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Repeated secondary loss of adaptin complex genes in the Apicomplexa
Parasitology International 58 (2009) 86–94
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Repeated secondary loss of adaptin complex genes in the Apicomplexa William D. Nevin a,b, Joel B. Dacks a,b,c,⁎ a b c
The Molteno Building, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK Corpus Christi College, Cambridge, CB2 1RH, UK Department of Cell Biology, University of Alberta, 6-30 Medical Sciences Building, Edmonton Alberta, T6G 2H7, Canada
a r t i c l e
i n f o
Article history: Received 26 August 2008 Received in revised form 9 December 2008 Accepted 9 December 2008 Available online 24 December 2008 Keywords: Plasmodium Golgi Evolution Endocytosis Rhoptry Degenerate
a b s t r a c t The Apicomplexa include parasites of devastating medical and economic consequence. While obviously essential for their parasitic mechanism, the molecular machinery underpinning membrane-trafficking in many apicomplexans is poorly understood. One potentially key set of players, the adaptins, selects cargo for incorporation into trafficking vesicles. Four distinct adaptin (AP) complexes exist in eukaryotes; AP1 and AP3 are involved in transport between the trans-Golgi Network (TGN) and endosomes, AP4 in TGN to cell surface transport, and AP2 in endocytosis from the cell surface. Of particular interest is the involvement of AP1 in Toxoplasma rhoptry biogenesis. The recent completion of several apicomplexan genomes should jump-start molecular parasitological studies and provide systems-level insight into the apicomplexan adaptin machinery. However, many of the encoded adaptin proteins are annotated conservatively and not to the necessary complex or subunit level. Prompted by previous evidence suggesting the lack of AP3 in Plasmodium falciparum, we undertook homology-searching and phylogenetic analysis to produce a rigorously annotated set of adaptin subunits encoded in diverse apicomplexan genomes. We found multiple losses of adaptins across the phylum; in particular Theileria, Babesia, and Cryptosporidium, but surprisingly not Plasmodium, appear to have lost the entirety of the AP3 complex. The losses correlate with a degenerate Golgi body structure and are reminiscent of recently reported secondary losses of additional endocytic components (i.e. the ESCRTs) in several Apicomplexa. These data may indicate a relaxation of the selective pressure on the apicomplexan endocytic system and, regardless, should greatly facilitate future molecular cell biological investigation of the role of adaptins in these important parasites. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The phylum Apicomplexa is home to some of the world's deadliest parasites. The most prominent is Plasmodium, which causes malaria in humans with approximately 500 million cases and a million deaths reported annually [1]. Infection with P. falciparum causes cerebral malaria, a multi-system condition leading to an unrousable coma and death. Another apicomplexan, Toxoplasma gondii, is believed to have infected up to one third of the world's population [2]. The infection is usually mild or asymptomatic, but in the immunocompromised it can lead to fatal encephalitis. Furthermore, congenital toxoplasmosis can cause death or neurological abnormalities in the foetus [2]. Cryptosporidiosis, caused by Cryptosporidium parvum, is a disease of the digestive tract. Self-limiting in healthy hosts, in the immunocompromised it can be a chronic and dangerous condition [3]. Other apicomplexan parasites, such as Babesia and Theileria, have serious
⁎ Corresponding author. Department of Cell Biology, University of Alberta, 6-30 Medical Sciences Building, Edmonton Alberta, T6G 2H7, Canada. Tel.: +1 780 248 1493; fax: +1 780 492 0450. E-mail address: [email protected] (J.B. Dacks). 1383-5769/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2008.12.002
economic consequences due to their infection of cattle herds in some of the poorest parts of the world [4,5]. Members of the supergroup Chromalveolata [6], the Apicomplexa have been recognised since the earliest microscopic studies. Today, over 2400 species have been described using microscopic techniques [7], and modern phylogenetic methods are showing the diversity of the Apicomplexa to be underestimated [8]. The Apicomplexa are so named because of an unusual set of membrane organelles that are crucial to the pathogenic mechanism of the parasites [9,10]. Together referred to as the apical complex, these include the conoid, the rhoptries and the micronemes. The conoid is a tightly wound tubulin polymer that is extended during invasion [11]. The rhoptries are specialised secretory organelles involved in release of enzymes key to the penetration and invasion of the host cell [12]. Finally, the micronemes are dense tubular bodies required for interaction with the host cell [9]. They appear to fuse with the neck of the rhoptry during invasion, leading to the discharge of the rhoptry contents. Secretory and endocytic processes are integral to the pathogenic mechanisms, not only of Apicomplexa (e.g. [13]) but most other parasites as well. Phytophthora, an important agricultural parasite, secretes adhesins required for invasion of plants [14]. Entamoeba (responsible for 111,000 fatalities a year) secretes cysteines proteases
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that are important both for invasion and evasion of the host immunesystem [15]. The endocytic system is commonly involved in immunesystem evasion. Trypanosoma brucei, responsible for African Sleeping Sickness, evades the immune-response via immunoglobulin degradation through the endocytic pathway and the recycling of cell surface receptors [16]. Endocytosis, vesicular trafficking, and secretion are enabled by the membrane-trafficking system. This set of dynamically interconnected organelles includes the endoplasmic reticulum (ER), Golgi apparatus, endosomes and lysosomes, as well as the plasma membrane. Common sets of protein machinery are responsible for the transport of material between the various locations, into and out of the cell [17]. Interestingly much of this machinery is derived from a few protein families whereby the various family members perform the same basic mechanistic function (whether in vesicle formation or vesicle fusion) but at discrete locations within the cell. This has allowed for evolutionary reconstruction to demonstrate that the Last Common Eukaryotic Ancestor (LCEA) had a complex endomembrane system including a stacked Golgi body and a sophisticated set of membranetrafficking machinery (reviewed in [18]). Not only does this provide insight deep into our cellular history, but along with a robust set of phylogenetic relationships, it enables the unusual cell biology of modern parasites to be interpreted as retained features, lineagespecific innovations or secondary losses because we have a reliable picture of the ancestral cellular configuration [19]. In the process of vesicle formation, specific sets of cargo are selected for transport and packaged into vesicle carriers destined for different locations in the cell [17]. This is performed by discrete protein coats and cargo adaptor proteins. One such set of cargo adaptor protein complexes is the Adaptins (AP). There are four AP complexes [20]. AP2 is found at the plasma membrane, and is involved with clathrin-mediated endocytic uptake. AP1 and AP3 are found at the trans-Golgi network (TGN) and endosomal membranes and have been implicated in transporting proteins between these locations [20]. AP4 is also localised mostly at the TGN but is thought to be involved with direct transport to the plasma membrane [21]. Each adaptin complex is composed of four subunits; two large subunits (gamma or GADE and beta), one medium (mu) and one small (sigma) (Fig 1A). The large subunits each contain N-terminal trunk regions, hinge and Cterminal ear regions [20]. Both the medium and small subunits contain domains distantly homologous to longins, a domain found in some SNARE proteins [22]. The four adaptin complexes are homologous to one another and to the F-COP cargo adaptor subcomplex of the COPI coat. The homology goes even deeper with the gamma and beta subunits being derived from an ancient gene duplication, as are the mu and sigma subunit families (Fig 1B) [23,24]. Comparative genomic and phylogenetic reconstruction have shown that all four AP complexes were present in the LCEA, as was
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the F-COP subcomplex [25,26]. Another set of cargo adaptor, the GGAs (Golgi-localising, Gamma-ear containing, ARF-binding proteins) are also homologous to the ear region of the gamma AP1 protein, but these proteins are restricted to animal and fungal lineages [25,27]. Lineage-specific innovation and secondary loss have both shaped the history of the adaptins. While the gamma, medium and small subunits of all four complexes had already evolved by the point of the LCEA, the beta subunits of the AP1 and AP2 complexes had not. These subsequently evolved convergently in animals, fungi and plants, but many eukaryotes have retained the ancestral feature of only a single beta subunit for AP1 and AP2 [26]. Secondary losses of adaptins have also occurred with Drosophila melanogaster, Caeanorhabditis elegans, Saccharomyces cerevisiae and Schizosaccharomyces pombe missing the entire AP4 complex, T. brucei missing the AP2 complex and Leishmania missing AP4 [27]. The adaptin proteins are of particular interest in the Apicomplexa as they are involved in rhoptry biogenesis, at least in T. gondii [28]. A broad survey of the endocytic machinery identified that P. falciparum might be missing the AP3 complex [27]. However, this was based on the failure to identify an AP3 large subunit gene in that organism: the full extent of the adaptin gene family complement in the Apicomplexa remains unexplored. We have performed comparative genomic and phylogenetic investigations of the adaptin family in a diverse array of apicomplexan genomes. This has revealed a wide-spread loss of the AP3 complex. Surprisingly this cannot be attributed to a single event but rather must be the result of several convergent losses. Losses of AP2 and degeneration of other subunits are also observed and the connections of this with apicomplexan endomembrane system structure are explored. 2. Materials and methods 2.1. Taxon sampling and databases Databases from a variety of apicomplexan taxa were searched [5,29–33]. The choice of databases was designed to encompass as broad a sampling of the Apicomplexa as possible, in order to get a picture of adaptin evolution across the entire phylum and also designed to focus in on a few well-sampled species to get a picture of adaptin evolution at a finer scale. Consequently three Plasmodium species (P. falciparum, P. vivax and P. yeolii yeolii) and two Theileria species (T. annulata and T. parva) were sampled, as well as T. gondii, C. parvum and Babesia bovis. The genome sequence of Phytopthora sojae, a soil born plant pathogen was used as an outgroup [34]. Since it is also a member of the super-group Chromalveolata [6], it can aid in the reconstruction of the ancestral adaptin complement and thus provide context for absences of adaptins in the Apicomplexa. Only genomes with predicted proteomes available were included, so as to increase the reliability of a predicted gene absence in a given
Fig. 1. Adaptin structure and domains. A) Cartoon of heterotetrameric adaptin structure showing the two large subunits, medium and small subunit. The N-terminal trunk region, hinge and C-terminal ear regions of the large subunits are shown. The interaction of the sigma and mu subunits with both large subunits is based on interaction data [57,58]. B) Relative size and notable domains of adaptin subunit proteins. The size of the subunit shown is based on the average length, in amino acids, of the H. sapiens subunits for the four AP complexes used for phylogenetic analysis and scaled relative to each subunit. The position of the domains is approximate. The vertical bars represent homology between the gamma/ alpha/delta/epsilon (GADE) and Beta, mu and sigma subunits respectively. LLD = Longin-like domain.
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Fig. 2. Phylogenetic analysis of beta adaptin subunits. This figure shows the resolution of the various sequences into clades corresponding to the adaptin complexes, denoted by the vertical lines to the edge of the page. The apicomplexan AP3 subunits appear to be evolving at a faster rate than the subunits of the other complexes from the same organisms, as illustrated by their longer branch-lengths. For this and all subsequent phylogenies the best Bayesian tree topology is shown and support values are given in the order of Posterior Probability values, PhyML and RAxML bootstrap values. Values for the nodes defining the adaptin complex subunits are shown in bold, regardless of their support. For all other nodes support values are shown when resolved by 0.80/50%/50% or better. In the case of nodes supported by 0.99/95%/95%, the values are replaced by a black dot. The taxon labels consist of the first letter of the genus, then the species and the AP complex designation. In all cases the locus tag is in brackets, with the exception of the P. sojae sequences whose labels list the database identifier and the H. sapiens sequences, whose labels list their accession numbers.
organism. Although the genomes of Eimeria tenella and Perkinsus marina were sampled, they were deemed at too preliminary a stage to be reliably included. The genome of the ciliate Tetrahymena thermophila was also sampled [35]. However, several unusual features of the adaptin complement in T. thermophila were identified that are beyond the scope of this analysis (e.g. multiple paralogues, subunits of unusual size, etc.). Consequently, it was decided not to include it as a general outgroup, opting instead for P. sojae. However, in one instance, the T. thermophila sequences were included as a closer outgroup for phylogenetics, in order to verify specific issues of subunit classification. Furthermore, while the genome of Plasmodium chabaudi was not included in the comparative genomic survey, the AP3S homologue (XP_743031) was included in phylogenetic analysis to clarify issues of AP sigma evolution. Data were retrieved from the following sources 1) NCBI — http://www.ncbi.nlm.nih.gov/ 2) ToxoDB — http://www. toxodb.org/toxo/ 3) PlasmoDB — http://plasmodb.org/plasmo/ 4)
TIGR — http://www.TIGR.org/tdb/e2k1/ttg/ 5) Sanger Institute database http://www.genedb.org/genedb/annulata/blast.jsp 6) Joint Genome Institute — http://genome.jgi-psf.org/Physo1_1/Physo1_1.home. html 7) EuPathDB — http://eupathdb.org/eupathdb/. The predicted protein databases for the C. parvum, T.annulata, T. parva, B. bovis and P. falciparum genomes were also downloaded from the above sites for searching via hidden markov model (HMM) algorithms. 2.2. Homology searching Homology searching was primarily performed using the BLASTp algorithm [36]. In all cases, the Homo sapiens homologue was used as the initial query against each database. Because of the functionally well-characterised nature of the human proteins, this choice of query avoided reliance on purely bioinformatically-derived inferences of homology for the initial query sequence. A candidate protein was
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deemed homologous based on several criteria. All candidate homologues from the queried genomes that were retrieved with a cut-off Evalue of 0.005 or better were subsequently verified by reciprocal blast analysis. This involved BLASTp analysis against the non-redundant genomic database using the identified sequences as queries and the use of the candidate proteins as queries against the available human genome, to find their closest orthologue in H. sapiens. Only candidates that retrieved the original query sequence or named homologues thereof were deemed positive. Candidates were also were subjected to manual inspection for size criteria and to deduce unique aspects of their structure. When the H. sapiens query failed to identify any apicomplexan homologues, the identified subunit from T.gondii was used as an
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alternate query to improve the reliability of the assessment that the subunit was missing, and not simply undetected due to divergence from the respective H. sapiens query. BLASTx searches with the H. sapiens queries against the nucleotide databases of the various genomes and hidden markov model searches against relevant predicted proteomes using HMMer version 2.3.2 were also performed to verify any “not identified” entries. The former were performed at the genome project websites above, while the latter were run locally. In these cases, the relevant databases were downloaded and HMM were created using the homologues of the complex and subunit of interest from H. sapiens, P. sojae and any apicomplexan sequences that had been identified. Any sequences retrieved with E-values of better than 3.0 were subject to reciprocal BLAST analysis as described above.
Fig. 3. Phylogenetic analysis of GADE subunits. This figure shows the resolution of the various gamma, alpha, delta and epsilon homologues into their respective clades. Here again the Plasmodium AP3 delta subunits appear to be rapidly evolving with respect to the other Plasmodium subunits.
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Fig. 4. Phylogenetic analysis of the mu subunits. This figure shows the resolution of the various mu homologues into their respective clades. The Plasmodium and T. gondii AP3 mu and the Theileria and Babesia AP2 mu subunits are characterised by long branch-lengths consistent with a relaxation of selection on those sequences.
2.3. Phylogenetics In order to confirm homology assessment of the candidates, down to the complex and subunit level, phylogenetic analysis was additionally performed. ‘MUSCLE’ was used to align the relevant adaptin amino acid sequences [37]. Following this, the alignments were analysed manually, and trimmed to remove any alignment positions where homology was ambiguous. The refined alignments were then subjected to analysis by Prot-test version 1.3 to find the optimal model of sequence evolution [38] including correction for unequal rates among sites, invariable sites and identification of the appropriate amino acid transition matrix. Details of all datasets used in the analysis, along with details of the models of sequence evolution implemented are listed in Table S1. All alignments are available upon request. Mr. Bayes version 3.2 [39] was used to determine tree topology and posterior probability values. 1 million Markov Chain Monte Carlo generations were used. The plateau was found by graphical repre-
sentation and the burn-in value was then calculated to remove those trees prior to the plateau, and thus suboptimal. PhyML version 2.44 and RAxML 7.0.0 were used to calculate maximum likelihood bootstrap values based on 100 pseudo-replicate datasets [40, 41]. 3. Results In order to obtain a more complete picture of the adaptin complement in the Apicomplexa, a comparative genomic survey was performed. Genomes spanning the diversity of the Apicomplexa were sampled in order to assess the trend across the phylum while several related sets of species were also sampled to assess finer-scale evolutionary trends. Phylogenetic analysis was used to confirm classification of the adaptin genes down to the complex and subunit level and to assess relative rates of evolution of the genes. The final annotated adaptin complement of the apicomplexan genomes sampled is found in Table S2. Subunit-specific results are described below.
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3.1. Beta Like many eukaryotes, the apicomplexans possess a single AP beta 1/2 subunit, which was easily identified in all organisms sampled. Clear AP4 beta subunits were also evident. However, AP3 genes were more difficult to identify. In Toxoplasma gondii, Plasmodium falciparum, Plasmodium yeolii yeolii, and Plasmodium vivax, candidate homologues were present. Nonetheless, the subunit was not found in either Theileria species, nor in Cryptosporidium parvum or Babesia bovis. Phylogenetic analysis robustly resolved clades of all three beta
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subunits (Fig. 2). Notably, despite their representing relatively long branches, the Plasmodium sequences did resolve with the AP3 beta subunits from T. gondii and the outgroups. 3.2. Gamma, alpha, delta, epsilon (GADE) The paralogues of the gamma adaptin family of large subunits each have complex-specific names; gamma (AP1), alpha (AP2), delta (AP3) and epsilon (AP4). Comparative genomic analysis was able to identify homologues for most of these subunits, aided in the classification by
Fig. 5. Phylogenetic analysis of the sigma subunits. This figure shows the resolution of the various sigma homologues into their respective clades. In this case the C. parvum AP2 sequence is a notably long branch.
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Fig. 6. Coulsen plot representation of the adaptin subunits in the Apicomplexa. AP1 is shown in blue, AP2 in green, AP3 in yellow and AP4 in purple. Missing subunits are denoted by black colouration. For the purposes of simplicity, the common apicomplexan AP1/2B has been shown as two separate subunits. Where multiple examples of the same subunit were found, this has been noted. The red stars indicate incidences of adaptin 3 complex loss, while the pink star indicates possible degeneration of adaptin 3 subunits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phylogenetic analysis. Homologues of the gamma subunit were identified in all cases, as were alpha subunits. The epsilon subunits were found to be well-conserved across all the organisms. However, delta subunits were not identified in several genomes. Both Theileria species, as well as Cryptosporidium parvum and Babesia bovis were missing the AP3 subunit. On the other hand, T. gondii, P. yeolii yeolii, P. falciparum and P. vivax were all found to possess AP3 delta subunits. The P. falciparum AP3D was not immediately retrieved by BLAST searches alone, but was eventually retrieved by HMM searches. The classification of the subunits was solidified by phylogenetic analysis, with robustly supported clades observed for the four adaptin complexes (Fig. 3). Again the Plasmodium AP3D sequences represented long-branches in the analysis. 3.3. Mu AP1 and AP4 mu subunits were identified in all genomes. Interestingly, while AP2 mu was found in almost all genomes sampled, we were unable to identify a candidate in C. parvum despite multiple query sequences, HMM searches and searching of the relevant nucleotide database. The pattern of AP3 mu subunits identified was similar pattern to that seen in the beta subunits, with clear presence in T. gondii, P. yeolii yeolii, P. falciparum and P. vivax. AP3 mu subunits were not identified in either Theileria species, Cryptosporidium parvum or Babesia bovis. Robust clades of AP1 and AP4 were observed upon phylogenetic analysis (Fig. 4), with the AP2 clade being slightly more moderately supported. The AP3 sequences that were identified represented particularly long branches indicative of rapid evolution in those gene sequences. Nonetheless, the clade was reconstructed with moderate support values (Fig. 4), regardless of inclusion of T. thermophila homologues (Fig. S1).
3.4. Sigma All organisms studied were shown to have all AP1, AP2 and AP4 sigma subunits present: the failure to identify AP3 subunits continued. While T. gondii, and the three Plasmodium species did have the protein, candidate homologues were not found for either Theileria species, C. parvum or B. bovis. The AP3 sigma of P. yeolii yeolii, found to be unusually short (50AA) and missing an N-terminal methionine, likely represents an incomplete predicted ORF, particularly as the protein is homologous to the C-terminus of other sigma adaptins, and is encoded on the plus strand near the 5′ end of its genomic contig (MALPY01052). This suggests that the remainder of the protein is likely encoded somewhere upstream but was not identifiable upon homology searching of the genomic sequence even with the closely related Plasmodium chabaudi homologue. Phylogenetic analysis of the sigma subunits with all sequences included provided robust support for the clades of AP3 and AP4, but poor resolution of the AP1 and AP2 clades (Fig. S2). This was likely due to the presence of partial sequences in the alignment and long-branch effects. Indeed when the fragmentary P. sojae AP1sx and P. yeolii AP3s sequences were removed with the latter replaced by the full length AP3 sigma homologue from P. chabaudi, the support for all four AP clades rose considerably (Fig. 5). 4. Discussion Our comparative genomic and phylogenetic analyses have identified the loss of the AP3 complex and possible degeneration of the AP2 complex in several apicomplexan lineages. These findings have implications for both the evolution and the biology of the parasites. Some of the adaptin complexes in the Apicomplexa sampled appear to be on a trajectory of degeneration, as demonstrated by a lack
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of clear homologues in a given genome or by rapidly-evolving sequences as compared to genes of other AP complexes, if the homologue was present. The most obvious was AP3. None of the four subunits were identified in either Theileria species, Cryptosporidium parvum, or Babesia bovis. While the AP3 subunits were identified in the Plasmodium species, in phylogenetic analyses the identified Plasmodium AP3 subunits seem to be faster-evolving than their AP1 and AP4 counter-parts, based on the long branch lengths observed for these sequences (Figs. 2,4,5). Even T. gondii appears to have moderately rapidly evolving AP3 genes as compared to its other AP complexes, despite it still retaining subunits for all four complexes (Fig. 2). AP3 is not the only complex to be degenerating, however. No AP2 mu sequence was identified in C. parvum and the branch length observed, particularly for the C. parvum AP2 sigma sequence suggests potential degeneration of this complex (Fig. 5). The Theileria and Babesia AP2 mu sequences also appeared to be rapidly evolving compared with the same sequences in Plasmodium and the outgroups (Fig. 4). The outgroup P. sojae appeared to possess all four subunits of all four complexes, even having expanded its adaptin complement slightly with multiple copies of several subunits. Counter to the general apicomplexan trend, Toxoplasma gondii seems also to have retained all four subunits. We do note that there may be various reasons why a homologue may not be present or identifiable in a given database, other than true absence. These could include sequence divergence from the query, or incompleteness of the database. Nonetheless, we did attempt to mediate these effects by using multiple query sequences, including an apicomplexan, and by including only completed genome sequences. As well, we used highly sensitive homology searching algorithms such as HMMer to additionally confirm our inability to identify potential homologues of particular subunits. We acknowledge that one or more of the predicted absences may be artifactual. However, we are relatively confident in ascribing inability to identify a sequence as likely absence. This is particularly the case when multiple subunits of the same complex are not found. Fig. 6 depicts the relationships of the sampled organisms, based on multi-gene concatenated phylogenies [42], and their respective adaptin complements. This enables us to more easily assess retention and loss and to deduce the evolutionary history of the adaptins in the Apicomplexa. On a fine-scale, the two Theileria species show the identical pattern of subunit retention and loss, as do the Plasmodium species. This suggests that the degenerative evolution of the adaptins is not occurring on a rapid (species-level) scale. Looking across the phylum, it appears as though the loss of AP3 has occurred twice, with a possible third trend toward degeneration (Fig. 6). Cryptosporidium is the most basal apicomplexan sampled and is known to be highly ‘stream-lined’ lacking many metabolic pathways and even organelles [43]. However, as T. gondii still possesses AP3, the loss of the complex must have occurred independently in Cryptosporidium and the single loss at the base of the Piroplasmida lineage that explains the absence in B. bovis and the two Theileria species (Fig. 6). The third and final loss appears to be incomplete, with the indication of rapid gene evolution in many of the AP3 Plasmodium subunits. Recent data specifically suggests that AP3 is involved in delivery of lysosomal proteins to the terminal digestive organelle in mammalian cells [44]. The lack of AP3 complexes in several apicomplexans, therefore, may indicate that this pathway is not active in these parasites. This, however, needs to be experimentally verified. The additional long branch-lengths of some adaptin subunit genes and the lack of AP2 mu in C. parvum all together suggest a relaxation of the selective pressure for retention of certain adaptins. Interestingly, the pattern of retention of AP3 correlated well with the structure of the Golgi apparatus in these organisms. In most eukaryotes, the Golgi body is composed of a set of flattened membrane stacks. This is the case in T. gondii [45,46], which has the full AP3 complement. However, in the remaining Apicomplexa, the Golgi body
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is either composed of unstacked membranes [47,48] or not clearly identifiable [49,50], indicative of a degenerate state. We believe it unlikely that the relationship between these two losses is causative. Firstly, the role of AP3 increasingly seems to be associated with endosomes rather than TGN [44] and the adaptins have not been proposed as involved in maintenance of Golgi structure. Secondly, the degeneration seems to be affecting AP2 as well, albeit to a lesser extent and AP2 is associated with clathrin-mediated endocytosis from the cell surface [20]. Thirdly, loss of adaptin complexes has been observed in other organisms and does not correlate in them with Golgi body structure. The LCEA likely possessed a stacked Golgi body with degeneration having occurred several times independently in the history of eukaryotes [51]. Giardia and the ascomycete fungi both lack stacked Golgi bodies and do, in fact, have reduced adaptin complements [25,52]; in the case of Fungi, AP4. However, the heteroloboseid Naegleria also lacks stacked Golgi bodies but possesses all subunits of all four complexes (Dacks, Field and Dawson unpublished). On the other hand, T. brucei lacks AP2 but has a stacked Golgi body, as does L. major which lacks AP4 [53,54]. A final piece of the puzzle to incorporate is the recent report of multiple secondary losses in a family of endocytic proteins in the Apicomplexa [55], reminiscent of the pattern observed here for the adaptins. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins form the machinery necessary for material to enter the multivesicular body, one of the key organelles of the endocytic pathway. Comparative genomics revealed, that while ESCRT complexes III and III-associated were retained, the ESCRT I and II complexes were missing in many of the Apicomplexa sampled [55]. In this case, P. falciparum, T. gondii and C. parvum were all found to be missing ESCRTs I and II. By contrast, these proteins were found in both species of Theileria. This again requires three separate secondary losses to explain. The fact that a broadly similar pattern was found to that with the adaptins, but with different taxa having lost the protein, suggests that these correlations are all effects of a larger cause, likely the adaptation of the organisms to a parasitic lifestyle and possibly a relaxation of selective pressure to retain an efficient endocytic system. The adaptin proteins are important in the Apicomplexa for the underlying cell biology of invasion via biogenesis of the rhoptries, and potentially other aspects involved with membrane trafficking [56]. The availability of genome sequences from a diverse array of apicomplexans will be tremendously helpful in the experimental study of these proteins. However, this is much more easily done when a well-annotated set of adaptins is easily accessible. Unfortunately many of the current databases have the proteins simply annotated by the protein family (adaptin) rather than at the complex or subunit level, making the choice of which specific proteins to study unclear. The carefully annotated, and comprehensive, listing of apicomplexan adaptin proteins provided here (Table S2) will hopefully help to jumpstart studies on these molecules in the Apicomplexa. The possible prediction of reduced endocytic trafficking based on the lack of AP3, as well as the general features of endocytic system degeneration and stream-lining detailed here have also hopefully added some interesting questions about the role of these proteins in organelle biogenesis, nutrient acquisition and parasitic mechanism to be addressed in future studies. Acknowledgements J. Ajioka, M. Field and the members of the Field lab are acknowledged for the helpful discussion, and L. Dacks is thanked for critical reading of the manuscript. We wish to thank the various genome projects for making their data publicly available and to the Camgrid computational resource on which these analyses were performed. Both JBD and WN wish to thank Corpus Christi College Cambridge for intellectual and financial support. JBD was also supported by a Parke Davis research fellowship.
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Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Repeated secondary loss of adaptin complex genes in the Apicomplexa William D. Nevin a,b, Joel B. Dacks a,b,c,⁎ a b c
The Molteno Building, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK Corpus Christi College, Cambridge, CB2 1RH, UK Department of Cell Biology, University of Alberta, 6-30 Medical Sciences Building, Edmonton Alberta, T6G 2H7, Canada
a r t i c l e
i n f o
Article history: Received 26 August 2008 Received in revised form 9 December 2008 Accepted 9 December 2008 Available online 24 December 2008 Keywords: Plasmodium Golgi Evolution Endocytosis Rhoptry Degenerate
a b s t r a c t The Apicomplexa include parasites of devastating medical and economic consequence. While obviously essential for their parasitic mechanism, the molecular machinery underpinning membrane-trafficking in many apicomplexans is poorly understood. One potentially key set of players, the adaptins, selects cargo for incorporation into trafficking vesicles. Four distinct adaptin (AP) complexes exist in eukaryotes; AP1 and AP3 are involved in transport between the trans-Golgi Network (TGN) and endosomes, AP4 in TGN to cell surface transport, and AP2 in endocytosis from the cell surface. Of particular interest is the involvement of AP1 in Toxoplasma rhoptry biogenesis. The recent completion of several apicomplexan genomes should jump-start molecular parasitological studies and provide systems-level insight into the apicomplexan adaptin machinery. However, many of the encoded adaptin proteins are annotated conservatively and not to the necessary complex or subunit level. Prompted by previous evidence suggesting the lack of AP3 in Plasmodium falciparum, we undertook homology-searching and phylogenetic analysis to produce a rigorously annotated set of adaptin subunits encoded in diverse apicomplexan genomes. We found multiple losses of adaptins across the phylum; in particular Theileria, Babesia, and Cryptosporidium, but surprisingly not Plasmodium, appear to have lost the entirety of the AP3 complex. The losses correlate with a degenerate Golgi body structure and are reminiscent of recently reported secondary losses of additional endocytic components (i.e. the ESCRTs) in several Apicomplexa. These data may indicate a relaxation of the selective pressure on the apicomplexan endocytic system and, regardless, should greatly facilitate future molecular cell biological investigation of the role of adaptins in these important parasites. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The phylum Apicomplexa is home to some of the world's deadliest parasites. The most prominent is Plasmodium, which causes malaria in humans with approximately 500 million cases and a million deaths reported annually [1]. Infection with P. falciparum causes cerebral malaria, a multi-system condition leading to an unrousable coma and death. Another apicomplexan, Toxoplasma gondii, is believed to have infected up to one third of the world's population [2]. The infection is usually mild or asymptomatic, but in the immunocompromised it can lead to fatal encephalitis. Furthermore, congenital toxoplasmosis can cause death or neurological abnormalities in the foetus [2]. Cryptosporidiosis, caused by Cryptosporidium parvum, is a disease of the digestive tract. Self-limiting in healthy hosts, in the immunocompromised it can be a chronic and dangerous condition [3]. Other apicomplexan parasites, such as Babesia and Theileria, have serious
⁎ Corresponding author. Department of Cell Biology, University of Alberta, 6-30 Medical Sciences Building, Edmonton Alberta, T6G 2H7, Canada. Tel.: +1 780 248 1493; fax: +1 780 492 0450. E-mail address: [email protected] (J.B. Dacks). 1383-5769/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2008.12.002
economic consequences due to their infection of cattle herds in some of the poorest parts of the world [4,5]. Members of the supergroup Chromalveolata [6], the Apicomplexa have been recognised since the earliest microscopic studies. Today, over 2400 species have been described using microscopic techniques [7], and modern phylogenetic methods are showing the diversity of the Apicomplexa to be underestimated [8]. The Apicomplexa are so named because of an unusual set of membrane organelles that are crucial to the pathogenic mechanism of the parasites [9,10]. Together referred to as the apical complex, these include the conoid, the rhoptries and the micronemes. The conoid is a tightly wound tubulin polymer that is extended during invasion [11]. The rhoptries are specialised secretory organelles involved in release of enzymes key to the penetration and invasion of the host cell [12]. Finally, the micronemes are dense tubular bodies required for interaction with the host cell [9]. They appear to fuse with the neck of the rhoptry during invasion, leading to the discharge of the rhoptry contents. Secretory and endocytic processes are integral to the pathogenic mechanisms, not only of Apicomplexa (e.g. [13]) but most other parasites as well. Phytophthora, an important agricultural parasite, secretes adhesins required for invasion of plants [14]. Entamoeba (responsible for 111,000 fatalities a year) secretes cysteines proteases
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that are important both for invasion and evasion of the host immunesystem [15]. The endocytic system is commonly involved in immunesystem evasion. Trypanosoma brucei, responsible for African Sleeping Sickness, evades the immune-response via immunoglobulin degradation through the endocytic pathway and the recycling of cell surface receptors [16]. Endocytosis, vesicular trafficking, and secretion are enabled by the membrane-trafficking system. This set of dynamically interconnected organelles includes the endoplasmic reticulum (ER), Golgi apparatus, endosomes and lysosomes, as well as the plasma membrane. Common sets of protein machinery are responsible for the transport of material between the various locations, into and out of the cell [17]. Interestingly much of this machinery is derived from a few protein families whereby the various family members perform the same basic mechanistic function (whether in vesicle formation or vesicle fusion) but at discrete locations within the cell. This has allowed for evolutionary reconstruction to demonstrate that the Last Common Eukaryotic Ancestor (LCEA) had a complex endomembrane system including a stacked Golgi body and a sophisticated set of membranetrafficking machinery (reviewed in [18]). Not only does this provide insight deep into our cellular history, but along with a robust set of phylogenetic relationships, it enables the unusual cell biology of modern parasites to be interpreted as retained features, lineagespecific innovations or secondary losses because we have a reliable picture of the ancestral cellular configuration [19]. In the process of vesicle formation, specific sets of cargo are selected for transport and packaged into vesicle carriers destined for different locations in the cell [17]. This is performed by discrete protein coats and cargo adaptor proteins. One such set of cargo adaptor protein complexes is the Adaptins (AP). There are four AP complexes [20]. AP2 is found at the plasma membrane, and is involved with clathrin-mediated endocytic uptake. AP1 and AP3 are found at the trans-Golgi network (TGN) and endosomal membranes and have been implicated in transporting proteins between these locations [20]. AP4 is also localised mostly at the TGN but is thought to be involved with direct transport to the plasma membrane [21]. Each adaptin complex is composed of four subunits; two large subunits (gamma or GADE and beta), one medium (mu) and one small (sigma) (Fig 1A). The large subunits each contain N-terminal trunk regions, hinge and Cterminal ear regions [20]. Both the medium and small subunits contain domains distantly homologous to longins, a domain found in some SNARE proteins [22]. The four adaptin complexes are homologous to one another and to the F-COP cargo adaptor subcomplex of the COPI coat. The homology goes even deeper with the gamma and beta subunits being derived from an ancient gene duplication, as are the mu and sigma subunit families (Fig 1B) [23,24]. Comparative genomic and phylogenetic reconstruction have shown that all four AP complexes were present in the LCEA, as was
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the F-COP subcomplex [25,26]. Another set of cargo adaptor, the GGAs (Golgi-localising, Gamma-ear containing, ARF-binding proteins) are also homologous to the ear region of the gamma AP1 protein, but these proteins are restricted to animal and fungal lineages [25,27]. Lineage-specific innovation and secondary loss have both shaped the history of the adaptins. While the gamma, medium and small subunits of all four complexes had already evolved by the point of the LCEA, the beta subunits of the AP1 and AP2 complexes had not. These subsequently evolved convergently in animals, fungi and plants, but many eukaryotes have retained the ancestral feature of only a single beta subunit for AP1 and AP2 [26]. Secondary losses of adaptins have also occurred with Drosophila melanogaster, Caeanorhabditis elegans, Saccharomyces cerevisiae and Schizosaccharomyces pombe missing the entire AP4 complex, T. brucei missing the AP2 complex and Leishmania missing AP4 [27]. The adaptin proteins are of particular interest in the Apicomplexa as they are involved in rhoptry biogenesis, at least in T. gondii [28]. A broad survey of the endocytic machinery identified that P. falciparum might be missing the AP3 complex [27]. However, this was based on the failure to identify an AP3 large subunit gene in that organism: the full extent of the adaptin gene family complement in the Apicomplexa remains unexplored. We have performed comparative genomic and phylogenetic investigations of the adaptin family in a diverse array of apicomplexan genomes. This has revealed a wide-spread loss of the AP3 complex. Surprisingly this cannot be attributed to a single event but rather must be the result of several convergent losses. Losses of AP2 and degeneration of other subunits are also observed and the connections of this with apicomplexan endomembrane system structure are explored. 2. Materials and methods 2.1. Taxon sampling and databases Databases from a variety of apicomplexan taxa were searched [5,29–33]. The choice of databases was designed to encompass as broad a sampling of the Apicomplexa as possible, in order to get a picture of adaptin evolution across the entire phylum and also designed to focus in on a few well-sampled species to get a picture of adaptin evolution at a finer scale. Consequently three Plasmodium species (P. falciparum, P. vivax and P. yeolii yeolii) and two Theileria species (T. annulata and T. parva) were sampled, as well as T. gondii, C. parvum and Babesia bovis. The genome sequence of Phytopthora sojae, a soil born plant pathogen was used as an outgroup [34]. Since it is also a member of the super-group Chromalveolata [6], it can aid in the reconstruction of the ancestral adaptin complement and thus provide context for absences of adaptins in the Apicomplexa. Only genomes with predicted proteomes available were included, so as to increase the reliability of a predicted gene absence in a given
Fig. 1. Adaptin structure and domains. A) Cartoon of heterotetrameric adaptin structure showing the two large subunits, medium and small subunit. The N-terminal trunk region, hinge and C-terminal ear regions of the large subunits are shown. The interaction of the sigma and mu subunits with both large subunits is based on interaction data [57,58]. B) Relative size and notable domains of adaptin subunit proteins. The size of the subunit shown is based on the average length, in amino acids, of the H. sapiens subunits for the four AP complexes used for phylogenetic analysis and scaled relative to each subunit. The position of the domains is approximate. The vertical bars represent homology between the gamma/ alpha/delta/epsilon (GADE) and Beta, mu and sigma subunits respectively. LLD = Longin-like domain.
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Fig. 2. Phylogenetic analysis of beta adaptin subunits. This figure shows the resolution of the various sequences into clades corresponding to the adaptin complexes, denoted by the vertical lines to the edge of the page. The apicomplexan AP3 subunits appear to be evolving at a faster rate than the subunits of the other complexes from the same organisms, as illustrated by their longer branch-lengths. For this and all subsequent phylogenies the best Bayesian tree topology is shown and support values are given in the order of Posterior Probability values, PhyML and RAxML bootstrap values. Values for the nodes defining the adaptin complex subunits are shown in bold, regardless of their support. For all other nodes support values are shown when resolved by 0.80/50%/50% or better. In the case of nodes supported by 0.99/95%/95%, the values are replaced by a black dot. The taxon labels consist of the first letter of the genus, then the species and the AP complex designation. In all cases the locus tag is in brackets, with the exception of the P. sojae sequences whose labels list the database identifier and the H. sapiens sequences, whose labels list their accession numbers.
organism. Although the genomes of Eimeria tenella and Perkinsus marina were sampled, they were deemed at too preliminary a stage to be reliably included. The genome of the ciliate Tetrahymena thermophila was also sampled [35]. However, several unusual features of the adaptin complement in T. thermophila were identified that are beyond the scope of this analysis (e.g. multiple paralogues, subunits of unusual size, etc.). Consequently, it was decided not to include it as a general outgroup, opting instead for P. sojae. However, in one instance, the T. thermophila sequences were included as a closer outgroup for phylogenetics, in order to verify specific issues of subunit classification. Furthermore, while the genome of Plasmodium chabaudi was not included in the comparative genomic survey, the AP3S homologue (XP_743031) was included in phylogenetic analysis to clarify issues of AP sigma evolution. Data were retrieved from the following sources 1) NCBI — http://www.ncbi.nlm.nih.gov/ 2) ToxoDB — http://www. toxodb.org/toxo/ 3) PlasmoDB — http://plasmodb.org/plasmo/ 4)
TIGR — http://www.TIGR.org/tdb/e2k1/ttg/ 5) Sanger Institute database http://www.genedb.org/genedb/annulata/blast.jsp 6) Joint Genome Institute — http://genome.jgi-psf.org/Physo1_1/Physo1_1.home. html 7) EuPathDB — http://eupathdb.org/eupathdb/. The predicted protein databases for the C. parvum, T.annulata, T. parva, B. bovis and P. falciparum genomes were also downloaded from the above sites for searching via hidden markov model (HMM) algorithms. 2.2. Homology searching Homology searching was primarily performed using the BLASTp algorithm [36]. In all cases, the Homo sapiens homologue was used as the initial query against each database. Because of the functionally well-characterised nature of the human proteins, this choice of query avoided reliance on purely bioinformatically-derived inferences of homology for the initial query sequence. A candidate protein was
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deemed homologous based on several criteria. All candidate homologues from the queried genomes that were retrieved with a cut-off Evalue of 0.005 or better were subsequently verified by reciprocal blast analysis. This involved BLASTp analysis against the non-redundant genomic database using the identified sequences as queries and the use of the candidate proteins as queries against the available human genome, to find their closest orthologue in H. sapiens. Only candidates that retrieved the original query sequence or named homologues thereof were deemed positive. Candidates were also were subjected to manual inspection for size criteria and to deduce unique aspects of their structure. When the H. sapiens query failed to identify any apicomplexan homologues, the identified subunit from T.gondii was used as an
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alternate query to improve the reliability of the assessment that the subunit was missing, and not simply undetected due to divergence from the respective H. sapiens query. BLASTx searches with the H. sapiens queries against the nucleotide databases of the various genomes and hidden markov model searches against relevant predicted proteomes using HMMer version 2.3.2 were also performed to verify any “not identified” entries. The former were performed at the genome project websites above, while the latter were run locally. In these cases, the relevant databases were downloaded and HMM were created using the homologues of the complex and subunit of interest from H. sapiens, P. sojae and any apicomplexan sequences that had been identified. Any sequences retrieved with E-values of better than 3.0 were subject to reciprocal BLAST analysis as described above.
Fig. 3. Phylogenetic analysis of GADE subunits. This figure shows the resolution of the various gamma, alpha, delta and epsilon homologues into their respective clades. Here again the Plasmodium AP3 delta subunits appear to be rapidly evolving with respect to the other Plasmodium subunits.
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Fig. 4. Phylogenetic analysis of the mu subunits. This figure shows the resolution of the various mu homologues into their respective clades. The Plasmodium and T. gondii AP3 mu and the Theileria and Babesia AP2 mu subunits are characterised by long branch-lengths consistent with a relaxation of selection on those sequences.
2.3. Phylogenetics In order to confirm homology assessment of the candidates, down to the complex and subunit level, phylogenetic analysis was additionally performed. ‘MUSCLE’ was used to align the relevant adaptin amino acid sequences [37]. Following this, the alignments were analysed manually, and trimmed to remove any alignment positions where homology was ambiguous. The refined alignments were then subjected to analysis by Prot-test version 1.3 to find the optimal model of sequence evolution [38] including correction for unequal rates among sites, invariable sites and identification of the appropriate amino acid transition matrix. Details of all datasets used in the analysis, along with details of the models of sequence evolution implemented are listed in Table S1. All alignments are available upon request. Mr. Bayes version 3.2 [39] was used to determine tree topology and posterior probability values. 1 million Markov Chain Monte Carlo generations were used. The plateau was found by graphical repre-
sentation and the burn-in value was then calculated to remove those trees prior to the plateau, and thus suboptimal. PhyML version 2.44 and RAxML 7.0.0 were used to calculate maximum likelihood bootstrap values based on 100 pseudo-replicate datasets [40, 41]. 3. Results In order to obtain a more complete picture of the adaptin complement in the Apicomplexa, a comparative genomic survey was performed. Genomes spanning the diversity of the Apicomplexa were sampled in order to assess the trend across the phylum while several related sets of species were also sampled to assess finer-scale evolutionary trends. Phylogenetic analysis was used to confirm classification of the adaptin genes down to the complex and subunit level and to assess relative rates of evolution of the genes. The final annotated adaptin complement of the apicomplexan genomes sampled is found in Table S2. Subunit-specific results are described below.
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3.1. Beta Like many eukaryotes, the apicomplexans possess a single AP beta 1/2 subunit, which was easily identified in all organisms sampled. Clear AP4 beta subunits were also evident. However, AP3 genes were more difficult to identify. In Toxoplasma gondii, Plasmodium falciparum, Plasmodium yeolii yeolii, and Plasmodium vivax, candidate homologues were present. Nonetheless, the subunit was not found in either Theileria species, nor in Cryptosporidium parvum or Babesia bovis. Phylogenetic analysis robustly resolved clades of all three beta
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subunits (Fig. 2). Notably, despite their representing relatively long branches, the Plasmodium sequences did resolve with the AP3 beta subunits from T. gondii and the outgroups. 3.2. Gamma, alpha, delta, epsilon (GADE) The paralogues of the gamma adaptin family of large subunits each have complex-specific names; gamma (AP1), alpha (AP2), delta (AP3) and epsilon (AP4). Comparative genomic analysis was able to identify homologues for most of these subunits, aided in the classification by
Fig. 5. Phylogenetic analysis of the sigma subunits. This figure shows the resolution of the various sigma homologues into their respective clades. In this case the C. parvum AP2 sequence is a notably long branch.
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Fig. 6. Coulsen plot representation of the adaptin subunits in the Apicomplexa. AP1 is shown in blue, AP2 in green, AP3 in yellow and AP4 in purple. Missing subunits are denoted by black colouration. For the purposes of simplicity, the common apicomplexan AP1/2B has been shown as two separate subunits. Where multiple examples of the same subunit were found, this has been noted. The red stars indicate incidences of adaptin 3 complex loss, while the pink star indicates possible degeneration of adaptin 3 subunits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phylogenetic analysis. Homologues of the gamma subunit were identified in all cases, as were alpha subunits. The epsilon subunits were found to be well-conserved across all the organisms. However, delta subunits were not identified in several genomes. Both Theileria species, as well as Cryptosporidium parvum and Babesia bovis were missing the AP3 subunit. On the other hand, T. gondii, P. yeolii yeolii, P. falciparum and P. vivax were all found to possess AP3 delta subunits. The P. falciparum AP3D was not immediately retrieved by BLAST searches alone, but was eventually retrieved by HMM searches. The classification of the subunits was solidified by phylogenetic analysis, with robustly supported clades observed for the four adaptin complexes (Fig. 3). Again the Plasmodium AP3D sequences represented long-branches in the analysis. 3.3. Mu AP1 and AP4 mu subunits were identified in all genomes. Interestingly, while AP2 mu was found in almost all genomes sampled, we were unable to identify a candidate in C. parvum despite multiple query sequences, HMM searches and searching of the relevant nucleotide database. The pattern of AP3 mu subunits identified was similar pattern to that seen in the beta subunits, with clear presence in T. gondii, P. yeolii yeolii, P. falciparum and P. vivax. AP3 mu subunits were not identified in either Theileria species, Cryptosporidium parvum or Babesia bovis. Robust clades of AP1 and AP4 were observed upon phylogenetic analysis (Fig. 4), with the AP2 clade being slightly more moderately supported. The AP3 sequences that were identified represented particularly long branches indicative of rapid evolution in those gene sequences. Nonetheless, the clade was reconstructed with moderate support values (Fig. 4), regardless of inclusion of T. thermophila homologues (Fig. S1).
3.4. Sigma All organisms studied were shown to have all AP1, AP2 and AP4 sigma subunits present: the failure to identify AP3 subunits continued. While T. gondii, and the three Plasmodium species did have the protein, candidate homologues were not found for either Theileria species, C. parvum or B. bovis. The AP3 sigma of P. yeolii yeolii, found to be unusually short (50AA) and missing an N-terminal methionine, likely represents an incomplete predicted ORF, particularly as the protein is homologous to the C-terminus of other sigma adaptins, and is encoded on the plus strand near the 5′ end of its genomic contig (MALPY01052). This suggests that the remainder of the protein is likely encoded somewhere upstream but was not identifiable upon homology searching of the genomic sequence even with the closely related Plasmodium chabaudi homologue. Phylogenetic analysis of the sigma subunits with all sequences included provided robust support for the clades of AP3 and AP4, but poor resolution of the AP1 and AP2 clades (Fig. S2). This was likely due to the presence of partial sequences in the alignment and long-branch effects. Indeed when the fragmentary P. sojae AP1sx and P. yeolii AP3s sequences were removed with the latter replaced by the full length AP3 sigma homologue from P. chabaudi, the support for all four AP clades rose considerably (Fig. 5). 4. Discussion Our comparative genomic and phylogenetic analyses have identified the loss of the AP3 complex and possible degeneration of the AP2 complex in several apicomplexan lineages. These findings have implications for both the evolution and the biology of the parasites. Some of the adaptin complexes in the Apicomplexa sampled appear to be on a trajectory of degeneration, as demonstrated by a lack
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of clear homologues in a given genome or by rapidly-evolving sequences as compared to genes of other AP complexes, if the homologue was present. The most obvious was AP3. None of the four subunits were identified in either Theileria species, Cryptosporidium parvum, or Babesia bovis. While the AP3 subunits were identified in the Plasmodium species, in phylogenetic analyses the identified Plasmodium AP3 subunits seem to be faster-evolving than their AP1 and AP4 counter-parts, based on the long branch lengths observed for these sequences (Figs. 2,4,5). Even T. gondii appears to have moderately rapidly evolving AP3 genes as compared to its other AP complexes, despite it still retaining subunits for all four complexes (Fig. 2). AP3 is not the only complex to be degenerating, however. No AP2 mu sequence was identified in C. parvum and the branch length observed, particularly for the C. parvum AP2 sigma sequence suggests potential degeneration of this complex (Fig. 5). The Theileria and Babesia AP2 mu sequences also appeared to be rapidly evolving compared with the same sequences in Plasmodium and the outgroups (Fig. 4). The outgroup P. sojae appeared to possess all four subunits of all four complexes, even having expanded its adaptin complement slightly with multiple copies of several subunits. Counter to the general apicomplexan trend, Toxoplasma gondii seems also to have retained all four subunits. We do note that there may be various reasons why a homologue may not be present or identifiable in a given database, other than true absence. These could include sequence divergence from the query, or incompleteness of the database. Nonetheless, we did attempt to mediate these effects by using multiple query sequences, including an apicomplexan, and by including only completed genome sequences. As well, we used highly sensitive homology searching algorithms such as HMMer to additionally confirm our inability to identify potential homologues of particular subunits. We acknowledge that one or more of the predicted absences may be artifactual. However, we are relatively confident in ascribing inability to identify a sequence as likely absence. This is particularly the case when multiple subunits of the same complex are not found. Fig. 6 depicts the relationships of the sampled organisms, based on multi-gene concatenated phylogenies [42], and their respective adaptin complements. This enables us to more easily assess retention and loss and to deduce the evolutionary history of the adaptins in the Apicomplexa. On a fine-scale, the two Theileria species show the identical pattern of subunit retention and loss, as do the Plasmodium species. This suggests that the degenerative evolution of the adaptins is not occurring on a rapid (species-level) scale. Looking across the phylum, it appears as though the loss of AP3 has occurred twice, with a possible third trend toward degeneration (Fig. 6). Cryptosporidium is the most basal apicomplexan sampled and is known to be highly ‘stream-lined’ lacking many metabolic pathways and even organelles [43]. However, as T. gondii still possesses AP3, the loss of the complex must have occurred independently in Cryptosporidium and the single loss at the base of the Piroplasmida lineage that explains the absence in B. bovis and the two Theileria species (Fig. 6). The third and final loss appears to be incomplete, with the indication of rapid gene evolution in many of the AP3 Plasmodium subunits. Recent data specifically suggests that AP3 is involved in delivery of lysosomal proteins to the terminal digestive organelle in mammalian cells [44]. The lack of AP3 complexes in several apicomplexans, therefore, may indicate that this pathway is not active in these parasites. This, however, needs to be experimentally verified. The additional long branch-lengths of some adaptin subunit genes and the lack of AP2 mu in C. parvum all together suggest a relaxation of the selective pressure for retention of certain adaptins. Interestingly, the pattern of retention of AP3 correlated well with the structure of the Golgi apparatus in these organisms. In most eukaryotes, the Golgi body is composed of a set of flattened membrane stacks. This is the case in T. gondii [45,46], which has the full AP3 complement. However, in the remaining Apicomplexa, the Golgi body
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is either composed of unstacked membranes [47,48] or not clearly identifiable [49,50], indicative of a degenerate state. We believe it unlikely that the relationship between these two losses is causative. Firstly, the role of AP3 increasingly seems to be associated with endosomes rather than TGN [44] and the adaptins have not been proposed as involved in maintenance of Golgi structure. Secondly, the degeneration seems to be affecting AP2 as well, albeit to a lesser extent and AP2 is associated with clathrin-mediated endocytosis from the cell surface [20]. Thirdly, loss of adaptin complexes has been observed in other organisms and does not correlate in them with Golgi body structure. The LCEA likely possessed a stacked Golgi body with degeneration having occurred several times independently in the history of eukaryotes [51]. Giardia and the ascomycete fungi both lack stacked Golgi bodies and do, in fact, have reduced adaptin complements [25,52]; in the case of Fungi, AP4. However, the heteroloboseid Naegleria also lacks stacked Golgi bodies but possesses all subunits of all four complexes (Dacks, Field and Dawson unpublished). On the other hand, T. brucei lacks AP2 but has a stacked Golgi body, as does L. major which lacks AP4 [53,54]. A final piece of the puzzle to incorporate is the recent report of multiple secondary losses in a family of endocytic proteins in the Apicomplexa [55], reminiscent of the pattern observed here for the adaptins. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins form the machinery necessary for material to enter the multivesicular body, one of the key organelles of the endocytic pathway. Comparative genomics revealed, that while ESCRT complexes III and III-associated were retained, the ESCRT I and II complexes were missing in many of the Apicomplexa sampled [55]. In this case, P. falciparum, T. gondii and C. parvum were all found to be missing ESCRTs I and II. By contrast, these proteins were found in both species of Theileria. This again requires three separate secondary losses to explain. The fact that a broadly similar pattern was found to that with the adaptins, but with different taxa having lost the protein, suggests that these correlations are all effects of a larger cause, likely the adaptation of the organisms to a parasitic lifestyle and possibly a relaxation of selective pressure to retain an efficient endocytic system. The adaptin proteins are important in the Apicomplexa for the underlying cell biology of invasion via biogenesis of the rhoptries, and potentially other aspects involved with membrane trafficking [56]. The availability of genome sequences from a diverse array of apicomplexans will be tremendously helpful in the experimental study of these proteins. However, this is much more easily done when a well-annotated set of adaptins is easily accessible. Unfortunately many of the current databases have the proteins simply annotated by the protein family (adaptin) rather than at the complex or subunit level, making the choice of which specific proteins to study unclear. The carefully annotated, and comprehensive, listing of apicomplexan adaptin proteins provided here (Table S2) will hopefully help to jumpstart studies on these molecules in the Apicomplexa. The possible prediction of reduced endocytic trafficking based on the lack of AP3, as well as the general features of endocytic system degeneration and stream-lining detailed here have also hopefully added some interesting questions about the role of these proteins in organelle biogenesis, nutrient acquisition and parasitic mechanism to be addressed in future studies. Acknowledgements J. Ajioka, M. Field and the members of the Field lab are acknowledged for the helpful discussion, and L. Dacks is thanked for critical reading of the manuscript. We wish to thank the various genome projects for making their data publicly available and to the Camgrid computational resource on which these analyses were performed. Both JBD and WN wish to thank Corpus Christi College Cambridge for intellectual and financial support. JBD was also supported by a Parke Davis research fellowship.
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