of intracellular ATP in those reconstituted normocyte ghosts are shown in Table 3. When the reconstituted normocyte ghosts were used as host cells for the cultivation of B. gibsoni, proportional increases of parasitemia were observed at the concentrations of 0.29, 1.7, and 2.8 mM ATP, while the level of parasitemia at 5.4 mM ATP was almost the same as that at 2.8 mM ATP (Table 3).
4. Discussion As described elsewhere, previous studies suggested that high concentrations of glutamate and GSH in reticulocytes might be a cause of the preferential multiplication of B. gibsoni in reticulocytes (Yamasaki et al.,
2000a). In the present study, however, B. gibsoni parasites multiplied well in reticulocytes even compared with HK dog erythrocytes possessing higher concentrations of glutamate and GSH than reticulocytes (Yamasaki et al., 2000a,b). This suggested that reticulocytes might contain other essential substances for multiplication of B. gibsoni. Reticulocytes are immature erythrocytes and differ from normocyte in their biochemical and morphologic characteristics. Reticulocytes have higher concentrations of ATP, GSH, amino acids and nucleic acids than do normocytes (Mons, 1990). The most prominent difference between reticulocytes and normocytes, as well as HK erythrocytes, is that reticulocytes still contain some micro-organelles, such as mitochondria in their cytoplasm, while both normocyte and HK erythrocytes have no micro-organelles. Thus, it was hypothesized that the preferential multiplication of B. gibsoni in reticulocytes might depend on those characteristics of reticulocytes, especially the presence of mitochondria in the cells, because mitochondria are an energy-converting organelles involved in the energy metabolism of the cell.
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Fig. 3. Electron microscopic observation of N[pR]. (a) The pellet, which was obtained from reticulocyte lysate by centrifugation, included many mitochondria 25,000. (b) N[pR] ghost. Mitochondria are observed in an N[pR] ghost (arrowheads) 20,000. Scale bar ¼ 500 nm.
Table 3 Concentrations of intracellular ATP and the level of parasitemia of B. gibsoni in reconstituted normocyte ghosts loaded with various concentrations of ATP Concentration of added ATP 1 mM (N[N]) 9
ATP concentration (lmol=10 ghosts) Parasitemia (%) with each ghostsa
0:59 0:38 2:15 0:54
2.5 mM 1:7 0:60 3:05 0:22
5 mM
10 mM
2:8 0:74 3:55 0:56
5:4 0:58 3:5 0:22
Data are expressed as means SD (n ¼ 3). Values are significantly (p < 0:05) higher than those of reconstituted normocyte ghosts with 1 mM Mg-ATP. a Babesia gibsoni was cultivated in reconstituted normocyte ghosts with various concentrations of Mg-ATP. *
The present study clearly showed that the mitochondria play a central role in the preferential multiplication of B. gibsoni parasites in reticulocytes. That is, the level of parasitemia in the culture using reconstituted normocyte ghosts loaded with mitochondria from reticulocytes (N[pR]) was increased to the same level as in the culture using the reconstituted reticulocyte ghosts (R[R]). In contrast, when mitochondria were removed from the R[R] ghosts, the level of parasitemia in the culture of those ghosts was decreased to that in the culture using the reconstituted normocyte ghosts (N[N]). In general, reticulocytes generate a considerable amount of ATP via the tricarboxylic acid cycle in mitochondria in addition to glycolysis, whereas ATP is chiefly generated by glycolysis in normocytes. As a result, reticulocytes have a higher concentration of intracellular ATP than normocytes. In the present study, the higher
concentration of intracellular ATP observed in N[pR] ghosts seemed to be generated by mitochondria embedded in the ghosts. Furthermore, the present study also showed that the level of parasitemia in the cultures with reconstituted normocyte ghosts was proportional to the intracellular ATP concentration of each reconstituted normocyte ghost. From these results, we concluded that the predominant cause of the predilection of B. gibsoni parasites for reticulocytes was chiefly the high concentration of ATP generated by mitochondria in the cells. In a previous study, we postulated that a high concentration of intracellular GSH, a tripeptide, which protects intracellular components from oxidative attack, might be an important factor in promoting the high multiplication of B. gibsoni in HK cells as well as reticulocytes. A recent study of de novo synthesis of GSH in malaria-infected erythrocytes has shown that the
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infection with Plasmodium falciparum causes drastic changes in the GSH metabolism of infected erythrocytes (Luersen et al., 2000). That is, infected erythrocytes lose GSH at a rate 40-fold higher than non-infected cells. It has also been reported that the formation of reactive oxygen species in P. falciparum-infected erythrocytes is increased as a result of hemoglobin digestion by the parasite, resulting in an enhanced requirement for GSH (Hunt and Stocker, 1990). Our recent study also showed that superoxide anions were produced in B. gibsoni-infected erythrocytes (Otsuka et al., 2001). These results indicated that the de novo synthesis of the tripeptide in erythrocytes was also essential for the parasiteÕs survival. In erythrocytes, even mature ones, GSH is synthesized from glutamate and 2 amino acids via 2 enzymatic reactions accompanied by conversion of ATP to ADP and inorganic phosphate. Thus, increased GSH synthesis may result in increased utilization of ATP in infected erythrocytes, indicating that a high concentration of ATP is of great advantage for the multiplication of B. gibsoni in erythrocytes. On the other hand, the penetration of host erythrocytes by the B. gibsoni parasite may be linked to intracellular ATP as reported for malaria parasites (Elford et al., 1995). It has been shown that the entry of the malaria parasite into erythrocytes requires the presence of ATP in the host cell cytoplasm (Dluzewski et al., 1983; Olson and Kilejian, 1982; Rangachari et al., 1986). The role of intracellular ATP in the multiplication of B. gibsoni, however, has not been elucidated. If it is clarified, it will undoubtedly be possible to unravel some of the more complex mechanisms concerning the invasion and development of the parasite in host erythrocytes. Acknowledgments This work was supported in part by a grant from the Science Research Fund of the Ministry of Education, Science, and Culture of Japan.
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