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Genome Res. 14:1686-1695, 2004 ©2004 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/04 $5.00 Letter Comparative Analysis of Apicomplexa and Genomic Diversity in Eukaryotes1 Department of Microbiology and Immunology, Weill Medical College and the Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021, USA 2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA 3 Department of Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota 55108, USA 4 Biomedical Genomics Center, University of Minnesota, St. Paul, Minnesota 55108, USA 5 Human Genome Sciences, Rockville, Maryland 20850, USA 6 Sanaria Inc., Rockville, Maryland 20852, USA
The apicomplexans Plasmodium and Cryptosporidium have developed distinctive adaptations via lineage-specific gene loss and gene innovation in the process of diverging from a common parasitic ancestor. The two lineages have acquired distinct but overlapping sets of surface protein adhesion domains typical of animal proteins, but in no case do they share multidomain architectures identical to animals. Cryptosporidium, but not Plasmodium, possesses an animal-type O-linked glycosylation pathway, along with >30 predicted surface proteins having mucin-like segments. The two parasites have notable qualitative differences in conserved protein architectures associated with chromatin dynamics and transcription. Cryptosporidium shows considerable reduction in the number of introns and a concomitant loss of spliceosomal machinery components. We also describe additional molecular characteristics distinguishing Apicomplexa from other eukaryotes for which complete genome sequences are available.
The availability of two apicomplexan complete genome sequences, Plasmodium (Gardner et al. 2002  ) and Cryptosporidium (Abrahamsen et al. 2004), provides a unique opportunity to understand the genome-scale trends accompanying adaptation to parasitic niches in the eukaryotes. All members of the apicomplexan clade are parasitic and share specific features related to parasitism, most notably a unique apical secretory structure mediating locomotion and cellular invasion. Despite general shared features, the apicomplexans have greatly diverged in many respects, including host specificities, tissue tropisms, and the requirement of multiple hosts. Hemosporidians, such as Plasmodium and the piroplasms Theileria and Babesia, infect blood cells and are transmitted to vertebrates by hematophagous arthropod definitive hosts. Most hemosporidians show multiple tissue trophisms and transformation through multiple developmental stages, such as the hepatocyte invasion and intrahepatocytic schizogony that is observed in Plasmodium. In contrast, Cryptosporidium and the gregarines have a relatively simple parasitic strategy involving a single host and invasion of a single cell type, primarily intestinal epithelial cells.
The current phylogenetic analysis of the characterized Apicomplexa suggest a basal position for Cryptosporidium and the gregarines with respect to a poorly defined "crown group" composed of hemosporidians and coccidians (Carreno et al. 1999
The Relationship of Apicomplexans to Other Eukaryotes and the Degree of Relatedness of the Apicomplexan Proteomes To obtain a robust phylogenetic model for the relationship of apicomplexans with other eukaryotes having complete genome sequences, we prepared a concatenated multiple alignment (see Supplemental data 1) of >30 conserved C. parvum proteins, such as ribosomal proteins, DNA and RNA polymerases, translation factors, and tRNA synthetases having orthologs in Plasmodium (Apicomplexa); Arabidopsis (plants); Caenorhabditis, Drosophila and Homo (animals); Neurospora, Saccharomyces, and Schizosaccharomyces (fungi); Giardia (Parabasalids); and Aeropyrum and Archaeoglobus (Archaea). This multiple alignment, spanning >4000 aligned positions, was used to compute maximum likelihood, maximum parsimony, neighbor joining, and least squares trees, all rooted using the archaeal sequences. These methods uniformly yield a tree topology with Plasmodium and Cryptosporidium forming a monophyletic lineage lying outside of a strongly supported "crown group" composed of animals, fungi, and plants (Fig. 1A). Giardia occupies a basal position amidst the eukaryotes included in this analysis. This topology is also supported by domain architecture analysis of  400 proteins belonging to different functional categories as discrete characters. Previous reports propose a weak association between the "plant clade" (comprised of green plants, rhodophytes, and glaucocystophytes) and a large assemblage of eukaryotes that include Stramenopiles and Alveolates (including Apicomplexa; Baldauf et al. 2000). We did not find significant support for a grouping between the alveolates and the plants with the above data set. This suggests that, against the vertical relationship of these eukaryotic taxa, apicomplexan proteins showing specific affinities to plants are likely contributions of the rhodophyte apicoplast progenitor (Fig. 1B). Additionally, several proteins with clear phylogenetic affinity to bacterial homologs were observed (Fig. 1C). These proteins appear to be derived from several distinct bacterial lineages, but the sources of lateral transfers were not always assignable.
To determine the relatedness of the apicomplexan proteome, and to provide a quantitative measurement of proteome similarity and divergence across protein functional categories, we used a simple measure termed the orthology coefficient (OC). For a set of proteins from two compared organisms, the OC represents the fraction occurring in orthologous groups. Thus, an OC = 1 indicates that all the proteins within a compared set have an orthologous relationship. Likewise, if only a fraction of the proteins in the set form orthologous groups, then the OC would fall between 1 and 0. Plasmodium and C. parvum shared 2000 orthologous groups, with an overall orthology coefficient of 0.41 (Fig. 2A). This suggests that both parasites possess a significant complement of genes that do not have any orthologous representatives in the other. We further defined OCs for protein sets classified into functional categories (Fig. 2A), revealing that orthology coefficients span a striking range from 0.2 to 0.4 for proteins related to extracellular adhesion and surface protein glycosylation, and 0.8 to 0.85 for core cellular functions such as translation, RNA processing, ubiquitination, DNA repair/replication, and chromatin dynamics. This suggests that evolutionary divergence of the two parasites has differentially affected various functional classes (Fig. 2A).
Many differences in ortholog distribution could be attributed to gene loss accompanying mitochondrion or apicoplast organellar degradation, or elimination of metabolic pathways in C. parvum. For example, versions of the tRNA synthetases and DNA repair enzymes, present in Plasmodium but lost in Cryptosporidium, likely represent forms with mitochondrion- and apicoplast-specific functions in Plasmodium. This prediction, based on patterns of gene loss, is corroborated by the presence of long N-terminal extensions mediating organellar targeting only in the Plasmodium versions of these proteins. Qualitative differences between the apicomplexan lineages occur even in the high-OC-value protein sets corresponding to core cellular processes. When the demography of the conserved domains in the two apicomplexan proteomes were compared to each other and against Saccharomyces cerevisiae, a unicellular eukaryote with a roughly comparable number of protein-coding genes, certain interesting large-scale trends were observed (Fig. 2B,C). The two apicomplexans show independent lineage-specific expansions of entirely different protease families, and C. parvum does not share the prominent lineage-specific expansion of RESA-type DnaJ domains that is encountered in Plasmodium falciparum (Fig. 2B,C). Relative to yeast, Cryptosporidium, like Plasmodium (Aravind et al. 2003 ), also shows a remarkable expansion of the calcium-binding EF hand domains, suggesting that a well-developed calcium-dependent signaling apparatus is likely to have been present in the ancestral apicomplexan. These differences involve both differential gene loss as well as lineage-specific innovations and are discussed further below in the context of specific functional systems. In general, proteins related to functional categories, such as chromatin structure and RNA processing, show fewer domains per protein in the apicomplexan with respect to animals. However, at least in the case of Cryptosporidium, the number of domains per protein in modular surface proteins is closer to those in animals (Fig. 2D). A case-by-case examination of these functional categories indicated more pronounced qualitative similarities and differences with protein architectures in other eukaryotes that are potentially correlated with the divergent adaptation of various eukaryotes.
Functional Categories With Low OC Values: Surface Proteins and the Glycosylation Machinery
We systematically investigated the affinities of the surface protein domains by searching the C. parvum proteome with a comprehensive library of PSI-BLAST-derived position-specific score matrices and hidden Markov models for surface protein domains. These profiles were previously used to detect such domains in adhesion proteins of Caenorhabditis elegans, Homo sapiens, and P. falciparum (Aravind and Subramanian 1999
The surface protein adhesion domains in the apicomplexan proteomes can be attributed to multiple distinct heritages: those originally derived from bacteria and animals and laterally transferred to Apicomplexa, and those "invented" within the Apicomplexa, typically in a lineage-specific manner. In principle, it is possible that the surface protein domains shared by animals and apicomplexans were present in the ancestral eukaryotes and secondarily lost in other lineages. Although gene loss occurs frequently in eukaryotes, most of these domains shared by both these lineages are often undetectable in the (nearly) completely sequenced genomes of multicellular (filamentous) fungi, plants, and other unicellular eukaryotes such as trypanosomes and parabasalids. Thus, if multiple gene losses were to be invoked, it would imply that the common ancestor of these lineages was probably more diverse in its protein complement than most of the descendents, and this is not consistent with the demography of the protein families encoded by eukaryotic genomes (Lespinet et al. 2002 ). Furthermore, in phylogenetic analysis, specific affinities between apicomplexan and animal versions were recovered (Fig. 1D; Pradel et al. 2004). The animal affinities of domains are also highly overrepresented in the category of surface proteins, as against intracellular proteins belonging to other functional categories. Taken together, these observations make lateral transfer from animals, followed by selective retention of functionally relevant protein domains involved in adhesion, as the most parsimonious explanation for these observations.
The plausibility of horizontal transfer from an animal source is also supported by the intracellular location of apicomplexan parasites for most of their life cycle and, in the case of Plasmodium, there is facile uptake and expression of DNA constructs introduced to the erythrocyte cytoplasm prior to parasite infection (Deitsch et al. 2001
For P. falciparum and C. parvum surface proteins, the OC is no more than 0.2 for proteins having conserved "animal-type" and "bacterial-type" domains, with few conserved architectures shared by the two lineages. Furthermore, the set of surface domains in these proteins is overlapping but not identical in the apicomplexans. For example, the MAC/perforin-type domain (Aravind et al. 2003
Comparison of the surface protein glycosylation apparatus reveals dramatic divergence between these two apicomplexans (Fig. 3B). Both possess a well-developed GPI anchor synthesis apparatus that is largely similar to the corresponding pathway in other eukaryotes. Unlike Plasmodium, Cryptosporidium lacks the canonical N-acetylglucosaminylphosphatidylinositol deacetylase that catalyzes the second step in the GPI anchor biosynthetic pathway. However, sequence analysis revealed the presence of an unrelated bacterial-type sugar deacetylase (cgd1_3060) that is likely to catalyze the same reaction. Whereas there is only a rudimentary N-linked glycosylation pathway present in Plasmodium, a more developed pathway is predicted in Cryptosporidium. N-linked glycosylation has been widely detected in other eukaryotes, including Toxoplasma (Odenthal-Schnittler et al. 1993
Low OC of Metabolic Pathway Components Suggest Life-Cycle-Specific Adaptations
The stem of the glycolytic pathway represents the most highly conserved metabolic pathway between Cryptosporidium and Plasmodium. However, unlike Plasmodium, Cryptosporidium also possesses enzymes for the terminal metabolism of pyruvate such as pyruvate:ferredoxin oxidoreductase, pyruvate decarboxylase, and malate dehydrogenase. Phylogenetic analysis of pathway components reveals a mosaic of strong affinities to enzyme versions of plants and bacteria. For example, the apicomplexan phosphoglucomutase, phosphofructokinase, and enolase enzymes grouped with the plant versions, whereas fructose bisphosphate phosphatase and phosphoglucomutase showed bacterial affinities. These affinities suggest displacements of the ancestral eukaryotic enzymes by versions derived from the apicoplast precursor and bacterial sources. Nevertheless, the current state of the data precludes us from determining the temporal point in alveolate evolution at which these displacements occurred. Interestingly, similar to the parasite Leishmania, C. parvum possesses a plant-type 2-phosphoglycerate kinase implicated in archaea in the synthesis of the possible denaturation protectant, 2-3 cyclic phosphoglyceric acid (Matussek et al. 1998
Differences Between Cryptosporidium and Plasmodium in Functional Classes With Moderate to High OCs, and Comparisons With Other Eukaryotes
The ratio of the total number of proteins in the proteome to the predicted specific transcription factors in Cryptosporidium and Plasmodium, 340 and 800, respectively, is in great contrast to the ratio of 29 in S. cerevisiae. The decreased ratio in C. parvum relative to Plasmodium is caused by a greater absolute number of specific transcription factors possessing a variety of conserved DNA-binding domains, such as E2F/DP1, bZip, and GATA DNA-binding domains, in conjunction with a lower overall gene count (Fig. 2B,C). Nevertheless, the numbers of specific transcription factors are far fewer than those encountered in yeast and other eukaryotes, suggesting major differences in the mechanisms of apicomplexan gene regulation. Recent microarray studies on P. falciparum indicate a continuous cascade of gene expression in the course of its intraerythrocytic stage cycle, in which groups of functionally related genes are coexpressed, with those involved in generalized functions being expressed first followed by those for increasingly specialized, lineage-specific functions (Bozdech et al. 2003 ; Le Roch et al. 2003). The relative paucity of transcription factors in both the apicomplexans suggests that the regulation of such transcriptional cascades may be dependent on the well-developed chromatin-remodeling apparatus (Fig. 4), which might work in conjunction with the small set of specific DNA-binding proteins detected in these genomes. In this context, it is of interest to note that both of these apicomplexans contain orthologs of the DNA cytosine methyltransferase DNMT2 (the P. falciparum, gi: 23612639, and C. parvum orthologs were recovered with e-values of 10-15 and 10-26, respectively, in searches of the nonredundant protein database). These apicomplexan DNMT2 orthologs showed a full-length alignment with the versions from other eukaryotes (see alignment, Supplemental Fig. 2), and the sequence similarity encompassed both the N-terminal catalytic Ado-Met binding domain and a C-terminal domain unique to the DNMT2 family of methylases (Tang et al. 2003). They also show absolute conservation of the active-site residues implicated in Ado-Met binding and catalysis, suggesting that they are active enzymes. Consistent with this, cytosine methylation has been previously reported in Plasmodium (Pollack et al. 1991); however, it remains to be determined if they participate in an epigenetic control mechanism related to transcriptional cascading.
Although there is a much higher correspondence between the two apicomplexans in chromatin proteins than specific transcription factors, interesting differences are found in both absolute numbers and architectures of these proteins (Fig. 4). C. parvum has 14 chromatin-remodeling SNF2/SWI2 ATPases, whereas Plasmodium has just 11. Comparisons of the apicomplexan Swi2/Snf2 ATPases with the other eukaryotes suggests that Plasmodium has lost the Rad26 and Swr1 orthologs, whereas Cryptosporidium appears to have lost one of the Rad16/Rad5-like Ring finger-containing forms and the version fused to a C-terminal Endonuclease VII domain. Cryptosporidium possesses a version of the Swi2/Snf2 ATPase, with a unique architecture containing two N-terminal chromo domains and one bromo domain. Likewise, Plasmodium possesses a unique predicted chromatin-associated protein, having an amine oxidase domain fused to a C-terminal PHD finger that is predicted to function as a novel enzyme that might modify histone amino groups or chromatin basic amines (Fig. 4). It may represent a case of an independently derived chromatin-associated oxidase, parallel to the amine oxidase fused to the SWIRM domain that is seen in the crown group eukaryotes (Aravind and Iyer 2002
The pellicle in several protozoans is supported by a distinctive fibrous cytoskeletal structure predominantly composed of low complexity, proline- and valine-rich proteins called articulins (Mann and Beckers 2001
Conclusions However, beyond the core set of genes, the evolution of parasitism has involved lineage-specific adaptation occurring through gene loss, additional lateral transfers, and lineage-specific expansions. Considerable lineage-specific gene loss is indicated by the absence of widespread eukaryotic proteins specifically in either one of the apicomplexan lineages (Fig. 4), suggesting that the common ancestral apicomplexan possessed a more complex genome encoding a greater repertoire of biochemical activities. The streamlining appears to correlate with increasing propensity of the parasite to use its host(s) for most of its metabolic requirements. Some of the losses are not easily explained: for example, in Cryptosporidium, the apparent elimination of Skp1p and Skp2p-like proteins, despite the presence of cullins, suggests potential differences in the cell cycle related ubiquitination complexes relative to other eukaryotes. Thus, as a consequence of gene losses and lineage-specific innovations, apicomplexans possess proteomes quite different from free-living unicellular eukaryotes that have similar overall gene numbers (Fig. 2A-D). Most notably, the apicomplexan proteomes have a large component devoted to pathogenesis, immune evasion, and adhesion rather than transcription, posttranscriptional regulation, or metabolism.
The apicomplexans confirm large-scale trends in the evolution of the eukaryotes, specifically the involvement of lineage-specific expansions in the generation of specific adaptations (Abrahamsen et al. 2004
Sequence Analysis and Phylogenetic Tree Constructions C. parvum genome sequence information and annotation supporting this manuscript are available on an in-house genome browser (http://134.84.110.219/cgi-bin/gbrowse/crypto909) and at (http://www.cryptodb.org). General methodologies supporting genome sequence annotation, including BLAST searches, multiple sequence alignments, protein structure determinations, gene family clustering, and phylogenetic analyses were performed as briefly follows. The nonredundant (NR) database of protein sequences (National Center for Biotechnology Information, NIH, November 21, 2003) was searched using the BLASTP program. Profile searches were conducted using the PSI-BLAST program (Altschul et al. 1997 ) with either a single sequence or an alignment used as the query, with a default profile inclusion expectation (E) value threshold of 0.01 (unless specified otherwise), and was iterated until convergence. Multiple alignments were constructed using the T_Coffee program (Notredame et al. 2000), followed by manual correction based on the PSI-BLAST results. Signal peptides were predicted using the SIGNALP program (http://www.cbs.dtu.dk/services/SignalP-2.0/; Nielsen et al. 1997). Transmembrane regions were predicted in individual proteins using the PHDhtm (Rost et al. 1996), TMHMM2.0 (Krogh et al. 2001), and TOPRED1.0 (Claros and von Heijne 1994) programs with default parameters. For TOPRED1.0, the organism parameter was set to "eukaryote" (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html). Additionally, the multiple alignments were used to predict TM regions with the PHDhtm program. The library of profiles for conserved protein domains were also prepared by extracting alignments from the PFAM database (Bateman et al. 2002; http://www.sanger.ac.uk/Software/Pfam/index.shtml) and updated by adding new members from the NR database. These updated alignments were then used to make HMMs with the HMMER package (Eddy 1998; Bateman et al. 2002) or PSSM with PSI-BLAST. All large-scale sequence analysis procedures were carried out using the SEALS package (http://www.ncbi.nlm.nih.gov/CBBresearch/Walker/SEALS/index.html). Similarity-based clustering of proteins was carried out using the BLASTCLUST program (ftp://ftp.ncbi.nih.gov/blast/documents/blastclust.txt).
Phylogenetic analyses were carried out using the maximum-likelihood, neighbor-joining, protein parsimony, and least-squares methods (Felsenstein 1989
Calculation of Orthology Coefficients (OC)
This work was supported in part by the Niarchos Foundation. The Department of Microbiology and Immunology at Weill Medical College acknowledges the support of the William Randolph Hearst Foundation. This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD. (http://biowulf.nih.gov). The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
7 These authors contributed equally to this work. 
8 Corresponding authors. [Supplemental material is available online at www.genome.org.] Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2615304.
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Received March 23, 2004; accepted in revised format June 14, 2004.  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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ABSTRACT
RESULTS AND DISCUSSION
-helical compositions, or cysteine-rich modules stabilized by disulfide bridges, suggesting that they have emerged rather late in evolution in particular lineages of apicomplexans. Extensive proliferation and divergence of these proteins are likely caused by selective forces such as host immune pressure driving antigenic diversity. Exemplifying the second class of proteins are MSP1, P25/28, CSP1, and TRAP from P. falciparum and the Toxoplasma gondii MIC micronemal proteins. These proteins contain conserved adhesion domains, such as the EGF domain, Thrombospondin type 1 (TSP1) domain, the von Willebrand Factor A (vWA) domain, and the APPLE domain, that are typically abundant in animal surface proteins but are either absent or rarely present in surface adhesion molecules in other eukaryotic lineages examined to date. 
-strand globular 12-cysteine domain (
correction for among site rate variation plus a correction for invariant sites (8 + 1 rate categories) from the data sets. ML distance analyses used TreePuzzle 4.02 to calculate ML distance matrices along with Puzzleboot for 1000 replicates (
