Parasitol Res (1992) 78:707-708
Parasitnlngy Research 9 Springer-Verlag1992
Demonstration of extraehromosmal D N A from Babesia equi merozoites Christian Schelp, Reinhard B6se, and Karl T. Friedhoff Institute of Parasitology, Hannover School of Veterinary Medicine, Bfinteweg 17, W-3000 Hannover 71, Federal Republic of Germany Accepted August 17, 1992
Abstract. A n extrachromosomal nucleic acid element was detected in high-molecular-weight D N A preparations form Babesia equi merozoites. This extrachromosomal element was shown to be D N A rather than R N A and had an apparent fragment size of about 9 kilobasepairs (kb). Hybridization experiments using purified 9kb D N A as a probe revealed sequence homologies with extrachromosomal D N A from two other Babesia species.
Babesia equi is a tick-borne p r o t o z o a n parasite and besides B. caballi, it is the causative agent o f equine babesiosis. B. equi differs significantly from B. caballi and most other babesial parasites. B. equi appears to be capable o f infecting lymphocytes in the vertebrate host and is not transmitted transovarially in the tick vector (Schein 1988). These characteristics are shared by Theileria species composing the closely related genus Theileria within the subclass Piroplasmia (Mehlhorn and Schein 1984). The erythrocytic stages of Theileria spp. and Babesia spp. have been well investigated and possess some interesting features, including the presence of mitochondrialike structures composed o f two concentric membranes (Weber 1978; Igarashi et al. 1988). The respiratory function of these organelles has long been in question because of the absence o f cristae. Cytochrome oxidase activity as an indication for an operative respiratory chain has been shown for T. annulata sporozoites from salivary glands o f ticks but not for erythrocytic stages (Weber 1978). More recently, an extrachromosomal D N A element of about 6 kb in T. annulata has been described that is transcribed during different stages of the parasite lifecycle (Hall et al. 1990) and has a coding capacity for apocytochrome b (Megson et al. 1991). Therefore, this D N A element is supposed to be part o f the mitochondrial genome. These are striking similarities to the 6-kb D N A element of malaria parasites, which likewise codes for mitochondrial proteins (Vaidya et al. 1989;
Correspondence to: C. Schelp
Feagin 1992). In the present report we describe an extrachromosomal D N A element from B. equi with sequence homologies to D N A from B. divergens and B.
ovis.
Materials and methods Parasites were isolated from the red blood cells of splenectomized and experimentally infected horses (Babesia equi, strain USDA), sheep (B. ovis, strain Ankara), and calves (B. divergens, strain Hullmann) essentially according to published procedures (Conrad et al. 1987). At the peak of parasitemia, blood was collected and washed three times with phophate-buffered saline (PBS; 1600 g, 10 min, 4~ C). The supernatant and part of the buffy coat was removed by suction after each centrifugation step. The final sediment was wanned to 37~ C and mixed with an equal volume of prewarmed (37~ C) sapoiain (1 mg/ml in H20). After incubation for 10 s at 37~ C, 4 vol. TEN [20 mM TRIS-HC1, 10 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaC1; pH 8.0] were added and the lysate was centrifuged at 15000 g, for 30 rain at 40 C. The parasite cells (cloudy sediment) were separated from leukocyte nuclei and unlysed host cells (hard sediment); the centrifugation step was repeated four times to minimize the contamination of parasite DNA preparations with DNA from leukocytes. High-molecular-weight DNA from horse lymphocytes and from B. equi merozoites was isolated by the salting-out procedure adopted from Miller et al. (1988), starting with 10 ml saponin lysate from each parasite preparation. Electrophoresis, restriction-enzyme digestion, and transfer of DNA to nylon membranes were performed according to standard procedures (Sambrook et al. 1989). The 9-kb element was purified by cutting the band out of an agarose gel and adsorbing the DNA to glass milk using a commercially available kit. The instructions given by the manufacturer were followed exactly (Quiaex, Quiagen, Dfisseldorf, FRG). The purified DNA was labeled with digoxigenin by the random priming method using the kit from Boehringer Mannheim (FRG). Hybridization was performed under conditions of reduced stringency at 55~ in saline sodium citrate (5 • 5% blocking reagent (Boehringer Mannheim, FRG), 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate (SDS) and was followed by washing in 5 x SSC/0A% SDS for several hours at hybridization temperature. Under more stringent conditions, .the DNA was subjected to hybridization at 68~ C in hybridization buffer followed by washing in 2 x SSC/0.1% SDS at room temperature and 0.1 x SSC/0.1% SDS at 50~ C. DNA hybrids were detected by enzyme-catalyzed color reaction using the detection kit from Boehringer Mannheim (FRG).
708
Results and discussion In D N A preparations f r o m Babesia equi merozoites, a nucleic acid fragment of a b o u t 9 kb could be separated f r o m the genomic D N A by agarose gel electrophoresis (Fig. 1, lane 3). This fragment was not seen in control preparations f r o m horse leukocytes (Fig. 1, lane 1), indicating that it was o f parasite origin and not an artefact. It was accessible to restriction-enzyme digestion with Eco R I and Hind I I I (Fig. 1, lanes 4, 6), leading to several fragments o f a b o u t 2 kb (lane 4) and two fragments of 3 and 6 kb (lane 6), respectively, Barn H I probably did not cleave the 9-kb b a n d (lane 5). Further evidence that the 9-kb band was D N A was provided by the observation that it was not affected by treatment with R N a s e A or electrophoresis under alkaline conditions. Moreover, it could be labeled by the standard r a n d o m - p r i m ing labeling technique w h e n isolated from a gel and could b e cloned into a plasmid (data not shown). D N A elements o f a size similar to that of the 9-kb band f r o m B. equi were also observed in D N A preparations f r o m B. divergens and B. ovis. The bands hybridized to the gel purified and labeled 9-kb D N A f r o m B. equi under conditions of low stringency. U n d e r m o r e stringent conditions, the probe hybridized solely to the 9-kb band of B. equi. A significant difference in fragment length was noted between the Babesia species. The ext r a c h r o m o s o m a l D N A f r o m B. equi was a b o u t 3 kb longer than the equivalents f r o m B. divergens and B. ovis as judged by agarose gel electrophoresis. The latter D N A s resembled the 6-kb D N A element of Plasmodium
Fig. 1. Detection of extrachromosomal DNA. DNA (5 ~tg) from horse leukocytes (lanes 1, 2) or Babesia equi merozoites (lanes 3-6) were subjected to electrophoresis through a 0.8% agarose gel and stained with ethidium bromide. The DNA was digested with Eco RI (lanes 2, 4), Bam HI (lane 5) and Hind III (lane 6) or remained uncleared (lanes 1, 3) Prior to electrophoresis. The bars on the left denote markers of the indicated size in kilobase pairs (kb).
spp. and Theileria annulata in terms of their length (Joseph et al. 1989; Hall et al. 1990). E x t r a c h r o m o s o m a l D N A can originate f r o m several different sources, the m o s t c o m m o n examples being organelle genomes, viral genomes, or amplified genomic D N A sequences. The existence of an e x t r a c h r o m o s o m a l D N A element of a b o u t 6 kb with conserved sequences coding for cytochrome b and cytochrome oxidase has been assumed to be a special feature o f members of the p h y l u m Apicomplexa (Joseph et al. 1989; Feagin et al. 1991). The 6-kb element is highly conserved a m o n g Plasmodium spp. and hybridizes to D N A from several apicomplexan parasites (e.g., Toxoplasma gondii, Eimeria tenella, T. parva, and B. microti) under low-stringency conditions (Joseph et al. 1989). This demonstrates that the extrachromosomal D N A elements f r o m apicomplexan parasites are related and m a y have the same origin. Our data suggest a relatedness of the 9-kb band of B. equi and the 6-kb band of B. divergens and B. ovis to the 6-kb D N A element o f T. annulata and other apicomplexan parasites. Sequencing of the cloned 9-kb D N A fragment will give further informations that might support this assumption.
References Conrad PA, Iams K, Brown WC, Sohanapal B, ole-MoiYoi OK (1987) DNA probes detect genomic diversity in Theileriaparva stocks. Mol Biochem Parasitol 25 : 213-226 Feagin JE (1992) The 6-kb element of Plasmodium falciparum encodes mitochondrial cytochrome genes. Mol Biochem Parasitol 52:145-148 Feagin JE, Gardener MJ, Williamson DH, Wilson RJM (1991) The putative mitochondrial genome of Plasmodiumfalciparum. J Protozool 38:243-245 Hall R, Coggins L, McKellar S, Shiels B, Tait A (1990) Characterisation of an extrachromosomal DNA element from Theileria annulata. Mol Biochem Parastiol 38 : 253-260 Igarashi I, Aikawa M, Kreier JP (1988) Host cell-parasite interactions in babesiosis. In: M Ristic (ed) Babesiosis of domestric animals and man. CCR, Boca Raton, pp 53-69 Joseph JT, Aldritt SM, Unnasch T, Puijalon O, Wirth DF (1989) 9Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaeeum. Mol Cell Biol 9:3621-3629 Megson A, Inman: GJ, Hunt PD, Baylis HA, Hall R (1991) The gene for apocytochrome B of Theiteria annulata resides on a small linear extrachromosomal element. Mol Biochem Parasitol 48:113-116 Mehlhorn H, Schein E (1984) The piroplasms: life cycle and sexual stages. Adv Parasitol 23:37-103 Miller SA, Dykes DD, Polesky JF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York Schein E (1988) Equine babesiosis. In: M Ristie (ed) Babesiosis of domestic animals and man. CCR, Boca Raton, pp 197-208 Vaidya AB, Akella R, Suplik K (1989) Sequences similar to genes for two mitochondrial proteins and portions of ribosomal RNA in tandemly arrayed 6-kilobase-pair DNA of a malaria parasite. Mol Biochem Parasitol 35:97-108 Weber G (1978) Atypical mitochondria in an intracetlular protozoan (Theileria annulata, Apicomplexa). Naturwissenschaften 65:601
Parasitnlngy Research 9 Springer-Verlag1992
Demonstration of extraehromosmal D N A from Babesia equi merozoites Christian Schelp, Reinhard B6se, and Karl T. Friedhoff Institute of Parasitology, Hannover School of Veterinary Medicine, Bfinteweg 17, W-3000 Hannover 71, Federal Republic of Germany Accepted August 17, 1992
Abstract. A n extrachromosomal nucleic acid element was detected in high-molecular-weight D N A preparations form Babesia equi merozoites. This extrachromosomal element was shown to be D N A rather than R N A and had an apparent fragment size of about 9 kilobasepairs (kb). Hybridization experiments using purified 9kb D N A as a probe revealed sequence homologies with extrachromosomal D N A from two other Babesia species.
Babesia equi is a tick-borne p r o t o z o a n parasite and besides B. caballi, it is the causative agent o f equine babesiosis. B. equi differs significantly from B. caballi and most other babesial parasites. B. equi appears to be capable o f infecting lymphocytes in the vertebrate host and is not transmitted transovarially in the tick vector (Schein 1988). These characteristics are shared by Theileria species composing the closely related genus Theileria within the subclass Piroplasmia (Mehlhorn and Schein 1984). The erythrocytic stages of Theileria spp. and Babesia spp. have been well investigated and possess some interesting features, including the presence of mitochondrialike structures composed o f two concentric membranes (Weber 1978; Igarashi et al. 1988). The respiratory function of these organelles has long been in question because of the absence o f cristae. Cytochrome oxidase activity as an indication for an operative respiratory chain has been shown for T. annulata sporozoites from salivary glands o f ticks but not for erythrocytic stages (Weber 1978). More recently, an extrachromosomal D N A element of about 6 kb in T. annulata has been described that is transcribed during different stages of the parasite lifecycle (Hall et al. 1990) and has a coding capacity for apocytochrome b (Megson et al. 1991). Therefore, this D N A element is supposed to be part o f the mitochondrial genome. These are striking similarities to the 6-kb D N A element of malaria parasites, which likewise codes for mitochondrial proteins (Vaidya et al. 1989;
Correspondence to: C. Schelp
Feagin 1992). In the present report we describe an extrachromosomal D N A element from B. equi with sequence homologies to D N A from B. divergens and B.
ovis.
Materials and methods Parasites were isolated from the red blood cells of splenectomized and experimentally infected horses (Babesia equi, strain USDA), sheep (B. ovis, strain Ankara), and calves (B. divergens, strain Hullmann) essentially according to published procedures (Conrad et al. 1987). At the peak of parasitemia, blood was collected and washed three times with phophate-buffered saline (PBS; 1600 g, 10 min, 4~ C). The supernatant and part of the buffy coat was removed by suction after each centrifugation step. The final sediment was wanned to 37~ C and mixed with an equal volume of prewarmed (37~ C) sapoiain (1 mg/ml in H20). After incubation for 10 s at 37~ C, 4 vol. TEN [20 mM TRIS-HC1, 10 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaC1; pH 8.0] were added and the lysate was centrifuged at 15000 g, for 30 rain at 40 C. The parasite cells (cloudy sediment) were separated from leukocyte nuclei and unlysed host cells (hard sediment); the centrifugation step was repeated four times to minimize the contamination of parasite DNA preparations with DNA from leukocytes. High-molecular-weight DNA from horse lymphocytes and from B. equi merozoites was isolated by the salting-out procedure adopted from Miller et al. (1988), starting with 10 ml saponin lysate from each parasite preparation. Electrophoresis, restriction-enzyme digestion, and transfer of DNA to nylon membranes were performed according to standard procedures (Sambrook et al. 1989). The 9-kb element was purified by cutting the band out of an agarose gel and adsorbing the DNA to glass milk using a commercially available kit. The instructions given by the manufacturer were followed exactly (Quiaex, Quiagen, Dfisseldorf, FRG). The purified DNA was labeled with digoxigenin by the random priming method using the kit from Boehringer Mannheim (FRG). Hybridization was performed under conditions of reduced stringency at 55~ in saline sodium citrate (5 • 5% blocking reagent (Boehringer Mannheim, FRG), 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate (SDS) and was followed by washing in 5 x SSC/0A% SDS for several hours at hybridization temperature. Under more stringent conditions, .the DNA was subjected to hybridization at 68~ C in hybridization buffer followed by washing in 2 x SSC/0.1% SDS at room temperature and 0.1 x SSC/0.1% SDS at 50~ C. DNA hybrids were detected by enzyme-catalyzed color reaction using the detection kit from Boehringer Mannheim (FRG).
708
Results and discussion In D N A preparations f r o m Babesia equi merozoites, a nucleic acid fragment of a b o u t 9 kb could be separated f r o m the genomic D N A by agarose gel electrophoresis (Fig. 1, lane 3). This fragment was not seen in control preparations f r o m horse leukocytes (Fig. 1, lane 1), indicating that it was o f parasite origin and not an artefact. It was accessible to restriction-enzyme digestion with Eco R I and Hind I I I (Fig. 1, lanes 4, 6), leading to several fragments o f a b o u t 2 kb (lane 4) and two fragments of 3 and 6 kb (lane 6), respectively, Barn H I probably did not cleave the 9-kb b a n d (lane 5). Further evidence that the 9-kb band was D N A was provided by the observation that it was not affected by treatment with R N a s e A or electrophoresis under alkaline conditions. Moreover, it could be labeled by the standard r a n d o m - p r i m ing labeling technique w h e n isolated from a gel and could b e cloned into a plasmid (data not shown). D N A elements o f a size similar to that of the 9-kb band f r o m B. equi were also observed in D N A preparations f r o m B. divergens and B. ovis. The bands hybridized to the gel purified and labeled 9-kb D N A f r o m B. equi under conditions of low stringency. U n d e r m o r e stringent conditions, the probe hybridized solely to the 9-kb band of B. equi. A significant difference in fragment length was noted between the Babesia species. The ext r a c h r o m o s o m a l D N A f r o m B. equi was a b o u t 3 kb longer than the equivalents f r o m B. divergens and B. ovis as judged by agarose gel electrophoresis. The latter D N A s resembled the 6-kb D N A element of Plasmodium
Fig. 1. Detection of extrachromosomal DNA. DNA (5 ~tg) from horse leukocytes (lanes 1, 2) or Babesia equi merozoites (lanes 3-6) were subjected to electrophoresis through a 0.8% agarose gel and stained with ethidium bromide. The DNA was digested with Eco RI (lanes 2, 4), Bam HI (lane 5) and Hind III (lane 6) or remained uncleared (lanes 1, 3) Prior to electrophoresis. The bars on the left denote markers of the indicated size in kilobase pairs (kb).
spp. and Theileria annulata in terms of their length (Joseph et al. 1989; Hall et al. 1990). E x t r a c h r o m o s o m a l D N A can originate f r o m several different sources, the m o s t c o m m o n examples being organelle genomes, viral genomes, or amplified genomic D N A sequences. The existence of an e x t r a c h r o m o s o m a l D N A element of a b o u t 6 kb with conserved sequences coding for cytochrome b and cytochrome oxidase has been assumed to be a special feature o f members of the p h y l u m Apicomplexa (Joseph et al. 1989; Feagin et al. 1991). The 6-kb element is highly conserved a m o n g Plasmodium spp. and hybridizes to D N A from several apicomplexan parasites (e.g., Toxoplasma gondii, Eimeria tenella, T. parva, and B. microti) under low-stringency conditions (Joseph et al. 1989). This demonstrates that the extrachromosomal D N A elements f r o m apicomplexan parasites are related and m a y have the same origin. Our data suggest a relatedness of the 9-kb band of B. equi and the 6-kb band of B. divergens and B. ovis to the 6-kb D N A element o f T. annulata and other apicomplexan parasites. Sequencing of the cloned 9-kb D N A fragment will give further informations that might support this assumption.
References Conrad PA, Iams K, Brown WC, Sohanapal B, ole-MoiYoi OK (1987) DNA probes detect genomic diversity in Theileriaparva stocks. Mol Biochem Parasitol 25 : 213-226 Feagin JE (1992) The 6-kb element of Plasmodium falciparum encodes mitochondrial cytochrome genes. Mol Biochem Parasitol 52:145-148 Feagin JE, Gardener MJ, Williamson DH, Wilson RJM (1991) The putative mitochondrial genome of Plasmodiumfalciparum. J Protozool 38:243-245 Hall R, Coggins L, McKellar S, Shiels B, Tait A (1990) Characterisation of an extrachromosomal DNA element from Theileria annulata. Mol Biochem Parastiol 38 : 253-260 Igarashi I, Aikawa M, Kreier JP (1988) Host cell-parasite interactions in babesiosis. In: M Ristic (ed) Babesiosis of domestric animals and man. CCR, Boca Raton, pp 53-69 Joseph JT, Aldritt SM, Unnasch T, Puijalon O, Wirth DF (1989) 9Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaeeum. Mol Cell Biol 9:3621-3629 Megson A, Inman: GJ, Hunt PD, Baylis HA, Hall R (1991) The gene for apocytochrome B of Theiteria annulata resides on a small linear extrachromosomal element. Mol Biochem Parasitol 48:113-116 Mehlhorn H, Schein E (1984) The piroplasms: life cycle and sexual stages. Adv Parasitol 23:37-103 Miller SA, Dykes DD, Polesky JF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York Schein E (1988) Equine babesiosis. In: M Ristie (ed) Babesiosis of domestic animals and man. CCR, Boca Raton, pp 197-208 Vaidya AB, Akella R, Suplik K (1989) Sequences similar to genes for two mitochondrial proteins and portions of ribosomal RNA in tandemly arrayed 6-kilobase-pair DNA of a malaria parasite. Mol Biochem Parasitol 35:97-108 Weber G (1978) Atypical mitochondria in an intracetlular protozoan (Theileria annulata, Apicomplexa). Naturwissenschaften 65:601
