Download A complete shikimate pathway in Toxoplasma gondii: an ancient

International Journal for Parasitology 34 (2004) 5–13
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A complete shikimate pathway in Toxoplasma gondii:
an ancient eukaryotic innovationq
S.A. Campbella, T.A. Richardsb, E.J. Muic, B.U. Samuelc,
J.R. Cogginsd, R. McLeodc, C.W. Robertsa,*
a
Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, Scotland G4 ONR, UK
b
Department of Zoology, The Natural History Museum, Cromwell Road, London, UK
c
Department of Ophthalmology and Visual Sciences, University of Chicago, Chicago, IL 60616, USA
d
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK
Received 19 September 2003; received in revised form 15 October 2003; accepted 16 October 2003
Abstract
The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and
Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the
shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic
fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and
apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or
replaced in a number of lineages.
q 2003 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Keywords: Apicomplexa; Toxoplasma; Plasmodium; Shikimate; AROM; DAHP synthase
The shikimate pathway consists of seven enzymes that
catalyse the sequential conversion of erythrose-4-phosphate
and phosphoenol pyruvate to chorismate, the common
precursor of the folates, ubiquinone, the aromatic amino
acids and many other aromatic compounds (Herrmann and
Weaver, 1999). Previously believed to be confined to
bacteria, plants and fungi, the shikimate pathway has
recently been shown to function in the apicomplexan
parasites, Plasmodium falciparum, Toxoplasma gondii and
Cryptosporidium parvum (Roberts et al., 1998). Inhibition
of this pathway by the herbicide glyphosate, a specific
inhibitor of 5-enolpyruvylshikimate-3-phosphate (EPSP)
synthase, restricts the growth of these parasite species in
vitro (Roberts et al., 1998). The absence of the pathway in
mammals, combined with its essential nature in certain
q
Nucleotide sequence data reported in this paper are available in the
GenBanke EMBL and DDBJ databases under the accession numbers
AY341375 and AY314743.
* Corresponding author. Tel.: þ44-141-795-4458; fax: þ 44-141-7954406.
E-mail address: [email protected] (C.W. Roberts).
microorganisms, makes the shikimate pathway enzymes
attractive targets for new anti-microbial agents.
The molecular organisation and structure of the shikimate pathway enzymes varies considerably between
taxonomic groups (Coggins et al., 1987). Bacteria have
seven individual polypeptides, each possessing a single
enzyme activity, which are encoded by separate genes.
Plants have a molecular arrangement similar to bacteria, i.e.
separate enzymes encoded by separate genes (Butler et al.,
1974), with the exception of dehydroquinase (DHQase) and
shikimate dehydrogenase which have been shown to be
present as separate domains on a bifunctional polypeptide
(Mousdale et al., 1987). Plant enzymes, although nuclear
encoded, are largely active in the chloroplast and accordingly possess an N-terminal transit sequence. In contrast, all
fungi examined to date have monofunctional 3-deoxy-D arabino-heptulosonate 7-phosphate (DAHP) synthases and
chorismate synthases and a pentafunctional polypeptide
termed AROM (Duncan et al., 1987). The AROM
polypeptide has domains analogous to the bacterial
enzymes: dehydroquinate (DHQ) synthase, EPSP synthase,
0020-7519/$30.00 q 2003 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2003.10.006
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S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
Fig. 1. Molecular arrangement of the shikimate pathway enzymes are the same in Toxoplasma gondii and fungi, but different from plants and bacteria. (A) The
T. gondii arom gene is 19,460 bp and is interrupted by 19 introns (black). Exons are coloured relative to the corresponding domain order in the T. gondii AROM
polypeptide (B), DHQ synthase (pink), EPSP synthase (green), shikimate kinase (red), dehydroquinase (yellow) and shikimate dehydrogenase (blue). The
entire polypeptide spans 3332 amino acids. (C) The five central shikimate pathway enzymes are fused in fungi (e.g. Saccharomyces cerevisiae), are
monofunctional in plants (e.g. Lycopersicuon escultentum), with the exception of dehydroquinase and shikimate dehydrogenase which are fused to form a
bifunctional protein. In general the bacterial enzymes are monofunctional (e.g. Escherichia coli). A gap indicates that the genes are not fused. (D) The DHQ
synthase domain of T. gondii has a high degree of sequence conservation with other species, a predicted secondary structure similar to Emericella nidulans and
all the residues known to be important for this enzyme. Sequences are: T. gondii (Accession no. AY314743); Pneumocystis carinii (Q12659); S. cerevisiae
S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
shikimate kinase, DHQase and shikimate dehydrogenase
(Fig. 1).
A number of apicomplexan parasites have a vestigial
plastid organelle called an apicoplast, most likely derived
from an ancient algal endosymbiont (Kohler et al., 1997;
McFadden et al., 1996). This prompted us to seek evidence
of plant-like biosynthetic pathways in these parasites and
led to the identification of the first apicomplexan shikimate
pathway enzyme, chorismate synthase. Chorismate synthase
was isolated from T. gondii and a number of Plasmodium
species and in all cases the proteins lacked an obvious
N-terminal transit sequence, suggesting that they are
cytosolically active and unlikely to be located in the
apicoplast (Roberts et al., 1998, 2002). P. falciparum
chorismate synthase has since been reported to be present in
the cytoplasm (Fitzpatrick et al., 2001). Consistent with this,
phylogenetic analysis inferred that these apicomplexan
chorismate synthases were most closely related to fungal
enzymes which also function in the cytoplasm (Keeling
et al., 1999). Despite the availability of the P. falciparum
genome sequence (Gardner et al., 2002), definitive identification of the genes for the other six shikimate pathway
enzymes has proven problematic. Taking advantage of the
studies of other apicomplexan genomes we sought to
identify the shikimate pathway enzyme genes from
T. gondii.
A search of the Toxoplasma genome project (ToxoDB
2.1) revealed two contigs (assembled genomic sequences)
containing regions that appeared to code for a number of
shikimate pathway enzymes. TGG 7014 contained
sequences homologous to EPSP synthase, shikimate kinase,
DHQase and shikimate dehydrogenase. The order of the
elements on this contiguous region of DNA, although
spanning some 20 kb, was identical to the genomic
arrangement for the same four enzymes of the fungal
AROM pentafunctional protein (Duncan et al., 1987). TGG
3535, a fragment of genomic DNA (gDNA) of approximately 5 kb, contained sequences homologous to DHQ
synthase, the remaining enzyme present in the fungal
AROM. PCR was used to amplify a region spanning the two
fragments, the sequence of which confirmed that the
fragments were contiguous. (This has since been confirmed
in ToxoDB 2.2, TGG 8613.) This established that these five
enzymes are clustered in the T. gondii genome. To
determine whether the genes were fused to form an
AROM-type arrangement the cDNA sequence was determined. Initially, a probe was generated from a region of
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the putative DHQ synthase to screen a T. gondii (RH strain)
tachyzoite cDNA library. This obtained the 50 -region of the
putative DHQ synthase gene including the initiation codon.
However, as this sequence was truncated, an alternative
approach was used. RNA was extracted from T. gondii
tachyzoites (RH strain) using Trizol reagent (Invitrogen)
and used to generate cDNA using Moloney Murine
Leukemia reverse transcriptase (Invitrogen) according to
the manufacturer’s instructions. A series of overlapping
clones were amplified by PCR and cloned into the pDRIVE
vector using the Qiagen PCR Cloning Kit (Qiagen)
according to the manufacturer’s instructions. Clones were
sequenced commercially (MWG Biotech, Milton Keynes,
UK) and assembled using Sequencher (Gene Codes, Ann
Arbor, MI). This revealed a 10 kb sequence that had a single
open reading frame encoding a polypeptide of 3332 amino
acids with a predicted molecular weight of 361.7 kDa.
Comparison of the cDNA with the gDNA sequence reveals
that the gene consists of 20 exons (Fig. 1A).
The predicted T. gondii AROM (TgAROM) polypeptide
has all the domains, known to be highly conserved in fungal
AROMs with all the enzyme domains arranged in the same
order as observed in fungi (Fig. 1B). Nonetheless,
TgAROM has a number of obvious differences from the
fungal counterparts. Notably the protein is considerably
larger than the fungal AROMs, which range in size from
1563 amino acids in Neurospora crassa to 1588 amino acids
in Saccharomyces cerevisiae. The T. gondii AROM protein
has a number of insertions not present in the fungal
counterparts. Analysis of the relative hydrophobicity and
charge of these regions, using the ExPASy ProtScale tool
(http://us.expasy.org/cgi-bin/protscale.pl), suggests that
these areas could form exposed surface loops. The functions
of these regions are not obvious although similar hydrophilic insertions have been noted in a number of
apicomplexan enzymes including chorismate synthase
(Roberts et al., 1998).
Early studies established that, although the fungal
AROM was highly susceptible to proteolysis, many of the
resultant individual domains retained their enzymatic
activity. This observation allowed biochemical characterisation of the various enzyme components of the AROM, and
encouraged the expression and characterisation of individual or bifunctional domains of the AROM. For example,
the DHQ synthase and shikimate dehydrogenase domains
from the Emericella nidulans AROM gene can be expressed
as individual enzymatically active proteins in Escherichia
R
(NP010412) and E. nidulans (P07547). Proteins were aligned using MacVector (Oxford Molecular Group). The predicted secondary structure of the T. gondii
enzyme domain was obtained by the program PredictProtein (Rost, 1996). The E. nidulans structure was previously determined (Carpenter et al., 1998).
Identical amino acids are marked in red, similar amino acids are coloured blue and variable residues are black. Dashes indicate gaps to maximise alignment.
The residues identified to be important in the E. nidulans enzyme and conserved in the T. gondii protein are marked by asterisks. The secondary structure
prediction of the T. gondii protein is given above the alignment, where arrows represent beta strands and cylinders, a-helical regions. This is compared to the
known structure of the E. nidulans DHQ synthase domain given below the alignment. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
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S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
coli (Moore and Hawkins, 1993). However, the EPSP
synthase domain is not active when expressed as a single
domain, but only shows activity when expressed as part of a
DHQ synthase – EPSP synthase bifunctional protein (Moore
and Hawkins, 1993). The DHQ synthase domain from
E. nidulans has been expressed in E. coli and the 3D
structure determined by X-ray crystallography (Carpenter
et al., 1998). As this is the only component of the AROM
polypeptide to have been studied in depth, we have
compared the DHQ synthase domains from both the
T. gondii and E. nidulans AROMs to determine if the key
features are conserved between both proteins (Fig. 1D). All
the key residues identified by Carpenter et al. (1998) which
are known to be involved in the mechanism of the
E. nidulans DHQ synthase are conserved within the
T. gondii protein. These include the residues corresponding
to E. nidulans Glu194, His271 and His287 which interact
with the pentacoordinate Zn2þ, and the residues involved in
providing a phosphate-binding pocket, Lys152, Asn162,
Asn268, His275 and Lys356. In addition, the residues
identified as important in the binding of the DAHP substrate
analogue, carbaphosphonate (Lys152, Asn268, His275 and
Lys356 and Arg130) are conserved within the TgAROM
protein. This provides insight into the rational design of
other possible inhibitors for the T. gondii DHQ synthase. A
secondary structure prediction of the TgAROM generated
by the PredictProtein programme (Rost, 1996) (http://cubic.
bioc.columbia.edu/predictprotein/) has been aligned with
the known secondary structure elements of the E. nidulans
enzyme (Fig. 1D). There is a general consensus in the
predicted positions of a-helices and b-strand regions
between both species.
DAHP synthase catalyses the first committed step in the
shikimate pathway. Two classes of this enzyme have been
described. Class I (AroAI) was originally described as
39 kDa proteins similar to the E. coli enzymes and
paralogues, but can now be subdivided into AroAIa and
AroAIb exemplified by the E. coli orthologues and the
Bacillus subtilis orthologues, respectively (Gosset et al.,
2001). Many fungal and one oomycete, Phytophtora
infestans, have had Class I (AroAI) genes sequenced,
suggesting a wide eukaryote taxonomic distribution. Class
II (AroAII) DAHPs were originally described as similar to
the 54 kDa higher plant enzymes (Walker et al., 1996), but
are now known also to exist in a number of divergent
microbes such as Streptomyces and in the fungus N. crassa
(Jensen et al., 2002). In plants, AroAII are feedbackinhibited by arogenate, a precursor of phenylalanine and
tyrosine. Many bacteria, including E. coli have three
paralogous AroAI, DAHPs designated AroF, AroG and
AroH that are inhibited by tyrosine, phenylalanine and
tryptophan, respectively. Interestingly, the fungi N. crassa
and several prokaryotes possess both Class I and II DAHP
synthases. Consequently, it has been suggested that the two
DAHP classes may have different functions, for example
N. crassa and the bacterium Streptomyces hygroscopicus
class II enzymes have been linked to secondary metabolism
such as the production of antibiotics (Gosset et al., 2001).
The tBLASTn alogrithim was used to search ToxoDB
2.1 for evidence of both Class I or/and II DAHP synthases.
A portion of Contig TGG_9597 was found to code for a
putative protein with similarity to Class II DAHP synthase,
but no likely candidates were identified for a Class I DAHP
synthase. This region was amplified by PCR and used as a
probe to screen a T. gondii cDNA library. This produced a
number of overlapping clones that assembled to give the
entire T. gondii DAHP synthase, which was confirmed by
reverse-transcriptase PCR amplification to produce a full
length clone (Genbank accession number AY341375).
Initial alignments revealed that the T. gondii DAHP
synthase was a member of the AroAII family. The T. gondii
DAHP synthase (TgDAHP) is 615 amino acids in length and
has a predicted molecular mass of 67.4 kDa, significantly
larger than the previously described Class II enzymes due to
the presence of a number of insertions (data not shown)
analogous to those observed in the other shikimate pathway
enzymes (Roberts et al., 1998).
Having identified the genes that encode all seven steps of
the shikimate pathway (Roberts et al., 1998 and the current
paper) we were intrigued to investigate the evolutionary
origins of these genes. Current sampling of shikimate
pathway genes is confined primarily to prokaryotes, fungi
and plants. Previous work had suggested that the plant
shikimate pathway is derived from gene transfer events
from prokaryotic genomes, most probably the cyanobacterial endosymbiont that became the plastid (Martin et al.,
2002). As such the plants and the fungi do not form a
monophyletic eukaryote group on the phylogenetic trees of
shikimate pathway genes. Step seven of the apicomplexan
shikimate pathway, chorismate synthase, had been demonstrated to cluster with fungal homologues on phylogenetic
trees (Keeling et al., 1999). The fungi and apicomplexa
lineages are distant relatives within the eukaryotic evolutionary tree (Stechmann and Cavalier-Smith, 2002). As
such there are at least two possible explanations for the
topology of the chorismate synthase tree, either the
shikimate pathway is ancestral to eukaryotes and has
evolved through vertical descent, or a horizontal gene
transfer (HGT) event has occurred between these two
lineages. While investigating the evolution of the six
remaining shikimate pathway genes, alternative evolutionary scenarios should also be considered. These include the
possibility that the shikimate pathway genes may have been
derived from independent HGT events or alternatively, from
endosymbiotic gene transfer from either the mitochondrial
or the apicoplast endosymbiont.
We undertook a full phylogenetic investigation of the
remaining six genetic units that encode the T. gondii
shikimate pathway. We aimed to test whether the T. gondii
shikimate pathway had a single common origin and analyse
whether these genes were derived from vertical descent or
had been inherited horizontally from either the apicoplast
S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
genome, mitochondrial genome or any other source.
Inheritance from the apicoplast would be evident if the
T. gondii shikimate pathway genes clustered with the plants
or the cyanobacterial taxonomic groups on phylogenetic
trees (Kohler et al., 1997). Inheritance from the mitochondria would be evident if the T. gondii shikimate pathway
genes clustered with the a-proteobacterial taxonomic
groups on phylogenetic trees. All six genes were aligned
with the available homologues from GenBank retrieved
using tBLASTn. The genes were aligned automatically
using the program Clustal_X (Thompson et al., 1997) and
refined manually using the program genetic data environment (GDE). The alignments were masked to exclude
sequence positions that could not be aligned with confidence
such as hyper-variable regions of the protein sequence. The
dehydroquinase portion of the AROM is highly variable
with few conserved characters identifiable; a reasonable
alignment and character sampling could not be achieved,
preventing phylogenetic analysis. However, BLAST
searches suggest that the T. gondii enzyme is most similar
to the type I enzyme normally associated with the AROM
protein. The DHQase domain is best aligned by focusing on
the lysine residue involved in the formation of the covalent
imine intermediate, which is characteristic of the type I
family of DHQases (Butler et al., 1974). This residue lies at
the centre of an eight-stranded a/b barrel which forms the
core of this domain. Secondary structure predictions of the
DHQase portion from the T. gondii AROM polypeptide can
identify many, but not all the components of this a/b-barrel
structure. Further analysis at the structural level maybe
required to fully determine if this sequence is capable of
forming the correct a/b-barrel structure.
The five remaining masked protein alignments were
analysed using Bayesian maximum likelihood phylogenetic
methods using the program MRBAYES 2.01 (Huelsenbeck
and Ronquist, 2001). Gamma distribution and the proportion of invariant site parameters were calculated using
the Bayesian Metropolis-coupled Markov chain Monte
Carlo (MCMCMC) parameter search. Tree and parameter
space was sampled using the MCMCMC method initiated
on a random tree, using the JTT matrix. The analysis was
run for 1,000,000 generations and sampled every 100
generations. All MCMCMC values reached a plateau within
the first 50,000 generations sampled, therefore the trees
were sampled from 50,000 to 1,000,000 generations
(Bayesian trees represent consensus of 9500 trees). All
other trees were excluded as burn in. The level of burn in
used was sufficient to guarantee that the parameter searches
had stabilised. Bayesian posterior probability values are a
product of sampling the MCMCMC plateau and are
therefore frequently less informative than bootstrap values,
often overestimating support values for phylogenetic tree
topologies. Therefore bootstrap support values from distance analysis were calculated with the program PuzzleBoot
providing a more rigorous analysis of the level of tree
topology support (Schmidt et al., 2002) (Holder, M., and
9
Roger, A.J. PuzzleBoot version 1.03; http://hades.biochem.
dal.ca/rogerlab/software/software.html). Gamma correction
and proportion of invariant-site values derived and averaged
from the plateau in the MCMCMC parameter space search
and the WAG model (Whelan-Goldman, 2000) of aminoacid evolution was used in the bootstrap analysis. The use of
two distinct methods, Bayesian and distance analysis with
different models of amino acid evolution for tree construction and tree evaluation, respectively, provides increased
confidence in tree topological relationships. Where both
methods are in agreement and tree topology is consistent
between these independent analyses, tree topologies are
unlikely to be artefacts of one method of phylogenetic
analyses or one model of protein evolution. Unrooted
analyses were performed to allow for all possible evolutionary scenarios, some of which may have been falsely
excluded if rooted analyses were performed. It has been
recognised that ‘the probabilities of obtaining the correct
rooted tree are considerably lower than the probabilities of
obtaining the correct unrooted tree’, therefore ‘a considerable amount of error in constructing a rooted tree occurs at
the time of rooting’ (Sourdis and Krimbas, 1987; Smith,
1994). Following our unrooted analyses that suggest the T.
gondii genes group with the fungi, a root was inferred
between the division of prokaryotes and eukaryotes and
trees were drawn accordingly. Currently it is impossible to
reliably identify the evolutionary root of these enzymes.
Consequently an ingroup/outgroup approach was used to
root these trees. The monophyletic fungi and T. gondii
clades supported in the unrooted analyses were used as the
ingroup, all bacteria and the plant genes (suggested to be
derived from horizontal gene transfer from a bacterial
source), were used as an outgroup. This rooting was only
possible as the unrooted analysis demonstrated that the
fungi and T. gondii eukaryotic clade was monophyletic and
separate from all the other groups, an evolutionary
relationship additionally supported by the AROM gene
arrangement. Interestingly, the plant enzymes were found to
group with the prokaryote enzymes, a relationship consistent with the proposal that the plant shikimate pathway is
cyanobacterial in origin and has been derived from the
bacterial ancestor of the chloroplast. However, the cyanobacteria did not consistently group with the plants in these
phylogenetic trees and as such the plant shikimate pathway
enzymes may have an alternative evolutionary ancestry.
The T. gondii DAHP gene sequence clustered with the
Class II homologue from N. crassa with 51% bootstrap
support in phylogenetic analysis (Fig. 2). This tree topology
is consistent with the phylogenetic relationships seen in the
chorismate synthase phylogeny, which show the T. gondii
gene clustering with the fungi, and similarly suggests that
the T. gondii and the N. crassa DAHP genes have a common
origin. These two eukaryotes group strongly (92% bootstrap
support) with a DAHP homologue from the d-proteobacteria
Stigmatalla aurantiaca. This relationship suggests that the
origin of the primitive eukaryote DAHP-synthase enzyme,
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S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
Fig. 2. Phylogeny of DAHP (II) shows the Toxoplasma gondii protein
clustering with the fungi, Neurospora crassa. The phylogeny was
calculated using MRBAYES (Huelsenbeck and Ronquist, 2001) and
bootstrap values were calculated using PuzzleBoot (Holder, M. and Roger,
A.J. PuzzleBoot version 1.03. http://hades.biochem.dal.ca/rogerlab/
software/software.html). Support values are shown when in excess of
49% in the order of Bayesian posterior probability/distance bootstrap value
(see text for further details of the methods used). The phylogeny was
calculated from a sampling of 42 taxa with a character sampling of 372
amino acids.
present in both T. gondii and N. crassa, is from the
d-proteobacteria. Increased sampling of prokaryote genomes may reveal an alternative sister group to the T. gondii/
fungi cluster. The presence of the two distantly related
S. auranitiaca homologues suggests that further prokaryote
DAHP-synthase genes remain unsampled or alternatively
there have been HGT events from a eukaryote to
S. auranitiaca.
The phylogeny for the DHQ synthase was poorly
supported and did not resolve a tree topology with any
confidence. Several attempts were made to adjust the
alignment and character sampling to improve the resolution
of the phylogenetic tree. The phylogeny did not resolve
whether the DHQ synthase was more closely related to
fungal homologues or clustered within the prokaryote
homologues (data not shown). This leaves unsolved the
evolutionary origin of the T. gondii DHQ-synthase AROM
domain, which plausibly may have evolved from a separate
HGT event from a prokaryote source. However, the
DHQ-synthase phylogeny confirmed that the T. gondii
DHQ-synthase gene did not originate from the apicoplast
genome as the T. gondii enzyme did not cluster with either
the plants or the cyanobacteria on our phylogenetic tree.
The remaining three genetic units of the AROM
indicated a monophyletic relationship between the fungi
and T. gondii. This relationship was supported with low to
moderate bootstrap values of 54, 78 and 64% in the
phylogenies of EPSP synthase (Fig. 3A), shikimate kinase
(Fig. 3B) and shikimate dehydrogenase (Fig. 3C), respectively. In the case of the EPSP synthase and shikimate
dehydrogenase, the T. gondii genes grouped at the base of
the fungal cluster consistent with these genetic units being
inherited by vertical descent from the common ancestor of
fungi and T. gondii. Interestingly, the shikimate kinase
Bayesian phylogeny recovered the T. gondii gene within the
fungal cluster (Fig. 3B); however, the bootstrap tree
topology is consistent with the EPSP synthase and shikimate
dehydrogenase phylogenies implying that T. gondii groups
at the base of the fungal cluster. This suggests that the
position of the T. gondii shikimate kinase gene within the
fungal cluster, rather than at the base is an artefact.
Overall the phylogenetic analyses are consistent with the
proposition that the shikimate pathway genes in fungi and
T. gondii are related by vertical descent, from a distant
eukaryotic ancestor of both lineages, in spite of the low to
moderate bootstrap values, symptomatic of this type of
study (Richards et al., 2003). This is not only supported by
five shikimate pathway gene phylogenies (this study and
Keeling et al., 1999) that show the grouping of T. gondii
with the fungi, but also with our demonstration that these
organisms have a homologous AROM arrangement.
Although HGT between the fungi and T. gondii lineages
could explain the tree topologies recovered, this explanation
is less parsimonious than the hypothesis of vertical descent
as it would require the transfer of three genetic units, the
AROM, DAHP synthase and chorismate synthase between
these two lineages. There is also an evidence that the
shikimate pathway is widespread through out the eukaryote
kingdom, for example the oomycete, P. infestans, encodes a
DAHP synthase protein (see Genbank accession number
AF424663.1). Additionally, there is biochemical evidence
of an AROM-like protein in Euglena gracilis (reviewed,
Roberts et al., 2002), supporting the hypothesis that the
AROM genetic arrangement in particular is both widespread and therefore probably of ancient derivation in the
eukaryotic kingdom. Although there are marked differences
in intron number and gene length between the T. gondii
AROM and the known fungal AROMs, it is highly unlikely
that this five-gene fusion would have evolved independently
on two separate occasions within the eukaryotic kingdom. It
is even more unlikely that the five-gene fusion, if it were to
occur independently in the eukaryote kingdom, would
produce a fused gene with the same domain order. Thus, the
most parsimonious explanation is that the AROM supergene
was an ancient eukaryotic innovation and probably occurred
by the fusion of the genes encoded on a previously evolved
prokaryotic operon (Andersson and Roger, 2002) donated
S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
11
Fig. 3. Phylogenetic trees showing the evolutionary relationships of three of the five functional domains encoded on the AROM polypeptide. All the
phylogenies were calculated using MRBAYES (Huelsenbeck and Ronquist, 2001) and bootstrap values were calculated using PuzzleBoot (Holder, M. and
Roger, A.J., PuzzleBoot version 1.03. http://hades.biochem.dal.ca/rogerlab/software/software.html). Support values are shown in excess of 49% in the order of
Bayesian posterior probability/distance bootstrap value (see text for further details of the methods used). (A) shows the EPSP phylogeny. (B) and (C) show the
phylogenies of shikimate kinase and shikimate dehydrogenase, respectively. The taxon and character sampling for these phylogenies is as follows: EPSP 69
taxa and 293 amino acid characters, shikimate kinase 44 taxa and 139 characters, and shikimate dehydrogenase 52 taxa and 166 characters. In all the
phylogenies the Toxoplasma gondii AROM domains cluster with the fungal homologues suggesting they are related, given the taxon sampling available. The
shikimate kinase phylogeny also revealed a potential cyanobacterial to plant gene transfer, consistent with this plant enzyme originating from the plant
chloroplast endosymbiont, although the cyanobacteria were not monophyletic in the Bayesian tree. The bootstrap tree shows a monophyletic cyanobacteria
clade sister to the plant clade with a bootstrap support value of 50%. Following preliminary phylogentic analysis nearest neighbour paralogues were excluded
unless sequence similarity was low as noted for Listeria moncytogenes (C) and Mesorhizobium loti (B).
12
S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
from the bacterial progenitor of the eukaryotes (Martin et al.,
2001). A survey of some 80 currently available completed
prokaryotic genomes found clustering of shikimate pathway
genes in a number of taxonomes, some of which are known
to be co-transcribed as an operon. The lack of a described
clustering in the precise order of the AROM functional
domains, may reflect lack of sampling or alternatively that
multiple sequential fusion events, coupled with rearrangements in domain order occurred in the evolution of an
efficient functional AROM protein.
In testing the evolutionary origin of the T. gondii
shikimate pathway we had to consider a number of possible
evolutionary scenarios that could have arisen during
apicomplexan evolution. These include the possibility of
direct vertical descent or the acquisition of genes that
encode plastid-located enzymes from the algal endosymbiont. In the latter case, these genes would have been
derived from the algal plastid genome and may or may not
have been transferred to the nuclear genome as proposed for
modern plants. We included homologues from the cyanobacteria and the plants, in an attempt to exclude an origin
from the plastid genome of the progenitor of the apicoplast.
We found no evidence to suggest that the T. gondii
shikimate pathways genes were inherited from the apicoplast genome. However, as these studies progressed and
with the realisation that the shikimate pathway may have
been an ancestral trait in eukaryotes another possibility had
to be considered. That is, the T. gondii genes may have been
derived from the nuclear genome of the algal endosymbiont
that became the apicoplast. Our analysis, however, could
not exclude this possibility. Given that the shikimate
pathway and the arom supergene appear to have a wide
eukaryotic distribution, it is plausible that the algal nucleus
may have contained the ancient eukaryotic shikimate
pathway genes with an AROM-like polypeptide. Eukaryotic
gene sampling, currently lacking shikimate pathway
homologues from algal groups, the likely progenitors of
the apicoplast (Kohler et al., 1997), means that it is
impossible to distinguish between vertical descent or
alternatively an origin from the nucleus of the algal
progenitor of the apicoplast.
The phylogenetic investigations fail to show a consistent
prokaryotic sister group to the fungi/Toxoplasma eukaryote
cluster, thus preventing the identification of a prokaryote
donor lineage. However, the phylogenies produced no evidence that the T. gondii genes were inherited from either the
mitochondria or the plastid. Our analysis also did not support
any incidence of prokaryote to T. gondii horizontal gene
transfer as all the phylogenies showed a common ancestor
with the fungi. This would suggest any transfer event would
have had to occur prior to the division of the fungi and
T. gondii lineages. However, the DHQ-synthase phylogeny is
currently unresolved and a prokaryote to T. gondii transfer
scenario is still possible for this enzyme domain.
Re-examination of the completed P. falciparum genome
did not provide evidence of an AROM-type protein.
However, a potential EPSP synthase/shikimate kinase
bifunctional protein is evident (accession no. NP472984)
and is likely to be the gene previously reported to have low
similarity with S. cerevisiae AROM polypeptide (Gardner
et al., 2002). Homologues of this potential EPSP synthase/
shikimate kinase bifunctional protein are present in a
number of other Plasmodium species (Plasmodium yoelii
accession no. EAA17633 and Plasmodium chrPch002449).
This raises the question as to why the remaining enzymes
are not readily identifiable. It seems unlikely that these
enzymes are absent, as we now have evidence for the final
three enzymes of the pathway, providing a route from
shikimate to chorismate. We also know that inhibition of
one of these enzymes, EPSP synthase, is capable of
restricting parasite growth (Roberts et al., 1998). There is
no known route to produce shikimate other than by the four
missing enzymes and shikimate would not be available
within the host. This suggests that there may be enzymes
with the same biochemical ability, but vastly different in
sequence, thus making them difficult to identify. Alternatively this highlights a potential ongoing challenge for gene
prediction and thus complete annotation of the P. falciparum and other Plasmodium genome projects.
We have provided the first evidence for the entire set of
seven shikimate pathway enzymes in any apicomplexan
parasite, their genetic and molecular arrangement and their
likely evolutionary origin. The results presented for
T. gondii provides the tools for functional studies, structural
determination and rational drug design. Phylogenetic
comparisons suggest that the AROM-gene fusion was an
innovation likely to have been present in the progenitor of
modern eukaryotes, as the distantly diverged T. gondii and
fungi lineages both posses a homologous arom supergene.
Thus, the shikimate pathway, rather than being confined to
bacteria, fungi and plants and at least some apicomplexans,
is likely to have been an ancient eukaryotic attribute. It has
been lost in many taxonomes, including mammals that are
now dependent on exogenous aromatic compounds. In
plants the ancient gene organisation has not survived and it
seems likely that the source of the shikimate pathway genes,
which are essentially bacterial like, has been through the
acquisition of the chloroplast, although this hypothesis
requires further testing. It also seems likely that the list of
taxonomes where this ancient pathway has been retained is
likely to grow as we see the completion of more eukaryotic
genome projects.
Acknowledgements
Preliminary genomic and/or cDNA sequence data was
accessed via http://ToxoDB.org and/or http://www.tigr.org/
tdb/t_gondii/. Genomic data were provided by the Institute
for Genomic Research (supported by the NIH grant no.
AI05093), and by the Sanger Center (Wellcome Trust). EST
sequences were generated by Washington University
S.A. Campbell et al. / International Journal for Parasitology 34 (2004) 5–13
(NIH grant no. 1R01AI045806-01A1). The work reported in
this manuscript was funded by NIH, USA RO1 AI-43228,
the Wellcome Trust, Koshland, Breenan, Blackmon, Langel
and Kiewit families. T.A.R. is supported by a BBSRC
studentship.
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