Wadsworth Center, New York State Department of Health, PO Box 22002, Albany, NY 12201-2002, USA1
Author for correspondence: Guan Zhu. Tel: +1 518 474 2187. Fax: +1 518 473 6150. e-mail: zhug{at}wadsworth.org
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ABSTRACT |
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Keywords: Cryptosporidium parvum, plastid genome, apicoplast, chloroplast
Abbreviations: FAS, fatty acid synthase; gDNA, genomic DNA; LSU, large subunit; SSU, small subunit
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INTRODUCTION |
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Cryptosporidium parvum is an emerging pathogen that causes one of the opportunistic infections in AIDS patients. Although C. parvum is currently assigned to the class Coccidia, evidence based upon SSU rRNA sequences indicates that Cryptosporidium does not form a monophyletic clade with either intestinal or cyst-forming coccidia, and that the genus is instead monophyletic to the Coccidia + Haematozoa (Escalante & Ayala, 1995 ; Gajadhar et al., 1991
; Van de Peer & De Wachter, 1997
). In fact, others have shown that the class Coccidia is monophyletic only if the genus Cryptosporidium is excluded (Morrison & Ellis, 1997
), and that the genus can even be used as an outgroup to determine the phylogenetic position of eucoccidia (Barta et al. 1997
).
In addition to its elusive phylogenetic position, several fundamental distinctions between C. parvum and other coccidia have been described, including: (1) the extracytoplasmic, but intracellular, location of the parasite just beneath the enterocyte apical membrane (Fayer et al., 1997 ); (2) polyamine synthesis by arginine rather than ornithine decarboxylation (Keithly et al., 1997
); (3) absence of introns in most genes; (4) insensitivity to anticoccidial drugs (Coombs, 1999
); and (5) sporulation of oocysts within the gut lumen, resulting in enterocyte reinvasion and prolonged, life-threatening infection in patients who are immunocompromised. Since there is currently no effective treatment against cryptosporidiosis, considerable effort has been and is still being expended toward the discovery of cytosolic or organellar metabolic pathways which could serve as leads for therapy (Coombs, 1999
).
Previously, others had observed both a plastid-like organelle in sporozoites of C. parvum (Tetley et al., 1998 ) and a low-molecular-mass band in pulse-field gels prepared from them (Blunt et al., 1997
). However, in contrast to work with P. falciparum and T. gondii, neither these nor other studies provided direct biochemical or molecular evidence for the presence of an apicoplast or its genome in C. parvum. Therefore, in this study both intracellular stages and free sporozoites of C. parvum were tested for the presence of a plastid genome. Specifically, gDNA from intracellular stages was amplified by PCR using primers specific for plastid LSU/SSU rRNA and tufA-tRNAPhe genes, whereas gDNA of sporozoites was tested by dot-blot hybridization using T. gondii LSU/SSU rRNA and tufA-tRNAPhe plastid genes as probes. A number of organisms with or without a plastid were used as controls for both experiments. Unexpectedly, no plastid genome was detected in C. parvum, whereas its presence in plants, a euglenoid and other apicomplexans used as positive controls was uniformly confirmed. This finding not only provides new molecular evidence for the divergence of C. parvum from its nearest relatives, but suggests that the plastid genome may be unavailable as a drug target in this opportunistic pathogen.
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METHODS |
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Detection by PCR.
Six pairs of degenerate, plastid-specific primers were used to amplify several highly conserved regions of known organellar genes, including three pairs of the LSU rRNA and two pairs of the SSU rRNA or tufA-tRNAPhe genes (Table 1). One pair of primers specific to a C. parvum P-type ATPase (Zhu & Keithly, 1997
) served as the positive control in all experiments. The DNA tested included that isolated from uninfected and C. parvum-infected HCT-8 cells, or that from the known plastid-containing eukaryotes T. gondii, Eimeria bovis, A. stellatum and S. oleracea. Each 50 µl PCR reaction consisted of hot-start TaqBead PCR reagents (Promega) plus 0·2 µM each of the primers and other appropriate reagents. Additional conditions of amplification are detailed in Table 1
.
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RESULTS AND DISCUSSION |
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Analysis by PCR indicates that C. parvum has no plastid genes
No plastid product was amplified from C. parvum regardless of the primers used (Fig. 1F). As expected, however, all six pairs of primers amplified products from the apicomplexans T. gondii and E. bovis (Fig. 1A
, B
). To confirm their identity as plastid genes, one amplicon each of the E. bovis LSU, SSU and tufA-tRNAPhe genes was cloned into a pCR2.1 vector using a TA-cloning kit (Invitrogen) and sequenced. Further support was obtained when an additional four pairs of primers amplified LSU and SSU genes from the plants wild scallions and spinach (Fig. 1C
, D
). Together these data suggest that degenerate primers, especially those for rRNA, can be used to amplify plastid genomes from a wide range of eukaryotes. That some non-specific priming of organellar rRNA genes can occur is shown by the single SSU rRNA product amplified from uninfected and C. parvum-infected HCT-8 cells (lane 6 in Fig. 1E
, F
). When this band was cloned and sequenced it was shown to be a human mitochondrial, rather than a plastid, rRNA gene. This is not especially surprising since it is well known that some regions of mitochondrial DNA have homologous regions within plastid DNA that can cause apparent cross-hybridization (Supplick et al., 1988
). The failure to amplify apicoplast genes was not due to denatured gDNA since consistent amplification of a C. parvum-specific calcium transporter (Zhu & Keithly, 1997
), which could not be amplified by PCR from a wide variety of organisms (Fig. 1A
E
), always occurred. Therefore, we propose that the most logical explanation is the absence of an organellar template for the rRNA and tufA-tRNAPhe genes in C. parvum.
Plastid probes do not hybridize to C. parvum gDNA
To further test the hypothesis that there is no plastid genome in C. parvum, the gDNA of a representative group of eukaryotes with and without a plastid, as well as a eubacterium, were examined by dot-blot hybridization using both a C. parvum nucleus-encoded rRNA and several T. gondii plastid genes as probes. As expected, the 7·8 kb C. parvum rRNA probe hybridized strongly to the gDNA samples of all Apicomplexa, less strongly to those of a euglenoid, kinetoplastids and yeast, slightly to those of mammals, and not at all to the prokaryote E. coli (Fig. 2). On the other hand, the T. gondii plastid probe containing >6·2 kb rRNA and tufA-tRNAPhe genes strongly hybridized to gDNA isolated from all plastid-containing organisms, including the apicomplexans E. bovis, P. falciparum and T. gondii, the plants A. stellatum and S. oleracea and the euglenoid E. gracilis, but not to those without plastids (mammals, yeast, kinetoplastids and E. coli [except for a slight hybridization in HCT-8 cells and E. coli]) (Fig. 2
). Thus, these observations support the PCR data showing the failure of plastid probes to hybridize with C. parvum gDNA, and suggest that C. parvum indeed lacks the apicoplast homologues for rRNA and tufA-tRNAPhe genes.
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Using sporozoites of C. parvum, molecular, ultrastructural and drug-testing data suggest that C. parvum has a mitochondrion (Riordan et al., 1999 ), thus supporting the hypothesis that no pre-mitochondrial eukaryotic species is extant today (Martin & Muller, 1998
). First, our data have shown that three signature genes for mitochondrial function are encoded by the nuclear genome of C. parvum, including the genes for adenylate kinase 2 (CpAK2), valyl-tRNA synthase and chaperonin 60. Phylogenetic analysis of CpAK2 robustly places this gene within the clade of organisms possessing mitochondria (Riordan et al., 1999
). Secondly, an unusual, ribosome-studded double-membrane-bound, acristate organelle has been identified posterior to the nucleus which resembles other protist mitochondria. Unlike others (Tetley et al., 1998
), we think it is unlikely this organelle is an apicoplast since it is located posterior to the nucleus and lacks the four enveloping membranes characteristic of plastids (Fichera & Roos, 1997
; Kohler et al., 1997
). Third, drug-testing indicates that this organelle may be a functional mitochondrion, since micromolar concentrations of naphthoquinone drugs known to inhibit respiration in other apicomplexans (Srivastava et al., 1997
) prevent growth of C. parvum in vitro.
It is now generally accepted that eukaryotes which have secondarily lost mitochondria and/or plastids once had these organelles, and that remnants of these symbiogenetic events persist in their genomes (Hashimoto et al., 1998 ; Muller, 1998
; Waller et al., 1998
). If confirmed, the loss of a plastid genome and its structure in C. parvum would be exciting, and could possibly indicate a second evolutionary fate for this organelle in the Apicomplexa. If, on the other hand, evidence for a plastid genome is eventually discovered, it might help determine whether the phylogenetic position of Cryptosporidium is truly ancestral to the Apicomplexa, and perhaps more nearly related to the dinoflagellates as some have proposed (Gajadhar et al. 1991
; Van de Peer & De Wachter, 1997
). The discovery that dinoflagellates have plastid genomes whose organization differs radically from those of their nearest relatives indicates that there may be two distinct types of plastid within the group Alveolata (Zhang et al., 1999
), which includes apicomplexans, ciliates and dinoflagellates. If C. parvum contains the one gene-one circle plastid, and perhaps an unusually reduced plastid genome like the dinoflagellates, this might help elucidate both its phylogenetic position within the Apicomplexa and among the Alveolata, as well as its lack of sensitivity to some antiplastid and most anticoccidial drugs. It is clear that the absence or presence of a plastid genome in C. parvum has valuable phylogenetic and therapeutic implications. The elucidation of these implications awaits further biochemical, molecular and phylogenetic analyses, which will require more inclusive datasets of genera within the Alveolata.
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ACKNOWLEDGEMENTS |
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Received 10 May 1999;
revised 19 August 1999;
accepted 18 October 1999.