Download Protein translation in Plasmodium parasites

TREPAR-1056; No. of Pages 10
Review
Protein translation in Plasmodium
parasites
Katherine E. Jackson1*, Saman Habib2*, Magali Frugier3, Rob Hoen4, Sameena Khan5,
James S. Pham1, Lluı́s Ribas de Pouplana6,7, Miriam Royo4, Manuel A.S. Santos8,
Amit Sharma5 and Stuart A. Ralph1
1
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of
Melbourne, Victoria, 3010 Australia
2
Division of Molecular and Structural Biology, Central Drug Research Institute–Council of Scientific and Industrial Research, PO
Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India
3
Architecture et Réactivité de l’ARN, Université de Strasbourg, Centre National de la Recherche Scientifique, Institut de Biologie
Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg CEDEX, France
4
Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, carrer Baldiri Reixac 10, 08028 Barcelona, Spain
5
Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali
Road, New Delhi 110 067, India
6
Institució Catalana de Recerca i Estudis Avançats, Passeig Lluı́s Companys 1, Barcelona 08010, Catalonia, Spain
7
Institute for Research in Biomedicine, Carrer Baldiri Reixac 10, Barcelona 08028, Catalonia, Spain
8
RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
The protein translation machinery of the parasite Plasmodium is the target of important anti-malarial drugs,
and encompasses many promising targets for future
drugs. Plasmodium parasites have three subcellular
compartments that house genomes; the nucleus, mitochondrion and apicoplast, and each requires its own
compartmentalized transcription and translation apparatus for survival. Despite the availability of the complete genome sequence that should reveal the requisite
elements for all three compartments, our understanding of the translation machineries is patchy. We review
what is known about cytosolic and organellar translation in Plasmodium and discuss the molecules that
have been identified through genome sequencing
and post-genomic analysis. Some translation components are yet to be found in Plasmodium, whereas
others appear to be shared between translationally
active organelles.
Plasmodium translation in the post-genomic era
Since the sequencing of the P. falciparum genome, and the
complete or near-complete genome sequencing of a handful
of other Plasmodium species, there have been tremendous
advances in the characterization of the molecules governing
host–pathogen interactions. Less striking has been the
pursuit in Plasmodium species of one of the basic processes
of life – the translation of RNA into protein. Perhaps the very
centrality of this process to biology has made it a less
attractive research topic in a parasite whose most captivating behaviors involve interplay with its host. Nevertheless,
protein translation is an important field of Plasmodium
research. Although translation is more easily studied in
model eukaryotes and prokaryotes, departures from these
*
Corresponding author: Ralph, S.A. ([email protected])
These authors contributed equally to this work.
models observed in Plasmodium and other apicomplexan
parasites reveal important aspects of diversity among translation systems. Most phylogenetic neighbors of Plasmodium
have poorly characterized protein translation and, where
mechanisms have been studied (in ciliates for example),
they appear to differ in important aspects from what is
Glossary
Apicoplast: non-photosynthetic plastid with its own circular genome, found in
most Apicomplexa. Arose via secondary endosymbiosis.
Cytosol: the intracellular fluid found within cells, excluding that within
membrane-bound organelles.
Isoacceptors: tRNAs that have different anticodons but are charged with the
same amino acid.
Mitochondrion: endosymbiotic organelle with its own small mitochondrial
chromosome. Generally plays a major role in energy conversion.
Peptide exit tunnel: the site, formed by proteins and rRNA, where the nascent
polypeptide chain leaves the ribosome.
P-granules: translationally silent storage particles containing high levels of
non-translated mRNA. Related to P-bodies, but differ in their inability to
degrade mRNA.
Plastid: endosymbiotic organelle found in plants, algae and some protists.
Houses photosynthetic machinery in some eukaryotes as well as several
biosynthetic pathways.
RNA polymerase III: the enzyme responsible for transcribing many non-coding
RNA molecules. These include the tRNA and 5S rRNA molecules essential for
translation, and also other structural, catalytic and regulatory RNA molecules.
Translation elongation factors: proteins that facilitate the steps involved in the
protein translation elongation. EF-Tu allows the correct aminoacyl-tRNA to
enter the ribosome, EF-Ts is the nucleotide exchange factor for EF-Tu, and EF-G
is the translocase that allows the mRNA and tRNA to move one codon along
the ribosome after each round of elongation.
Translation initiation factors: a large group of proteins responsible for effective
initiation of protein synthesis. In eukaryotes, eIF-4A (eukaryotic initiation
factor) is a helicase that prepares the mRNA for ribosome binding eIF-4B is
responsible for the recognition of the mRNA cap, eIF-4E and eIF-4G direct
ribosomes to the mRNA cap, eIF-5A stimulates formation of the first peptide
bond in translation, and eiF1 prevents premature association of the large and
small ribosomal subunits to increase specificity in initiation codon recognition.
tRNA synthetase: one of a family of enzymes that catalyze the ligation of an
amino acid to its cognate tRNA to form an aminoacyl-tRNA (also referred to as
charged tRNA) ready for use in protein translation. In most cases a distinct
tRNA synthetase is required for each amino acid.
1471-4922/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2011.05.005 Trends in Parasitology xx (2011) 1–10
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known for Plasmodium. A more pressing reason to study
Plasmodium translation is its role as the target for several of
the very few successful antimalarial drugs and a suite of
promising inhibitors. This precedent for successful drugs
urges more research in this field to improve our understanding of the mechanisms of action and resistance to existing
drugs and to explore rationally the potential for new translation-blocking drugs.
Three compartments, three translation machines?
Plasmodium, as do plants, algae, and the majority of other
Apicomplexans, possesses three active compartments of
translation: the cytosol, mitochondrion and a relic plastid
termed the apicoplast (see Glossary) or apicomplexan plastid. Apicomplexans contain, therefore, a mixture of translation machinery, with eukaryotic components for cytosolic
mRNA translation and prokaryotic-like components for
mRNA translation in the mitochondria and apicoplast.
The apicoplast is non-photosynthetic and appears to supply the parasite with precursors of heme, isoprenes, fatty
acids, and iron–sulfur clusters [1] whereas the mitochondria is involved in energy conversion and pyrimidine
synthesis [2].
The cytosolic protein translation machinery is
responsible for translation of an estimated 5385 genes
encoded by the nuclear genome, whereas the apicoplast
machinery translates less than 50 genes encoded by the
35 kb circular apicoplast genome [3]. The plastid encodes
genes relevant for its own translation, including rRNAs,
tRNAs, ribosomal proteins, and the translation elongation factor Tu (EF-Tu), and also a handful of other
poorly characterized proteins unrelated to translation
(Figure 1). Most proteins resident in the apicoplast are
encoded by the nuclear genome, translated in the cytoplasm, and transported into the apicoplast using specific
targeting signals [4]. Detection of plastid-encoded EF-Tu
by western blot and immunofluorescence has provided
evidence for translation in P. falciparum plastids [5].
Indirect evidence of apicoplast translation also comes
from the identification of ribosome-like particles carrying
plastid-specific rRNA and mRNA [6]. The efficacy of
inhibitors targeting apicoplast translation components
also confirms the importance of this machinery (reviewed
in [7,8]).
Plasmodium spp. have the smallest mitochondrial genome known to date (6 kb), and this encodes only three
proteins, namely cytochrome c oxidase subunits I and III
(Cox1 and Cox3) and cytochrome b (Cytb) [2,9]. The mitochondrial genome does not encode tRNA genes but encodes
small fragmented rRNAs [10]. Despite the apparent
incompleteness of the mitochondrial translational machinery and lack of direct evidence for translation in mitochondria, indirect evidence suggests that mitochondrial
translation is active and is essential. A major antimalarial
drug (atovaquone) targets the mitochondrial cytochrome
bc1 complex, and point mutations in the mitochondrial cytb
gene correlate with resistance to this drug [11]. Cytochrome c oxidase activity, the requirement of the complex
for mitochondrial electron transport and its inhibition by
cyanide also suggest that the mitochondrial cox genes are
translated in the mitochondrion [12,13].
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Amino acids
P. falciparum parasites have multiple means of obtaining
amino acids for protein synthesis, but the most important
for growth in vivo remain unclear. Of the 20 canonical
amino acids, Plasmodium possesses biosynthetic pathways for Asn, Gln, Gly, Pro, Asp and Glu [14]. However,
very low amounts of these amino acids are incorporated
into Plasmodium proteins [15]. This limited capacity for de
novo amino acid synthesis means that, during in vitro
culture at least, to charge its own tRNAs Plasmodium
relies largely on amino acids released from the digestion
of host hemoglobin in the parasite food vacuole and on
amino acid uptake from the extracellular space [16].
Human hemoglobin is rich in most amino acids, but is
low in Cys, Glu, Gln and Met, and lacks Ile; these could be
rate-limiting for protein synthesis. Supplementation of
Plasmodium with these five essential amino acids is sufficient for short-term parasite development [17], and recent
evidence has shown that Ile alone is sufficient to support
parasite growth [16]. However, for some Plasmodium
strains Met supplementation is also required for optimal
growth [16]. The dependence on such exogenous amino
acids means that parasite protein synthesis relies on amino acid transport across the erythrocyte plasma membrane, food-vacuole membrane, and its own plasma
membrane [18,19].
In addition to the canonical 20 amino acids, P. falciparum incorporates two others. The first is the redoxregulating selenocysteine, which is incorporated into very
few proteins (only four have been identified so far) [20,21].
The second is formyl-methionine (fMet), that is probably
used as the initiator amino acid in the apicoplast. The
transferase responsible for adding the formyl group to the
charged fMet tRNA is apicoplast-targeted in the related
parasite Toxoplasma [22]. The peptide deformylase required to process such amino acids is also trafficked to
the apicoplast in Plasmodium [23,24], and inhibition of this
enzyme results in parasite death [25]. No corresponding
enzymes appear to be targeted to the mitochondrion [22].
Components of the translation machinery
tRNA
Of the three Plasmodium genomes, nuclear, apicoplast and
mitochondrial, only the first two encode tRNAs [3,26,27]
and correspond to compartments in which translation has
been demonstrated [5]. A total of 46 tRNA genes, coding for
45 tRNA isoacceptors (initiator and elongator tRNAMet are
encoded by two different genes), are found in the nuclear
genome whereas the apicoplast genome contains 35 genes
encoding 26 tRNA isoacceptors. With the exception of the
apicoplast initiator tRNAMet, characterized by a unique
variable region (11 nucleotides), they all resemble other
eukaryotic tRNAs [28]. Indeed, they have the potential to
adopt a canonical tertiary fold because they contain all
conserved and semi-conserved nucleotides necessary to
form the well-known L-shaped structure [28,29]. Some
but not all Plasmodium species have very low G/C contents
in their genomes (less than 20% G/C for P. falciparum [3]).
Cytosolic tRNAs have a G/C content of 56% whereas apicoplast-encoded tRNAs have 26% G/C [28]. Higher G/C
content in tRNA and rRNA species has been associated
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Amino acids sourced by:
• Hemoglobin breakdown
• De novo synthesis
• Import from circulation
35 kb apicoplast genome
encodes:
• All rRNAs (save 5s rRNA)
• 35 tRNAs (a full set)
• 17-18 ribosomal proteins (number
varies between apicomplexans)
• EF-Tu
• No aaRSs
23 Mb nuclear genome
encodes:
• Asexual and sexual
stage rRNA molecules
• 46 tRNAs (a full set)
• Ribosomal proteins
• 36 aaRSs
Apicoplast targeted
molecules:
• Some ribosomal proteins
• 8 initiation, elongation
and release factors?
• 20 aaRSs?
Mitochondrially targeted
molecules:
• Some rRNAS?
• All tRNAs (aminoacylated)
• All ribosomal proteins?
• 5 initiation, elongation
and release factors?
3 kb Mitochondrial genome
encodes:
• Fragmented rRNA molecules
• No tRNA
• No ribosomal proteins
• No translation initiation,
elongation or release factors
• No aaRSs
TRENDS in Parasitology
Figure 1. Translation in Plasmodium: a tale of many compartments. Translation takes place in the Plasmodium apicoplast, mitochondrion and cytosol (including at the
rough ER). The apicoplast genome, like that of most plastids, encodes its own tRNA and rRNA molecules, and also many ribosomal proteins, but imports many other
nuclear-encoded proteins needed for its translation. The mitochondrion appears to import all proteins and tRNAs needed for translation of mitochondrial-encoded
transcripts, and it is unclear if its fragmented rRNA is functional. Some of the amino acids necessary for translation in these three compartments can be synthesized by the
parasite, but others are imported from the extracellular milieu, or can be sourced from hemoglobin degraded in the food vacuole.
with higher optimum growth temperatures or increased
structural stability, although it is hard to see why these
should vary between the apicoplast and cytosol. The consequences for translational efficiency of the unusual G/C
content of the genomes and tRNAs of P. falciparum are
completely unknown. Finally, only two introns were identified in the anticodon domain of these tRNAs [30]; one
intron is located in the nuclear tRNATyrGTA and another in
the apicoplast tRNALeuTAA.
The most striking observation is that there is only one
gene copy per tRNA isoacceptor in the nuclear genome, and
therefore Plasmodium has the fewest known tRNA genes of
any eukaryote. Whether this limits translation efficiency in
the parasite or whether its RNA polymerase III machinery
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is regulated by as yet unknown factors is unclear. If the
different tRNAs are equally abundant, the most-used
codons in the genome will have relatively fewer tRNA
molecules available per codon. Codons for Asn, in particular,
are highly used in low-complexity regions of Plasmodium
proteins, and it has been proposed that a relative limitation
of tRNAAsn might slow down local translation of these
regions, with implications for translation accuracy and
efficiency and for proper cotranslational folding of the
relevant proteins [31]. Nuclear, plastid and mitochondrial
genomes show differences in codon usage, but every amino
acid is used at least once in each of the three compartments,
and therefore a full complement of tRNAs and aminoacyltRNA synthetases should be necessary for translation of
each genome.
tRNA synthetases
Aminoacyl-tRNA synthetases (aaRSs) are the enzymes
responsible for the esterification of a specific amino acid,
or its precursor, to its compatible cognate tRNA to form
aminoacyl-tRNAs. These enzymes belong to two structurally distinct classes, apparently evolving from two distinct
ancestors via convergent evolution [32]. The increase in
complexity of the aaRSs matches that of their tRNA substrates, whose structure can also be divided into two
distinct structural domains, namely the acceptor-arm domain and the anticodon-arm domain [33].
Plasmodium has genes for only 37 aaRSs, and these are
apparently sufficient to translate the nuclear, apicoplast,
and mitochondrial genomes [34]. The reduction from
a theoretical maximum of more than 60 to 37 aaRS genes
implies that several Plasmodium aaRSs aminoacylate
tRNAs that are active in more than one subcellular
compartment. Studies in Toxoplasma gondii suggest that
in this species all mitochondrial tRNAs are in fact aminoacylated externally and transported into the mitochondria
for use in protein synthesis [22,35]. Such a mechanism
presumably suffers from the deficiency that imported
tRNAs would be unable to be recharged readily, and it
is currently unknown if these tRNAs are in some way
recycled or are degraded after a single use. To our knowledge this is the first example of a translation machinery
that depends fully on transport of charged tRNAs from
another cellular compartment. The mitochondria of unrelated trypanosomatid parasites also import all tRNAs [36],
but in that case are charged by mitochondrial-targeted
tRNA aaRS enzymes.
In P. falciparum the subcellular distribution of most
aaRSs remains unclear, although computational studies
suggest that most of these proteins are either targeted to
the apicoplast or the cytosol, but not to the mitochondria
[34]. The two isoleucyl-tRNA synthetases have been shown
to be targeted to the apicoplast and cytosol, respectively [37].
Several aaRS enzymes are present only once in the genome,
despite their apparent requirement in the apicoplast and
the cytosol. In those cases we predict that these will be
dually targeted to both compartments (Table 1). In each case
the gene models start with a predicted apicoplast targeting
sequence [4], but whether all isoforms contain this is unknown. Some aaRSs in Toxoplasma are dual-targeted to the
Table 1. Analysis of the full complement of Plasmodium aaRSs and related enzymesa
Enzyme
Alanine tRNA synthetase
Arginine tRNA synthetase
Asparagine tRNA synthetase
Asparagine tRNA synthetase
Cysteine tRNA synthetase
Glutamate tRNA synthetase
Glutamine tRNA synthetase
Glutamate tRNA amidotransferase subunit A
Glutamate-tRNA amidotransferase subunit B
Glycine tRNA synthetase
Histidine tRNA synthetase
Isoleucine tRNA synthetase
Leucine tRNA synthetase
Lysine tRNA synthetase
Methionine tRNA synthetase
Methionine tRNA formyltransferase
Phenylalanine tRNA synthetase subunits
Proline tRNA synthetase
Serine tRNA synthetase
Threonine tRNA synthetase
Tryptophan tRNA synthetase
Tyrosine tRNA synthetase
Valine tRNA synthetase
a
Apicoplast
Cytosol
PFI0680c b
PFE0475w
PFE0715w
PFL0900c b
PFB0525w
PFA0145c
MAL13P1.281
not used
PFD0780w
PFF1395c
PF13_0257
PF13_0170
not used
not used
PFI1645c
PFL1210w
PF08_0011
PF14_0166
PF10_0053
MAL13P1.67
PFL1540c
PFI1240c
PFL0770w
PF14_0428
PF13_0179
PFF1095w
PF13_0262
PF10_0340
not used
PFF0180w
PF11_0051
PFA0480w
PFL0670c
PF07_0073
PFL2485c
PF11_0181
PFC0470w
PF13_0205
MAL8P1.125
PF14_0589
Single copy; dual targeted?
PF13_0354
PF10_0149
PF14_0198
PF11_0270
The number of enzymes predicted in Plasmodium, and their supposed cellular localization, are shown. Subcellular localization is predicted on the basis of possession of
trafficking sequences for the apicoplast or mitochondrion, or from experimental localization in the case of the isoleucyl tRNA synthetases [37]. We postulate that the aaRS
enzymes that are found only once in the genome are dual-targeted to the cytosol and the apicoplast by mechanisms yet to be characterized.
b
Systematic gene designation.
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cytosol and apicoplast, but the underlying mechanisms are
unclear [22]. Dual-targeted enzymes of apicoplast origin are
particularly attractive drug targets because these enzymes
are often similar to bacterial versions and significantly
different from the human enzymes. Molecules that block
such enzymes should effectively arrest translation of the
main genomes of the parasite without significantly affecting
orthologous human aaRSs.
L–D amino acid discrimination
In many cases, aaRSs do not exclude D-amino acids when
charging tRNA. The stereospecificity of these enzymes is
therefore not absolute, and this charging error can be toxic
to cells [38]. Early studies on bacterial tyrosyl-tRNA
synthetases, and later on S. cerevisiae tyrosyl-, tryptophanyl- and aspartyl-tRNA synthetases, demonstrated transfer of the D-isomer of their cognate amino acid to the
corresponding tRNA species. This activity can be compensated for by D-tyrosyl-tRNATyr deacylase (DTD), which
hydrolyzes the misacylated tRNA back into D-tyrosine
and free tRNA [38,39]. DTD can also recognize other
tRNAs charged with corresponding D-amino acids [39]
and could function as a general proofreading mechanism
against D-amino acid incorporation. Disruptions of dtd in
bacteria and yeast are viable, but lead to increased toxicity
of D-amino acids [38]. Similarly, inhibitors of Plasmodium
DTD enhance the toxicity of D-amino acids, suggesting
that D-amino acids from the host cell would indeed present
a potential challenge for the parasites [40]. Recently, the
structure of the P. falciparum DTD enzyme (PfDTD) was
solved, shedding light on the mechanism for its deacylation
reaction [40,41]. The availability of a PfDTD structure and
the inhibitory nature of drugs modeled on PfDTD suggest
that hydrolysis of misacylated tRNA should be explored
further as a novel and unique drug target.
Ribosomes
The ribosome is a large complex of macromolecules that
manufactures proteins in all eukaryotic and prokaryotic
cells. Substantial information on this molecular giant has
been generated to atomic resolution, and its intricate mechanisms have been unraveled using X-ray crystallography
and electron microscopy in model organisms [42]. Very little
has been published on the protein members of the Plasmodium ribosomes, but the rRNA has received considerable
attention, and has several features not found in other
eukaryotes. As with other plastid-bearing eukaryotes, Plasmodium possesses three sets of rRNAs: cytoplasmic rRNAs,
encoded in the nucleus and responsible for the majority of
protein synthesis, as well as mitochondrial and plastid
rRNAs that are encoded by the corresponding genomes
and are responsible for protein synthesis in these organelles.
Apicomplexan parasites contain rRNA gene units that
are comprised of 18S, 28S, and 5.8S genes separated by the
internal transcribed spacer regions and flanked by external transcribed spacer regions [43–48]. Many apicomplexan parasites contain a small number of structurally
distinct rRNA gene units that are dispersed in their
genomes. These rRNA gene units are transcribed in a
stage-specific manner, thereby reducing the total number
of simultaneously active gene units [43–48]. Based on
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differences in expression patterns and differences in the
rRNA sequences, the existence of three structurally different ribosome types has been proposed. These are referred
to as A-type (asexual liver and blood stages), O-type (ookinete) and S-type (mosquito sporozoite stages). Controlling
expression of different sets of rRNA genes could help the
parasite to regulate ribosomes as a function of the host
environment in which the parasite finds itself [49]. In
addition to the nuclear genome, both the apicoplast and
mitochondrial genomes contain rDNA sequences [9,50,51].
The latter appear to be polycistronically transcribed and,
as with the cytosolic forms, appear to be developmentally
regulated [52].
Initiation, elongation and release factors
All translation factors required for initiation, elongation
and release of the polypeptide chain in the cytosol, apicoplast and mitochondrion are encoded by the parasite nucleus, with the exception of the apicoplast EF-Tu that is
encoded by the apicoplast genome. Most of the translation
factors required for cytosolic translation are currently
annotated in the P. falciparum genome (Table 2), however,
the majority of their functional interactions await confirmation. Among these cytosolic factors, mRNA cap-binding
by eIF-4E has been reported in P. falciparum, and also
interactions of eIF-4G with eIF-4E, eIF-4A (PfH45), and
poly(A)-binding protein, PABP [53–55].
Identification of native protein interactions in the parasite have indicated contacts between the phosphoprotein
eEF-1b and the G protein eEF-1a (the functional homologs
of prokaryotic EF-Ts and EF-Tu, respectively) with two
additional proteins, eEF-1d and eEF-1g, also associated
with eEF-1b [56,57]. We have been unable to find an eIF4B coding sequence in the P. falciparum genome; however,
the sequence of this molecule is poorly conserved between
distant eukaryotes. The translation initiation factor, eIF5A from P. falciparum, contains the unique amino acid
hypusine and has been shown to be post-translationally
modified by parasite deoxyhypusine synthase and hydroxylase [58–60].
The presence of an active translation machinery is established in the P. falciparum apicoplast [5], and a case for
mitochondrial translation in apicomplexans has been made
[12,22]. Putative prokaryotic-like initiation factors for both
of these organelles are apparent in the genome, in some
cases with clear targeting presequences (Table 2). Recent
data show that peptide chain elongation in the apicoplast is
facilitated by plastid-encoded EF-Tu and nuclear-encoded
EF-Ts, presumably in conjunction with an apicoplast-targeted EF-G [61]. The nuclear-encoded EF-Tu and an EF-G
are predicted to contain mitochondrial transit peptides,
but mitochondrial EF-Ts is lacking in all Plasmodium species. In prokaryotes, two release factors RF-1 and RF-2
decode the stop codons UAA/UAG and UAA/UGA, respectively [62]. Apicoplast open reading frames (ORFs) terminate with either UAA or UGA. The apicoplast RF-2
candidate (PFD0480w) has a strong targeting prediction
but lacks a conserved stop-codon-specificity recognition
domain. Thus, the plastid RF-1 candidate (PF14_0265),
with a PXN (Pro-x-Asn) motif replacing the conserved
bacterial PXT (Pro-x-Thr) codon recognition motif, could
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Table 2. P. falciparum apicoplast and mitochondrion translation factorsa
Translation process
Initiation
Elongation
Termination
Ribosome recycling
Factor
IF-1
IF-2
Apicoplast
PF14_0658 b
PFE0830c
IF-3
EF-Tu
EF-Ts
EF-G
RF-1
RF-2
RF-3
RRF
MAL8P1.27
Apicoplast-encoded (X95276)
PFC0225c
PFF0115c
PF14_0265
PFD0480w
Not found
PFB0390w
Mitochondrion
Not found d
PF08_0018
PF13_0069
(only cytb has an AUG start
codon and can require
tRNAfMet for initiation)
Not found
MAL13P1.164
Not found
PFL1590c
MAL7P1.20
Not found
Not found
a
Organellar assignment is in nearly all cases predicted from possession of appropriate trafficking signals. The apicoplast EF-Tu is encoded on the apicoplast genome, and
the localization of the nuclear-encoded apicoplast EF-Ts has been verified [61].
b
Systematic gene designation.cSome mitochondria lack an IF-1
function with an altered decoding specificity for UAA/UGA.
Each of the three mitochondrial ORFs terminates at a
UAA stop codon, and a single RF (MAL7P1.20) could
therefore suffice for peptide chain release in this organelle.
A prokaryotic RF-3, which would normally help RF1 and
RF2 to dissociate from the ribosome, is lacking in many
bacteria [63], and is not apparent in the P. falciparum
genome.
Apicoplast genes, transcribed as mono- or polycistrons,
have short intergenic spaces and lack the purine-rich
prokaryotic ribosome-binding Shine–Dalgarno (SD) sequence upstream of their AUG codons, with the corresponding apicoplast sequences being generally A/T-rich.
This is similar to the green alga Euglena gracilis where a
group of chloroplast mRNAs lacking the SD sequence are
characterized by a 50 -UTR with a high A/T content [64]. The
complementary pyrimidine-rich anti-SD sequence at the 30
end of the bacterial small ribosomal subunit, 16S rRNA, is
also replaced by an A/T-rich sequence in the apicoplastencoded ssu16S rRNA. Similar to mammalian mitochondrial genes that have very short 50 -UTRs, the three P.
falciparum mitochondrial mRNAs do not use the SD interaction to recognize the start codon and presumably have
other mechanisms for interaction with the ribosome and
fragmented rRNA of the organelle for selection of the
correct AUG.
Control and regulation of translation
A large number of proteomic studies have examined individual life stages of Plasmodium, or compared transitions
from one stage to another (reviewed in [65]). Although
these studies show shifts in protein composition between
stages or treatments, the lack of corresponding transcriptome analyses for the same parasites makes it unclear
whether expression regulation takes place at the transcriptional, translational or post-translational levels. The earliest mRNA microarrays indicate that the timing of mRNA
production is generally well correlated with known timing
of protein function [66]. This suggests that Plasmodium
transcripts are produced as needed, or just before the time
they are required to be translated. Despite a strong correlation between transcriptome and proteome, several studies
6
have found a significant delay between maximum mRNA
abundance and peak protein expression, indicating that
post-transcriptional regulation is involved [67–71]. During
asexual replication in the erythrocyte, consensus motifs in
the 5’- and 3’-UTRs are proposed to be involved in posttranscriptional regulation of gene expression [67,69],
however, very few transcriptional regulators have been
identified so far. This absence has lead to theories that
Plasmodium transcription is ‘hard-wired’ [72] and regulation of expression depends on post-transcriptional and
post-translational mechanisms such as proteolytic cleavage,
glycosylation, phosphorylation, myristoylation, acetylation
and ubiquitination [73]. Recent studies on Plasmodium
eIF2a and its interacting kinases have shed some light on
specific post-transcriptional mechanisms regulating the
initiation of translation. The Plasmodium eIF2a kinase,
IK1, was demonstrated to mediate the response to amino
acid starvation by phosphorylating eIF2a [74]. Intriguingly,
the Plasmodium salivary gland sporozoites have been
successful at exploiting the eIF2a kinase, IK2, classically
associated with stress responses in other eukaryotes, to
repress translation. This IK2-mediated repression of
translation maintains the parasites in a latent state in
preparation for differentiation into liver stages [75].
In recent years there have been major developments in
our understanding of translation regulators during zygote
development. Translation repression in P-granules has
been implicated in the regulation of over 370 proteins
during the sexual development of Plasmodium. Mair
and colleagues [68,71] have demonstrated that transcripts
produced in the gametocyte stage are silenced and stabilized in P-granules with the aid of the conserved DEAD-box
RNA helicase, DOZI, and the Sm-like factor, CITH, in
preparation for translation following gamete fertilization.
It is noteworthy that the cores of Plasmodium P-granules
do not contain any of the RNA degradation factors annotated in the genome (e.g. XRN1, Lsm1–7, UPF1–3, DCP1
and 2) [71]. This suggests Plasmodium P-granules are not
mRNA degradation depots, but are predominantly involved in stable storage of translationally repressed transcripts to be co-coordinated for transcription following
fertilization.
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Trends in Parasitology xxx xxxx, Vol. xxx, No. x
Drug targets
Translation is a longstanding and major focus for drug
development in Plasmodium. The relevant enzymes in the
apicoplast and mitochondria in particular are attractive
targets for malaria chemotherapy due to their prokaryotic
origin. The promise of anti-translation inhibitors as antimalarials is highlighted by the number of inhibitors of
protein translation already used for the clinical treatment
of malaria (Table 3), of which doxycycline is perhaps the
most widely known. Despite its potency and efficacy, doxycycline and other tetracyclines, and also clindamycin, exhibit an inhibition behavior known as the ‘delayed-death’
phenomenon [76]. In delayed death, parasites show no
growth inhibition in the first asexual cycle during treatment, but die in the second asexual cycle, even if drug is
washed out in the first cycle. This effect is observed for
some but not all translation-blocking inhibitors [77,78].
Although the cause of delayed death is unknown, indirect
evidence suggests that delayed-death inhibitors target the
apicoplast. For example, tetracyclines block expression of
the apicoplast genome, producing defects in apicoplast
genome replication, apicoplast segregation and protein
trafficking into the apicoplast [76].
Delayed death raises concerns about using apicoplast
inhibitors as antimalarials; however, in combination these
compounds are effective against malaria. Two combination
therapies that each include one inhibitor of apicoplast
translation, fosmidomycin/clindamycin [79] and azithromycin/chloroquine [80], are currently in Phase III clinical
trials. Clindamycin and azithromycin inhibit by binding at
the peptide exit tunnel formed by the interaction of the 23s
rRNA with the 50s ribosomal subunit. Some antibiotics
that attack ribosomal RNA rather than ribosomal proteins
themselves [42,81] have antimalarial activity. Specifically,
the ribosome-blocking antibiotics telithromycin and quinupristin–dalfopristin target the parasite apicoplast and
result in delayed death, suggesting inhibition of apicoplast
translation [82]. Recent P. berghei studies show that probable apicoplast translation inhibitors (azithromycin, clindamycin and doxycycline) also block development of the
apicoplast during exo-erythrocytic schizogony in the liver,
leading to impaired parasite maturation [83]. Azithromycin also inhibits of apicoplast replication in different stages
of Plasmodium berghei (blood, liver and mosquito) [84].
These data strengthen the rationale for prophylactic use of
such drugs.
The Plasmodium translation systems have a number of
other potentially interesting drug targets, namely translation elongation factors, tRNA synthetases and S-adenosylmethionine-decarboxylase. For example, amythiamicin-A
and kirromycin are effective against EF-Tu and demonstrate anti-malarial activity [61,85] and the EF-G inhibitor, fusidic acid, also inhibits P. falciparum growth [86].
In addition to organellar translation factors, proteins
involved in post-translational modification of cytosolic
translation initiation and elongation factors have been
proposed as targets for antimalarials. Plasmodial S-adenosyl-methionine-decarboxylase, required for biosynthesis
of spermidine and a precursor for hypusine synthesis
on eIF-5A, and deoxyhypusine hydroxylase have been
Table 3. Molecular targets of some known inhibitors of Plasmodium protein translation
Target
Apicoplast LSU 23s rRNA
Compounds
Thiostrepton
Clindamycin
Comments
Thiostrepton has been shown to bind 23s rRNA [89,90]
Clindamycin-resistant Toxoplasma and Plasmodium have mutations in the small
subunit of the apicoplast rRNA [91], although it has not been established that these
mutations are causal for Plasmodium [92]. Clindamycin has been used in humans
in combination with fosmidomycin [79]
Azithromycin
The polypeptide exit tunnel formed by the 23s rRNA, ribosomal proteins L4 and L22
is thought to be the site of action of azithromycin. Mutations in apicoplast 23s rRNA
confer azithromycin resistance [93]
Chloramphenicol
Chloramphenicol has some delayed death effect [94] but has not been specifically
shown to block apicoplast translation
Telithromycin
Telithromycin has some delayed death effect [82] but has not been specifically
shown to block apicoplast translation
Quinupristin–dalfopristin The quinupristin–dalfopristin combination has some delayed death effect [82] but
has not been specifically shown to block apicoplast translation
The polypeptide exit tunnel of the ribosome formed by the 23s rRNA, ribosomal
Azithromycin
Ribosomal protein L4 (PfRpl4)
proteins L4 and L22 is thought to be the site of action of azithromycin. Mutations in
apicoplast targeted L4 confer azithromycin resistance [93]
The polypeptide chain formed by the 23s rRNA, ribosomal proteins L4 and L22 is
Ribosomal protein L22 (PfRpl22) Azithromycin
thought to be the site of action of Azithromycin [93]
Tetracyclines
The tetracycline antibiotic doxycycline is used as an antimalarial prophylactic and
Apicoplast 16s rRNA
inhibits translation in the apicoplast [76,78,95]. To our knowledge tetracyclines have
not formally been demonstrated to inhibit via interaction with 16s rRNA, but this
is the probable site of action
Mutations in EF-Tu bestow resistance to amythiamicin (EF-Tu is encoded by the
Amythiamicin
Apicoplast EF-Tu
apicoplast genome) [85]
Kirromycin
Kirromycin-EF-Tu complex observed [85]
Actinonin
Actinonin is assumed to inhibit apicoplast translation via inhibition of deformylation
Peptide deformylase
of the initiator fMet [25]
Treatment with tetracycline was found to lead to decreased transcription of
Tetracyclines
Mitochondrial SSU rRNA
apicoplast-encoded genes as well as mitochondrion-encoded transcripts [95].
Tetracycline also modifies mitochondrial morphology [78]
7
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Review
identified as potential targets [60,87]. A serine/threonine
protein phosphatase 2C that regulates nucleotide exchange activity of EF-1b in P. falciparum has also been
suggested as a candidate chemotherapeutic target [88].
Unanswered questions
Plasmodium has three translationally active compartments, the nucleus, apicoplast and mitochondrion, each
with distinct evolutionary origins. These origins are
reflected in differing mechanisms controlling translation,
presenting three separate biological stories, and three
potential sets of targets for inhibition. Several aspects of
these stories remain to be revealed: ribosomal proteins for
each compartment are generally poorly annotated, and the
recent structural elucidation of ribosomes in model organisms provides a template to improve the organization and
understanding of Plasmodium ribosomal proteins. The
Plasmodium translation initiation, elongation and release
factors are also incompletely understood. Several components appear to be missing, and more work will be required
to clarify whether or how translation proceeds in their
absence. Mitochondrial translation in particular remains
a black box. We have no understanding of how tRNAs used
in the mitochondria are charged, of how charged tRNAs are
imported, nor what happens to these tRNAs after use.
Indeed, direct evidence for translation in mitochondria is
absent. The mitochondrion of Plasmodium could be the
target of important inhibitors of bacterial-type translation,
but the lack of a direct mitochondrial translation marker
complicates the assessment of the effects of inhibitors on
this organelle, and thus impedes the development of future
inhibitors. Future work on these topics should provide not
only a better understanding of how protein translation
proceeds in a eukaryote very distant from well-characterized models, but also opportunities for drug development
on the most solidly established of anti-infective drug targets.
Acknowledgments
The authors are funded by an European Union FP7 Collaborative Project
Grant HEALTH-F3-2009-223024 – Mephitis. S.A.R. is funded by
Australian Research Council Future fellowship FT0990350. The
authors acknowledge and apologize to the authors of many important
studies in this field whose findings had to be omitted or referred to only
via review papers owing to space constraints.
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