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RES E A R C H A R T I C L E Open Access
Horizontal gene transfer of epigenetic machinery
and evolution of parasitism in the malaria
parasite Plasmodium falciparum and other
apicomplexans
Sandeep P Kishore
1
, John W Stiller
2
and Kirk W Deitsch
1*
Abstract
Background: The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the
transition of the anc estor of apicomplexans from a free-living organism to an obligate parasite. The ability to
control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated
forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding
question in protistan biology.
Results: In this wor k, we study SET domain bearing genes that are implicated in mediating immune evasion,
invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first
conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal
host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes
involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36
methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to
the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of
cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of
transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust
and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in
ciliates, the nearest free-living organisms with complete genome sequences available.
Conclusions: Animal and plant sources of epigenetic machinery provide new insights into the evolution of
parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and
key transcription factors, the acquisition of SET domain meth yltransferases marks a key transitional event in the
evolution to parasitism in this important protozoan lineage.
Keywords: Protozoa, Plasmodium, Apicomplexa, Transcription, Parasitism, SET domain, Horizontal gene transfer
* Correspondence: kwd2001@med.cornell.edu
1
Department of Microbiology and Immunology, Weill Cornell Medical
College, New York, NY 10065, USA
Full list of author information is available at the end of the article
© 2013 Kishore et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Kishore et al. BMC Evolutionary Biology 2013, 13:37
http://www.biomedcentral.com/1471-2148/13/37
Background
The e volutionary processes whereby free-living or sym-
biotic organisms made the tr ansition to full-fledged
obligate, intracellular parasites remain unclear. This is
perha ps best exemplified by the case of the Apicomplexa,
a group of ~5000 spe cies that includes several major
human disease-causing agents including Toxoplasma
gondii, Cryptosporidium spp.andPlasmodium falciparum,
the most lethal of human malari a parasites [1]. Presumably,
this transition involved the development of novel cellu-
lar differentiation pathways that enabled infection and
replication with in different ho sts , inva sion schema,
cytoadhesion to host substrates and immune evasion
strategies including antigenic vari ation.
The newly acquired lifestyle complexity m ight al so
require the acquisition of new mechanisms to control
gene expression. For example, more sophisticated tran-
scriptional regulation and epigenetic machinery would
enable the evolution of complex life cycles involving
multiple hosts and stages , and facilitate developmental
changes accompanying the transition to parasitism.
This could involve either de novo innovations or the
horizontal acquisition of transcriptional and epigenetic
machinery from other eukaryotes. Examples of both
mechanisms of innovation have been reported. For
instance, we p reviously described the unusual and
rapid evolution of the C-terminal domain of RNA
polymerase II within the Pla smodium lineage, and
more spe cifically the expansion of this dom ain in para-
sites that infect primates [2]. This domain is crucial for
controlling m any aspect s of gene expression, including
epigenetic mechanisms of control, and the rapid de
novo evolution of host-specific modifications demon-
strates how important aspe cts of gene expression are
for parasitism.
In contrast, it is now well established that the pri-
mary class of transcription factors in apicomplexan
parasites ( the ApiAP2 family) was acquired through an
ancient horizontal gene transfer (HGT) e vent [3]. It is
known that the ancestor to apicomplexans engulfed an
alga, endowing it with photosynthetic abilities and
enabling it to synthesize several important products ,
including fatty acids [4,5]. The engulfed alga later
degenerated into a relic , the apicoplast; this accompan-
ied the loss of photosynthetic ability and the e volution
of apicomplexans into obligate intracellular para sites
metabolically dependent on their animal hosts. The
ApiAP2 cla ss of transcription factors was acquired
horizontally from the relic alga and now contributes to
controlling parasite gene expre ssion. Similarly, protein
domains involved in cytoadhesion and O-linked glyco-
sylation appear to have been acquired through HGT, in
these ca ses from the parasites’ hosts rather than the
algal endosymbiont [6,7]. The origin of the innate
immune system in early animals, the likely hosts of
newly e volved apicomplexan parasites , also dates to
this evol utionary peri od. Thus , the hallmarks of para-
sitism, including nutritional dependence, inva sion, and
immune escape likely developed as part of the same
evolutionary process during which photosynthetic
ability wa s lost.
In the case of the Apicomplexa, therefore, two major
potential sources for HGT have been established: i)transfer
from an algal endosymbiont (fatty acid synthases or
ApiAP2 transcription factors) [3,8], or ii) transfer from
an animal host (domains involved in cytoadhesion a nd
O-linked glycosylation) [6,7]. Here, we consider the
putative acquisition of epigenetic machinery in the an-
cestor of apicomplexans with a focus on histone lysine
modifiers, which are central to pathways of cellular
differentiation, c ell invasion and immune e va sion in
apicomplexan parasites.
Histone lysine methyltransferases, characterized by a
SET domain, play a fundamental role in gene activation
and epigenetic regulation across all eukaryotes [9]. These
domains modify histone lysine residues at Histone H3 Ly-
sine 4, 9, 36, and Histone H4 Lysine 20. These modifica-
tions are crucial for the establishment and maintenance of
epigenetic memory, including in P. falciparum, and are
involved in imprinting genes involved in invasion and
immune evasion [10-15]. Among these SET domain bear-
ing modifiers, the epigenetic modifier Set8 is known to
participate in mitosis and is thought to facilitate the trans-
mission of heterochromatic marks through the cell cycle
in higher eukaryotes as well as in the Apicomplexa [16].
In recent work, Sautel et al. (2007) sampled a small set of
animal sources and found strong homology to apicom-
plexan Set8, raising the possibility that this gene was
acquired by an ancient apicomplexan ancestor from its
eukaryotic host. That study, however, did not explicitly
address the likelihood of HGT or, if such an event did
transpire, when in the course of apicomplexan evolution it
likely occurred.
An extensive examination of SET doma in containing
proteins (and the corresponding demethylases) found in
the P. falciparum genome was reported by Cui et al.
[17]. One of these genes displays significant similarity to
Set2, a chromatin modifier known to deposit methyl
groups during active transcription by RNA polymerase
II [18]. This protein is present in primate malaria para-
sites but c onspicuously missing in the closely related
rodent parasites. In this work, we provi de evidence fo r
horizontal transfer of these me thyltransfera ses. Intri-
guingly, the proposed transfer event s occur on the
branch of the phylogenetic tree on which the para sitic
lifestyle of a picomplexans appears to have e volved,
including the acquisition of cytoadhesion domains and
their O-glycosyltransferase modifiers. The acquisition of
Kishore et al. BMC Evolutionary Biology 2013, 13:37 Page 2 of 12
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these various capabilities were essential steps in equipping
these organisms to their new host-dependent lifestyles and
therefore marks a key transitional event in the evolution to
parasitism in this important protozoan lineage.
Results
Apicomplexan Set8 is derived from animal Set8
The histone methyltransfera se Set8 of apicomplexan
parasites was recently shown to display strong sequence
similarity to the orthologous protein in animals [16],
suggesting the possibility of an ancient horizontal acqui-
sitionofthegeneencodingthisprotein.Therefore,we
were interested in more extensive phylogenetic a nalyses
of the origins of apicomplexan Set8. Using a canonical
SET domain from Homo sapiens Set8 (PR-7) as the
query, we searche d for the closest seque nces to the Se t8
domain across all major eukaryotic phyla. In many
organisms, no con clusive Set8 ortholog coul d be identi-
fied, and in all taxa other than animals and apicomplex-
ans, sequences retrieved using human Set8 as query
proved to be most similar to other Set fa milies in recip-
rocal bla st searches of the complete NC B I protein data-
base, suggesting t hat they might not be bona fide Set8
orthologs. Nonetheless, to avoid erroneously excl uding
highly diverge nt sequences , the nearest bla stp matches
to human Set8 were included in our analyses. Our
complete dataset therefore represented p otential Set8
sequences from all major eukary otic lineages for use in
phylogenetic analyses.
A phylogenetic tree was derived from combined
maximum-likelihood (ML) and Bayesian inference using
Set8 orthologs across this broad sampling of eukaryotic
organisms with the results shown in Figure 1. This tree
differs significantly with current consensus phylogenies
derived from larger data set s [19], which clearly show
the great evolutionary distances between apicomplexans
and the animal and plant kingdoms. Spe cifically, we
recovered apicomplexan Set8 sequences as a monophy-
letic grouping that does not occur in the expected
position on the tree. Instead of grouping with closely
related p rotist s such a s Perkinsus marinus and ciliates
[19], apicomplexan Set8 sequences nest strongly within
the animal Set8 clade. Consistent with this, no other
phyla or taxa show any phylogenetic affinity for apicom-
plexan Set8; this inc ludes algal and plant sequences,
which represent alternative potential sourc es for HGT.
Moreover, the other SET domain bearing sequences
(the lower clade in Figure 1) do n ot appear to represent
bonafideSet8proteins.Tetrahymena proteins are
found in this clade, for instance, and Tetrahymena is
known to lack Set8 [16]; a s noted above, none of these
sequences retrieved Set8 when used as queries in recip-
rocal blastp searches.
The recent completion of the P. marinus genome
provided a particularly valuable resource for investigat-
ing whether the Set8 transfer likely occurred spe cific-
ally in the ancestor of extant apicomplexans. Perkinsus
marinus, a parasite of oysters, i s the nearest known
relative of dinoflagellates and therefore s er ves as a
representative sister group of apicomplexans for o ur
analysis. Be cause dinoflagellates are predominantly
free-living, Perkinsus mu st have evolved its para sitic
lifestyle independently of the apicomplexans. The
nearest SET domain bearing sequence to human Set8
from the P. mar inus genome (E ≤ 10
-13
)didnotreturn
a match to Set8 when used as a query in a reciprocal
blastp search, and nested deeply within the clade of
non-Set8 sequences in phylogenetic analyses (Figure 1).
While caution must always be used when concluding
that a sequence is absent from any particular genome,
the P. marinus genome database includes over 23,000
inferred proteins , suggesting a relatively complete data
set for p roteomic comparisons.
In an expanded survey of eukaryotes, including partial
genomic resources available to date, blast queries with
human Set8 failed to identify any significant hits (E ≤ 10
-5
)
in other organisms closely related to the apicomplexans,
namely dinoflagellates, colpodellids, and Chromera velia,a
photosynthetic autotroph discovered recently. The same
was true for a variety of other protists including kinetoplas-
tids, Trichomonas and Giardia,aswellasfungi(including
budding yeast), and also certain amoebozoans including
Acanthamoeba.
As shown in Figure 1, statistical support for apicom-
plexan Set8 grouping with animals is very strong (Bayesian
probability of 1.0 and ML bootstrap of 86%), while the clos-
est sequences to Set8 in all other non-animal species
grouped in a separate clade (also with very strong support).
TheonlyexceptionistheslimemoldDictyostelium,which
also groups within the animal Set8 clade. Given that
complete genomes of other members of the Amoebozoa
phylum (e.g. Entamoeba and A canthamoeba) lack Set8, this
provides evidence of a similar horizontal transfer of animal
Set8 into slime molds. Considering the vast evolutionary
distances between animals, the Apicomplexa and slime
molds, and the absence of Set8 from all other eukaryotic
lineages, it appears almost certain that the latter two groups
acquired Set8 independently via HGT from animals. The
highly unlikely alternative would be direct descent of api-
complexan Set8 from a common ancestor with animals,
followed by gene loss repeatedly and independently from
all other eukaryotes except some cellular slime molds.
A nematode appears to be the source of the host transfer
to an ancient apicomplexan
The finding that apicomplexan Set8 is likely of animal
origin raises questions about th e approxim ate time of
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Figure 1 Phylogenetic tree of eukaryotic organisms based on Set8 orthologs. This tree based on Set8 shows a phylogenetic incongruence
with respect to trees recovered by broad scale eukaryotic phylogenomics. Displayed in blue is the animal clade of Set8 denoted by a strongly
supported node (large arrow). Apicomplexan Set8 groups within the metazoan/animal clade with strong posterior probability support that is
unexpected given accepted eukaryotic relationships. Set8 is absent in other lower eukaryotes based on current genome sequences. Where the
closest Set8 hits were found, the proteins group together in the yellow shaded box (with strong support). These are likely not Set8 as
Tetrahymena, a representative ciliate known to lack Set8, is featured in this clade. Based on these observations, it is likely that the apicomplexan
Set8 is derived from animals. A horizontal gene transfer provides the simplest, most parsimonious explanation.
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the transfer e vent , and the source of the acqu ired
sequence. A s noted above, the time of the transfer
appearstobeafterthedivergenceofapicomplexans
from dinoflagellates and the colo podellid s [20] since
these organisms appear to lack Set8, but prior to the
radiation of the known apicomplexans , which all have
Set8. To more accurately explore which spe cific animal
taxon was the likely source of the gene, Set8 sequences
from extant apicomplexans (P. falciparum, T. annulat a,
T.gondii, C. par vum and B. bov is)wereusedinphylo-
genetic analyses with only the animal and D ictyostelium
sequences. To promote more accurate re covery of
phylogenetic associations within the Set8 clade, we
discarded highly divergent, non-orthologous SET
sequences from other eukaryotes that could produce
phylogenetic artifacts within the Set8 clade.
As shown in Figure 2, we consistently obser ved that
apicomplexan sequences branch within the nematodes ,
and specifically with Set8 from Tr ichonella spiralis.
The ML bootstrap support for this relationship is weak
(32%) but the Bayesian posterior probability is strong
(0.99). Importantly, th is tree recovers the same rela-
tionship between nematodes and Apicomplexa as did
the expanded analysis, indicating this topology is stable
regardless of the ta xa sampled.
Furthermore, it is telling that both the slime mold
Dictyostelium and the A picomplexa branch with nema-
todes, but separate from each other. If the positions of
the Dictyostelium and apicomplexan s equences were
long-branch attraction artifacts, they would be expected
to attract each other as the two most divergent branches
of the Set8 tree. M oreover, neither is attracted to the
base of the Set8 clade, or even to the ba se of the nema-
tode clade, but rather to individ ual sub-clades within
the nematodes. This contrast s with the c lear artefac tual
rooting of the overall Set8 clade, wherein long-branch
outgroups are attracted to the rapidly evolving sequence
from the parasitic trematode Schistosoma m ansoni.
Thus, although statistical support for a specific relation-
ship between apicomplexan and nematode sequences is
Vertebrates
Arthropods
Nematodes
Apicomplexa
Figure 2 A deeper look at Set8: Apicomplexa, animals and slime molds. A phylogenetic analysis with only animal, apicomplexan and slime
mold Set8. Set8 from Apicomplexa, nematodes and arthropods strongly group together (0.81 posterior probability) in a sub-clade apart from
vertebrates. A Set8 ortholog from an animal, the parasitic nematode Trichinella spiralis, groups with strong Bayesian posterior probability with the
Apicomplexans orthologs. Similarly, soil nematodes group with the soil-based slime mold Dictyostelium, implying a horizontal gene transfer event
in this taxon as well. These proposed events are denoted by black arrows. As all apicomplexans studied feature Set8, the most likely scenario is a
horizontal gene transfer to an ancestral apicomplexan.
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not strong , there appears to be no basis for concluding
it is a typical phylogenetic artefact. While the precise
timing of the emergence of nematodes is a subject of de-
bate, all estimates place the ir origin between 600– 1200
million years ago [21]. Thus , nematodes were extant at
the time of the apicomplexan radiation, making the
proposed transfer plausible.
Analysis of the reciprocal H3K36 modifiers Set2 and
JmjC1 in Apicomplexa
Similar t o Set8, Set2 is a histone methyltransferase that
contributes to the regulation of higher order chromatin
assembly and the epigenetic control of gene expression.
Set2 and it s cognate demethylase JmjC1 work b y adding
and removing methyl groups f rom H3K36, respe cti vely
[22]. With the exceptio n of the rodent plasmodia, all
apicomplexan parasit es, regardless of host , possess an
apparently orthologous (within apicomplexans) protein
similar to Set2. At least two ci liates (Tet rahy mena and
Paramecium) also possess putative Set2 homo logs,
suggesting that the Set2 family could have been present
in the alveolate lineage before the divergence of the
Apicomplexa from ciliates.
Aravind and colleagues have argued that a major fam-
ily of transcription factors (ApiAP2) in Apicomplexa
were laterally transferred from the algal endosymbiont
harbored intracellularly by all members of this group of
parasites [3]. This notion led us to question whether the
chromatin modifiers Set2 and JmjC1 also could have
originated from a similar horizontal transfer event. To
determine if Set2 and JmjC1 might have been acquired
from an algal endosymbiont, or any othe r higher
eukaryotic cell, we performed bioinformatic and phylo-
genetic analyses as described previously for Set8.
Horizontal transfer of a H3K36 modifier into the
Apicomplexa
In addition to Set8 disc ussed above, phylogenetic
analyses clearly define Plasmodium proteins similar to
the Set2 subfamily, as well as to Set1 and Set3 (Figure 3).
Interestingly, although a Set2 subfamily including ani-
mal, yeast and higher plant exemplars is well resolved in
our analyses (Figure 3), the putative Set2 sequence from
Plasmodium (PF3D7_1322100) groups with Ashr3, a
related H3K36 methyltransferase from green plants. An
initial broader analysis performed with putative Set2
Figure 3 Phylogenetic tree and domain characteristics of SET domain proteins from P. falciparum. SET proteins were retrieved through
reciprocal blastp searches and analyzed using maximum-likelihood and Bayesian inference. The upper, dark gray box indicates the strong
grouping of P. falciparum ‘Set2’ with Ashr3 sequences from green plants. The larger, lighter gray box below defines canonical Set2 sequences
defined in model organisms. Although both are included in a larger clade of putative H3K36 modifying proteins, apicomplexan ‘Set2’ homologs
do not group with their ostensible alveolate sister group, the ciliates (represented by Tetrahymena and Paramecium). The strong phylogenetic
association with plant Ashr3 proteins is further supported by overall domain architecture, including (and unlike canonical Set2 and ciliate
homologs) the shared C-terminal location of the SET domain, as well as a highly conserved PHD region that is proximal to the SET domain in
both. In the alignment of this region included, residues that are invariable among plant and apicomplexan sequences are shaded green, those
with conservative substitutions are in yellow. The only SET-containing sequence from Perkinsus marinus with significant similarity to Set2, is
strongly supported as a member of the Set1 subfamily, and (as expected) as sister to the P. falciparum Set1 paralog.
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sequences from all apicomplexans re covered the same
topology, but with weaker support (Additional file 1).
To clarify the SET protein subfamily to which apicom-
plexan Set2 belongs, we produced a tree with more
balanced sampling, using only sequences of P. falciparum
along with Set family exemplars from plants, animals and
yeast, and Set proteins from ciliates and P. marinus.The
resulting tree is shown in Figure 3, and provides strong
support in both ML and Bayesian analyses for an associ-
ation b etween the Pla smodium sequence and Ashr3
homologs from green plant s. In contrast, the nearest
SET protein from P. marinus , t he expected sister group
to apicomplexans , is defined clearly as a member of the
Set1 subfamily.
Because only the SET domain itself can be aligned
across all these diverse sequences, a relatively short
alignment (231 aa) is available for tree re construction;
potentially this could lead to spurious phylogenetic
associations. In this case, howe ver, there is additional
corroborating evidence outside the SET domain for a
close relationship between PF3D7_1322100 and the
plant Ashr3 family. In both cases , the SET domain is
positioned at the C-terminal end of the protein. In
contrast, Set2 orthologs from animal, yeast and green
plant models have a SET domain in more N-terminal
locations [23]. Potential Set2 homologs from ciliates ,
the nearest relatives of apicomplexans present in the
Set2 sub-clade, also have SET domains positioned at
their extreme N-termini. More significantly, Simple
Modular Architecture Research Tool (SM ART; [24])
identifies a shared plant homeodomain (PHD) as the
first identifiable d omain upstream of the SET domain
(Figure 3). Although the complete gene from Plasmodium
has undergone a dramatic expansion, this PHD region is
conser ved strongly enough between plant Ashr3 and
Plasmodium genes to be found in reciprocal blastp
searches; in contrast, no comparable match is found
among numerous PHD and other ring domains in various
SET proteins in the NCBI database.
The combination of this conserved PHD in comparable
synteny with the SET domain, similar overall domain
architecture, and strong phylogenetic support for SET
domain monophyly between Plasmodium and plant
Ashr3 are unlikely to be coincidental. Rather, they rep-
resent credible e vidence of an orthologous relationship.
The A shr3 Set subfamily is not found in animals or
fungi [23] and, based on our sur vey of complete N C BI
protein databases, appe ars to be restricted to green
plants and apicomplexans. Although w e were unable to
identify Ashr3 genes in either green or red algae, they
are present in early land plants, and phylogenetic
analyses, both ours and previous [23], indicate that
Ashr3 is a relatively ancient SET protein family. In
addition, all apicomplexan A shr3/Set2 sequences are
recovered a s a monophyletic group (Additional file 1),
indicating a single transfer event before the radiation of
extant apicomplexans. Therefore, it is possible that
Ashr3 was present in the a lgal ancestor of the apicopla st
and moved into apicomplexans via endosymbiotic gene
transfer.
At present, whether the apicoplast is descended from
thesameendosymbiontthatgaverisetootherphoto-
synthetic alveolates remains under debate, as does the
taxonomic affiliation of that algal endosymbiont [25,26].
Given this uncertainty, and the relative paucity of genomic
data from dinoflagellates and red algae (a possible source
of alveolate plastids), HGT of Ashr3 from an algal endo-
symbiont is purely speculative at this juncture, and
remains one of several possibilities for the acquisition of
this prote in. Nevertheless, the known presence of an
algal endosymbiont in the ancestor of apicomplexans
provides a r ea sonable biological explanation for the
presence of a plant-specific chromatin remodeling pro-
tein in the lineage. Whatever the ve ctor, the horizontal
transfer appears to predate the origin of extant apicom-
plexans, as Cryptosporidum contains a four-PHD SE T
protein that groups with PF3D7_1322100 and all related
apicomplexan sequences in expande d p hylogenetic
analyses (Additional file 1).
We also completed similar analyses for JmjC1 homo-
logs (Figure 4). In this ca se, however, we recovered an
apicomplexan association with sequences from ciliates ,
although not with the sequence from P. marinus.
Although support for these relationships is rea sonably
strong, we should point out that it depends on how
JmjC1 genes are sampled. For the analyses shown in
Figure 4, we chose the closest blastp match to JmjC1
from P. falciparum or T. gondii from each taxon. In
contrast, when we sampled sequences using human
and yeast exemplars as queries , phylogenetic analyses
tended to group apicomplexan JmjC1 with sequences
from green algae. Nevertheless , we could find no com-
pelling evidence at present to indicate that the history
of JmjC1 in apicomplexans is complicated by HGT.
Discussion
The putative horizontal transfer of nematode derived
Set8 genes to both slime molds and an ancestor of
apicomplexans is both interesting and biologically
plausible. Both nematodes and arthropods (the next
closest ta xon in Set8 trees) we re extant at the time the
ancestor of apicomplexans acquired the Set8 ortholog .
Whereas arthropods are known definitively a s hosts for
many apicomplexans today, apicomplexan parasitism of
nematodes has not been documented (perhaps be cause
parasites of nematodes have been under-studied).
Although Trichenella is a modern parasite of mammals ,
the transfer of Set8 to an ancestral apicomplexan during
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co-infection of a mammalian host is highly unlikely,
given that mammals had not yet appeared at the
proposed time of the acquisition. It is thus more likely
that the ancestral apicomplexan parasitize d nematodes
and acquired Set8 in a gene transf er event. The timing
of this transfer e vent is more precisely defined through
comparison to P. marinus. The absence of Set8 in t he
complete genome of this sister taxon to apicomplexans
adds confidence to the proposal of a transfer event that
took place after thedivergenceofapicomplexans.
What is the function al consequence of this transfe r?
Given the adaptation s of apicomplexans toward intra-
cellular niches, the acquisition of a major epigenetic
regulator that governs and faithfu lly affirms chromatin-
silencing marks could have been a critical step by proto-
zoans for parasitism in diverse hosts with increasingly
complex immune systems. H4K20Me1, the epigenetic
mark deposite d on h istones by canonical Set8, is known
to participate in gene silencing , heterochromatin f orma-
tion and m itotic regulation as well as DNA damage
checkpoint repair [27-29]. We propose that the acqui-
sition of Set8 enabled higher order gene regulation,
including cellular d ifferentiation (for invading different
hosts) as well a s for an imal immune s ystem eva sion.
Acquisition of this type of regulation would also enable
the evolution of complex life cycles that involve
multiplehostsaswellasdormantcyststages.
The observation that extant malaria parasites are cap-
able of internalizing and incorporating exogenous host
DNA spontaneously in animal cells is consistent with a
potential mecha nism of transfer [6,30]. Of special note
is the observation that two other instances of horizontal
gene transfer from animals are theorized to have
occurred at a similar time in the evolution of these
Aspergillus (EDP51191)
Ustilago (XP_758729)
Candida (EEQ45431)
Oreochromis (XP_003445112)
Mus (BAC97906)
Drosophila (NP_523486)
Anopheles (XP_314337)
Amphimedon (XP_003385970)
Perkinsus (XP_002787231)
Micrococcus (XP_002500485)
Ostreococcus (XP_003080681)
Phytophthora infestens (XP_002909520)
Phytophthora sojae (EGZ26944)
Thalassiosira (XP_002287652)
Phaeodactylum (XP_002183428)
Ectocarpus (CBN78828)
Selaginella (XP_002989181)
Arabidopsis (NP_181034)
Tetrahymena (XP_001024156)
Ichthyophthirius (EGR33188)
Paramecium (XP_001433812)
Neospora (CBZ54889)
Toxoplasma (XP_002371874)
Theileria
Babesia (XP_001609219)
P. falciparum ( XP_001349427)
P. vivax (XP_001617218)
P. knowlesi (XP_002262191)
Cyanidioschyzon (CMT461C)
0.1
0.87
77
1.0/100
1.0/100
1.0
100
1.0
87
1.0
91
0.94
95
Ciliates
Apicomplexans
(XP_954636)
Animals
Fungi
Stramenopiles
Green algae
Land plants
0.79
59
1.0/100
1.0/100
100
1.0
Rhodophyte
Alveolate
Figure 4 Phylogenetic tree of JmjC1. JmjC1 domains retrieved using P. falciparum and T. gondii orthologs as queries, and analyzed
phylogenetically using Bayesian inference and maximum-likelihood. Support values for the respective analyses are shown above or to the right of
key nodes important to the relative positions of apicomplexan, ciliates and potential sources of HGT. Although alternative sampling strategies,
and inclusion of resulting exemplars from various green algae, separated ciliate and apicomplexan sequences in other phylogenetic analyses, in
no case was phylogenetic support for a scenario of HGT as strong as support for the ciliate-apicomplexan sister relationship on this tree.
Sequences from additional organisms could alter this tentative conclusion, however, given that JmjC1 from Perkinsus, an organism believed to be
much closer than ciliates to apicomplexans, branches separately from other alveolates.
Kishore et al. BMC Evolutionary Biology 2013, 13:37 Page 8 of 12
http://www.biomedcentral.com/1471-2148/13/37
parasites (that is, into ancestors of modern apicomplex-
ans). Domains involved in cytoadhesion and invasion, as
well as genes required for O-linked glycosylation (GDP-
fucose protein O-fucosyltransferase 2), are both proposed
products of HGT dating from this time [21]. Notably,
these are also missing from P. marinus, the nearest avail-
able relative of apicomplexans, consistent with a proposed
HGT coincident to the Set8 transfer. The animal sources
of the cytoadhesion domain and O-linked glycosylation
HGTs, however, remain unidentified [7,31]. Although
sampling of appropriate genomes remains relatively
sparse, these results, taken together, suggest that during
the transition to parasitism, in addition to acquiring the
adhesion and glycosylation capabilities that underlie many
of the intercellular interactions required for invasion and
survival within their hosts, apicomplexan parasites also
acquired additional transcriptional regulatory capabilities.
These include both the ApiAP2 class of transcriptional
regulators and the proteins needed for the epigenetic
control utilized in cellular differentiation pathways and
immune evasion. Moreover, these acquisitions appear to
have occurred through HGT events at a similar time in
evolutionary history, marking a key point in the transition
of apicomplexans from a free-living to parasitic lifestyle
(Figure 5).
Similarly, the ancestor of m odern soil-ba sed nema-
todes (e.g. C. ele gans ) could have transferred Set8 to
the immediate ancestor of social amoebae and slime
molds. Set8 orthologs are found in additional colonial
slime m olds (e.g. Polysphondylium pallidum) but are
missing from other amoebae. Thus , the most parsimo-
nious solut ion is an ani mal-derived HGT spe cific t o
slime molds. C aenorhabditis elegans is known to feed
on Dictyostelium, ingesting and dispersing Dictyostelium
spores [32]. This suggests that the incorporation of genes
encoding nematode epigenetic machinery into the slime
mold genome also is biologically feasible. Moreover, slime
molds are known to adapt to predation pressure exerted
by nematodes. This predation could provide a source of
strong positive selection on enhanced control of gene
expression in slime molds [32]. As evidence of the utility
of DNA transfer, there are 16 known cases of horizontal
gene transfer from bacterial sources into Dictyostelium
[33,34]. Slime molds are well known to scavenge bacteria
from the soil and recent reports indicate that bacteria
are consumed by slime molds and incorporated into
slime mold fruiting bodies and spores during asexual
reproduction [35]. Thus it appears that over the course of
evolution, slime molds have acquired DNA from both
their predators and prey.
500
250
50
0
500-750
390-600
340-520
280-510
Set8 (nematode)
Cytoadhesion domains
O-linked glycosylation
Algal
endosymbiont source?
Ciliates
Dinoflagellates
Perkinsids
Colpodellids
Chromera velia
Apicomplexa
Alveolates
ApiAP2 transcription factors
Ashr3 (Set2)
Animal host
Plant genes
Figure 5 The timing and source of horizontal transfer of genes that facilitate parasitism. The evolution of alveolates is shown, with
approximate times (million years ago) of major branching events denoted in gray type. Ancestral apicomplexa are thought to have appeared
during the transition from the Neoproterozoic to the Paleozoic era, a time when nematodes and arthropods were extant, providing a possible
source for transfer of Set8, cytoadhesion protein/invasion proteins and enzymes involved in O-linked glycosylation (red arrow). The proposed
acquisition of the histone modifier Ashr3 and ApiAP2 transcription factors also appear to have occurred at or near this point in evolutionary
history. The acquisition of an algal endosymbiont that is thought to be a source of ApiAP2 proteins and possibly Ashr3 is more controversial
(dashed green arrows), but could also have coincided with the divergence of apicomplexan parasites. The apparent convergence of these distinct
events within a narrow evolutionary window suggests that this represented a key moment in the development of apicomplexan parasitism
(denoted by asterisk). Adapted from: Okamato, N. 2008. The mother of all parasites. Future Microbiology 3:391–395.
Kishore et al. BMC Evolutionary Biology 2013, 13:37 Page 9 of 12
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Notably, while the phylogenetic analyses described
here suggest horizontal acquisition of both Set8 and a
Set2 functional analog at or near the origin of the
apicomplexan clade, there is significant flexibility in the
requirements and functions of these p roteins in various
extant organisms. This mirrors and extends our earlier
investigations on transcriptional machinery in Plasmodium.
We r ecently identified an expanded motif w ithin the
tail of the RNA polymerase II C TD that is only other-
wise found in animal (specifically, mammalian) host
polymerases [2]. Intriguingly, this ex pansion is p resent
only in primate malaria para sites and is absent in para-
sites of rodents and birds. In model eukaryotes, the
CTD is connected to epigenetic memory through
direct recruitment o f S ET-bearing methyltransfera ses
that mark chromatin during active transcription [18].
Further demonstrating the flexibility in this a spect of
transcriptional regulation, ‘Set2’ (Ashr3) is present in
Cryptospor idium, wherea s JmjC1 is absent , r aising the
intriguing possib ility that they did not act tog ether
initially.
In Plasmodium specifically, the presence of additional
RNA polymerase II CTD heptapeptides in primate para-
sites , a presumed requireme nt for recruitment of ‘Set2 ’,
suggests that a cooperative interaction between ‘Set2 ’
andJmjC1couldhaveco-evolvedwithexpansionofthe
CTD in these organisms. Consistent with this hypoth-
esis, Pla smodium species that parasitize rodents do not
have an expanded C TD and genes encoding Set2 and
JmjC1 a re missing from their genomes. Moreover, the
deletion of both S et2 an d JmjC1 in rodent plasmodia
suggests tight and specific association between these
chromatin modifiers in Plasmodium; this association is
consistent with a recent integration into the genome,
rather than with ancient, conserved functions that
would have been harder to lose. The absence of e pigen-
etic memory at rodent para site surface-exposed variant
antigens [36] ( best studied in the yir family in P. yoelii)
suggests a functional conse quence of this deletion.
Our analysis also represents the first report of HGT of
proteins involved in epigenetic c ontrol from animal
provenance into slime molds. It is worth noting that
among the amoebae, multicellular development a nd
complex cellular communication and differentiation are
found only in slime molds , the group that also acquired
Set8 from animals [35,37]. We do not argue causa lity
but simply raise the point that acquisitions of epigenetic
machinery accompanied t he evolution of higher order
cellular processes in both apicomplexan parasites and
social amoebozoans. Therefore, our work suggest s that
synergies between transcription machinery and epi-
genetic modifiers are at the nexus of the evolut ion of
complex intracellular interactions, be they multicellular
development or host/parasite interactions.
Conclusions
We hope this work will shed light on the curious fate of
lateral transfers from animal hosts into apicomplexans
and other organisms, particularly SET-bearing chroma-
tin modifiers and those molecules involved in epigenetic
memory and immune evasion. Furth er investigations
could reveal other novel, underappreciated acquisitions
by protists at the dawn of apicomplexan parasitism that
were functionally exp loited as the immunological arms
race between animal host s and their parasites intensified.
Targeting these factor s that accompanied the transition
to apicomplexan parasitism may prove crucial for
advances in efforts to disa ble parasitism.
Methods
Identification, curation and phylogenetic analysis of Set8,
Set2 and JmjC1 orthologs
The queries from canonical orthologs of Set8 from Homo
sapiens were used in BlastP searches of the NCBI non-
redundant protein databases for sequences from the Api-
complexa, dinoflagellates, ciliates, gregarines, diplomonads,
rhodophytes, plants, Fungi, Microsporidia/Encephalitzoa,
kinetoplastids, Entamoeba, heterokonts/stramenopiles,
Amoebozoa (Dictyostelium), and various metazoan taxa
including sponges, nematodes, trematodes, arthropods,
trochozoans, echinoderms, birds, reptiles and primates.
NCBI accession numbers for both query and retrieved
sequences used for this and other phylogenetic analyses
are included on the respective tree figures in this paper.
Comparative evolutionary analyses were carried out
on all sequences using the following approach.
Sequences were aligned using the program MUSCLE
[38] and trimmed to include only the core SET domain
that could be aligned clearly across all ta xa included
in the analyses. Phylogenetic relati onships among
sequences were resolved by both Bayesian inference
(MrBayes) [39] and ma ximum-likelihood (PhyML)
[40]. Parameters for phylogenetic analyses were deter-
mined using ‘Model Selection (ML) in MEGA 5.05
[41], and found to be the WAG substitution matrix
and a gamma + I model for rate variation across sites
(estimated from the d ata in both ML and Bayesian
analyses) for all alignments used in this study. Both
ML and Bayesian inference resulted in the same ba sic
tree topologies in all analyses , with only mi nor rearran-
gements in sub-clades unrelated to the well-supported
position of apicomplexans within the trees. The ML
topology is shown in a ll tree figur es. Relative support
for tree nodes was inferred from Bayesian posterior
probabilities estimated from all trees sampled once the
average standard deviation of split frequencies had
converged on a stable value, as well as through 100
nonparametric ML boot strap replicates.
Kishore et al. BMC Evolutionary Biology 2013, 13:37 Page 10 of 12
http://www.biomedcentral.com/1471-2148/13/37
Comparable analyses were carried to test the phylogen-
etic affinity of Plasmodium Set2 and JmjC1 sequences. In
these cases, we sampled NCBI protein databases with
exemplars from both human and budding yeast, as well as
sequences from P. falciparum and T. gondii.Ifdifferent
proteins were recovered using the alternative search strat-
egies, both were included in initial respective phylogenetic
analyses. For global phylogenetic analyses of SET domains,
exemplars from model organisms (animals, yeasts and
higher plants) were chosen to define established SET pro-
tein subfamilies. The ciliate and Perkinsus sequences
included were the closest match to both Set2 exemplars
from model eukaryotes and to putative ‘Set2’ homologs
from the two apicomplexans. Because results from both
search strategies were comparable, sequences were
assembled into a single alignment for phylogenetic recon-
struction. Since different closest matches tended to
emerge in blastp searches using apicomplexan versus
human and yeast JmjC1 queries, separate phylogenetic
analyses were carried out on the two data sets. With
human and yeast queries, apicomplexan JmjC1 tended to
group with various green algal sequences rather than with
Perkinsus or ciliates, their nearest alveolate relatives with
complete and well-annotated genomes. In contrast, query-
ing with apicomplexan sequences resulted in trees that
grouped the apicomplexans and ciliates together. The
latter were considered to be the most conservative esti-
mates of JmjC1 sequence relationships and, therefore,
were included in as our primary phylogenetic analyses
(see results).
Additional file
Additional file 1: Figure S1. Expanded phylogenetic analyses of Set
domain containing proteins showing that all putative Set2 (Ashr3)
homologs from apicomplexans are recovered as a monophyletic group.
Competing interests
The authors declare no competing financial, political, personal, religious,
ideological, academic, intellectual, commercial or any other interests related
to the work described in this publication.
Authors’ contributions
SPK, JWS and KWD designed the study. SPK identified and assembled the
datasets for Set2, Set8 and JmjC1 sequences. JWS conducted the maximum-
likelihood and Bayesian inference analyses and generated the phylogenetic
trees. SPK, JWS and KWD wrote the paper. All authors read and approved
the final manu script.
Acknowledgements
This work was supported by grant AI 52390 from the National Institutes of
Health to KWD and the Medical Scientist Training Program grant GM07739
and the Paul and Daisy Soros Fellowship for SPK.
Author details
1
Department of Microbiology and Immunology, Weill Cornell Medical
College, New York, NY 10065, USA.
2
Department of Biology, East Carolina
University, Greenville, NC 27858, USA.
Received: 5 September 2012 Accepted: 5 February 2013
Published: 11 February 2013
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doi:10.1186/1471-2148-13-37
Cite this article as: Kishore et al.: Horizontal gene transfer of epigenetic
machinery and evolution of parasitism in the malaria parasite
Plasmodium falciparum and other apicomplexans. BMC Evolutionary
Biology 2013 13:37.
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