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Apicoplast translation, transcription and genome replication: targets for antimalarial antibiotics
Review
Apicoplast translation, transcription and genome replication: targets for antimalarial antibiotics Erica L. Dahl1,2 and Philip J. Rosenthal1 1
Department of Medicine, Division of Infectious Disease, Box 0811, University of California, San Francisco, San Francisco, CA 94143, USA 2 Institute for In Vitro Sciences, Inc., 30 West Watkins Mill Road, Suite 100, Gaithersburg, MD 20878, USA
Several antibiotics possess antimalarial properties, although the mechanisms by which they kill malaria parasites have been poorly understood. Recent data suggest that the target for multiple antimalarial antibiotics is the apicoplast, a chloroplast-like organelle of uncertain function. Translation inhibitors (such as tetracyclines, clindamycin and macrolides) and gyrase inhibitors (such as ciprofloxacin) cause modest antimalarial effects initially but are much more potent against the progeny of treated parasites. These progeny inherit nonfunctional apicoplasts, suggesting that blocking production of apicoplast proteins causes the ‘delayeddeath effect’. Interestingly, the antibiotics thiostrepton and rifampin are fast acting and might target additional processes outside the apicoplast. Antibiotics are effective antimalarial drugs The antimalarial activities of antibiotics were observed first in the late 1940s when animal studies and clinical trials demonstrated that chloramphenocol and tetracyclines had antimalarial properties, although they were slow acting and required up to a week of treatment to clear parasites. A review by Coatney and Greenberg in 1952 [1] concluded that these drugs were too slow to be useful clinically. The worldwide spread of chloroquine-resistant Plasmodium falciparum led to a re-evaluation of the use of antibiotics to treat malaria in the 1970s [2,3]. Though the term ‘antibiotic’ originally referred to microbial metabolites with antibacterial activity, it is used here to include naturally occurring antibacterial compounds and their synthetic derivatives and novel synthetic antibacterial drugs. This article focuses on antibiotics that inhibit the prokaryotic housekeeping functions of translation, transcription and genome replication. Compounds targeting folate metabolism (such as proguanil, pyrimethamine, sulfas and dapsone) [4] and fatty acid synthesis (such as fosmidomycin and triclosan) [5] will not be included. Because Plasmodium spp. are eukaryotes, it is unlikely that inhibitors of prokaryotic translation, transcription or genome maintenance disrupt these processes in the cytosol or nucleus. Indeed, several recent studies suggest that these antibiotics exert antimalarial effects by disrupting these processes in the apicoplast, a unique organelle of Corresponding author: Rosenthal, P.J. ([email protected]).
apicomplexan parasites. This review will summarize these studies and discuss how their findings have expanded our knowledge of apicoplast biology. In vitro studies: delayed death, the mitochondrion and the apicoplast In 1983 Geary and Jensen observed that antibiotics that inhibit protein or nucleic acid synthesis possessed antimalarial properties, whereas those that disrupted cellwall synthesis (penicillins) did not [6]. Moreover, they found that tetracycline and erythromycin were effective at clinically achievable concentrations only after cultured parasites were treated for 96 h, or two full erythrocytic cycles, consistent with the slow antimalarial activities observed clinically. It was thought that these antibiotics inhibited mitochondrial protein synthesis because of the prokaryotic ancestry of this organelle. Supporting this, prolonged exposure to tetracycline caused abnormal rhodamine 123 staining of mitochondria [7] and depressed Glossary Antibiotics: compounds having antibacterial activity, including naturally occurring antibacterial compounds and their synthetic derivatives and novel synthetic antibacterial drugs. Apicoplast: a nonphotosynthetic plastid organelle unique to apicomplexan parasites. Delayed-death effect: this specifically refers to a phenomenon observed in Toxoplasma and Plasmodium in which treatment with antiparasitic drugs does not kill the treated parasites directly but cause them to produce progeny that are unable to complete a replicative life cycle. The death of the progeny occurs even if the drugs are removed before the completion of the parents’ life cycle. Ef-Tu: a GTP/GDP-binding protein required for correct placement of the aatRNA into the acceptor site of the ribosome during elongation of the peptide strand. Gyrase: a topoisomerase enzyme that can introduce or relax supercoils in a DNA molecule by temporarily introducing double-strand breaks to wind or unwind the strand. Merozoites: free-swimming stage of the parasite asexual life cycle. Nuclear-encoded polymerase (NEP): a viral-like, nuclear-encoded RNA polymerase commonly found in plant chloroplasts and/or mitochondria that is encoded on the nuclear genome. Plastid-encoded polymerase (PEP): a bacterial-like RNA polymerase commonly found in plant chloroplasts that is often encoded on the plastid genome. Polysomes: ribosomes bound to an mRNA molecule. Schizont: intracellular stage of the parasite asexual life cycle, during nuclear and cellular division, before the release of merozoites. Topoisomerase IV: a topoisomerase enzyme that can introduce double-strand breaks to relax supercoils but also is responsible for unlinking circular DNA molecules after replication. Trophozoite: intracellular stage of the parasite asexual life cycle, before nuclear and cellular division begins.
1471-4922/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.03.007 Available online 29 April 2008
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Review activity of the mitochondrial enzyme dihydrooritate dehydrogenase [8]. Understanding the antimalarial mechanisms of antibiotics has been facilitated by studies in the related apicomplexan parasite Toxoplasma gondii. Several antibiotics are effective against cultured T. gondii, but as with P. falciparum, activity at clinically achievable concentrations was seen only after prolonged treatment [9]. This delayed action of antibiotics against T. gondii was observed to occur even after a short course of treatment [10]; parasites treated for as little as 2 h replicated normally and reinvaded host cells but died soon after reinvasion. This action against the progeny of treated parasites rather than the parasites exposed to the drugs was termed ‘delayed death’. In the early 1990s it became clear that Plasmodium and Toxoplasma species had a second genome-containing organelle in addition to the mitochondrion. In P. falciparum, sequencing demonstrated that a 35 kb extrachromosomal genome previously presumed to be mitochondrial had features of chloroplast genomes [11,12], whereas a smaller 6 kb genome carried genes encoding mitochondrial cytochromes [13,14]. The 35 kb genome of T. gondii was localized to a distinct organelle by in situ hybridization [15], confirming that these parasites contain a nonphotosynthetic, plastid-like organelle, named the ‘apicoplast’, which appears to have descended from an algal plastid after a secondary endosymbiotic event [16–18]. Because both the mitochondrion and the apicoplast are of prokaryotic origin, either was a logical target for antimalarial antibiotics. Sequencing of both genomes revealed putative antibiotic targets [12,14]. The apicoplast genome encodes components of prokaryotic 70 S ribosomes and subunits for a bacterial-type RNA polymerase; the ribosomes and RNA polymerases are likely targets of antibiotic inhibitors of translation or transcription, respectively. The mitochondrial genome also encodes fragments of 70 S ribosomes. In 1997 Fichera and Roos demonstrated that the plastid genome of T. gondii began to degrade in the cycles after antibiotic treatment [19], linking the effects of antibiotics to the apicoplast. In a transgenic T. gondii line possessing an apicoplast segregation defect [20], offspring that inherited an apicoplast reinvaded host cells and replicated normally, whereas those that did not inherit an apicoplast died shortly after invasion. This observation led to the hypothesis that antibiotics exert their effects against T. gondii and P. falciparum by blocking apicoplast segregation. However, a specific effect of antibiotics against apicoplast segregation was not demonstrated, and the nature of the apicoplast defect elicited by antibiotics in P. falciparum remained elusive. A delayed-death effect of antibiotics in P. falciparum, similar to that observed previously in T. gondii, was recently described by several groups. Parasites treated with clinically relevant concentrations of tetracyclines [21–23], clindamycin [22,23] or azithromycin [24,25] progressed through the erythrocytic life cycle and ruptured erythrocytes to release merozoites; these merozoites invaded new erythrocytes and completed most of a second cycle of development, forming multinucleated schizonts. However, these schizonts were unable to form functional 280
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merozoites capable of rupturing the host cell and appeared grossly abnormal [21]. These effects were observed even if antibiotics were present only during the first cycle and removed before merozoite rupture and reinvasion of host cells. Interestingly, during the first cycle of incubation with antibiotics, both the apicoplast and the mitochondrion, visualized by using targeted fluorescent proteins in transgenic parasites, appeared morphologically normal. Both organelles elongated, branched and segregated normally into developing progeny [21,22,25], demonstrating that the delayed-death effect was not due to blocking segregation of either the apicoplast or mitochondrion. How can the paradox of antibiotics affecting only the life cycle after incubation with malaria parasites be explained? Characterization of parasite transcription by microarray identified a specific decrease in apicoplast gene expression in doxycycline-treated parasites [21]. Apicoplasts inherited by the progeny of doxycycline-treated parasites were nonfunctional because they could not replicate their genomes, were unable to process apicoplast-targeted transgenic proteins and were unable to elongate into the branching pattern typically observed in schizont-stage parasites. By contrast, the mitochondrion displayed normal gene expression, genome replication and branching morphology [21,22]. Results similar to those obtained with doxycycline have been seen with tetracycline, azithromycin and clindamycin [22,24,25]. It appears likely that all the antibiotics that cause a delayed-death effect in P. falciparum target the apicoplast. However, rather than blocking apicoplast segregation, these antibiotics block apicoplast housekeeping functions, resulting in the distribution of nonfunctional apicoplasts into developing progeny. Multiple antibiotics with different structures and mechanisms of action display antimalarial effects (Table 1, and see Supplementary Data online). They can be grouped into three functional categories: inhibitors of translation, inhibitors of transcription and inhibitors of genome replication. The apicoplast genome The full sequence of the apicoplast genome, reported in 1996, provided surprisingly little information regarding the function of this organelle [12]. Phylogenetic analysis suggested the plastid descended from either a red [16] or green [17] algal symbiont. Most of the genes apparently are involved in organelle transcription and translation, and there are just nine exceptions: a putative component of protein import machinery (ClpC), a protein involved in iron-sulfur cluster biosynthesis (SufB) and seven open reading frames with products of unknown function. The sequencing of the full P. falciparum genome and the identification of an apicoplast-targeting sequence [26] allowed identification of several hundred nuclear-encoded proteins that are probably targeted to this organelle, leading to the assignment of several biochemical pathways including the biosynthesis of fatty acids, isoprenoids and heme [27] to the apicoplast. Translation Many of the antibiotics that cause a delayed-death effect in P. falciparum interact with prokaryotic 70 S ribosomes to
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Table 1. Antibiotics displaying antimalarial activity Antibiotic (class) Bacterial target Location of putative target in P. falciparum 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Doxycycline (peptidyl tRNA binding) mitochondrial genomes; nuclear-encoded components contain (Tetracycline) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Tetracycline (peptidyl tRNA binding) mitochondrial genomes; nuclear-encoded components contain (Tetracycline) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Clindamycin (peptidyl tRNA transfer) mitochondrial genomes; nuclear-encoded components contain (Lincosamide) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Azithromycin (peptide exit tunnel) mitochondrial genomes; nuclear-encoded components contain (Macrolide) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Thiostrepton (GTPase turnover) mitochondrial genomes; nuclear-encoded components contain (Cyclic either apicoplast- or mitochondrial-targeting signals thiopeptide) RNA polymerase Partially encoded on apicoplast genome; putative nuclearRifampin encoded subunit contains an apicoplast-targeting signal (Rifampin) DNA gyrase Nuclear encoded but contains apicoplast-targeting signals Ciprofloxacin (Fluoroquinoline)
inhibit translation. Macrolides block the peptide exit tunnel, preventing elongation of nascent peptides [28]. Clindamycin and chloramphenocol both interfere with peptidyl-transferase activity by blocking translocation of the peptidyl tRNAs [28]. Tetracyclines primarily interrupt translation by blocking the binding of the peptidyl tRNA to the acceptor site on the small subunit [28]. Thiopeptides, such as thiostrepton, interfere with the GTPase turnover that provides energy for translation [28,29]. The cytosolic ribosomes of Plasmodium spp. are the 80 S type typically found in eukaryotes and are unaffected by antibiotics targeting prokaryotic 70 S ribosomes. Chloramphenocol, doxycycline, tetracycline and thiostrepton, as well as the eukaryotic ribosomal inhibitor cycloheximide, all block incorporation of radiolabeled amino acids into cytosolic proteins after relatively brief incubations but only at concentrations well above the reported IC50s for growth inhibition [30,31]. Thus, the delayed effects observed at pharmacologically relevant concentrations probably involve targets other than the cytosolic ribosomes. The apicoplast genome encodes RNA for the large and small ribosomal subunits, 17 ribosomal proteins and the elongation factor Tu (ef-Tu). Several ribosomal proteins and elongation factors are encoded in the nucleus [32] and are likely targeted to the apicoplast [33]. The apicoplast genome also encodes 25 tRNAs, which is sufficient for a minimal translation system for apicoplast proteins [34]. Studies have suggested the presence of distinct 70 S polysomes that include rRNA enocoded by the apicoplast [12,35]. Much of the evidence linking antibiotic effects to the apicoplast ribosome has involved identifying mutations that would be expected to disrupt their interactions. Thiostrepton bound to a fragment of isolated apicoplast rRNA in vitro, and this interaction was abolished by inserting a base change found in thiostrepton-resistant ribosomes [36,37]. Clindamycin-resistant T. gondii [38] and azithromycin-resistant P. falciparum [24] generated in the laboratory accumulated mutations in ribosomal rRNA sequences and/or proteins, although additional mutations in nuclear-encoded genes could not be ruled out. These studies link the antibiotic actions to the
Delayed death Yes
Refs [21,25,65,66]
Yes
[6,21–23,65]
Yes
[6,22,23,25,67]
Yes
[24,25,66]
No
[22,24,31,68]
No
[6,22,23,25,65,67]
Strain dependent [22,25,59,69]
apicoplast ribosome, but a direct effect on apicoplast translation has been difficult to prove. Efforts to assess apicoplast translation have been problematic because reliable antibodies directed against apicoplast-encoded proteins are not available. Three studies have reported experiments using antibodies against the apicoplast-encoded ef-Tu [35,39,40]. However, P. falciparum encodes a copy of the gene encoding ef-Tu (tufA) on the nuclear genome that is 48% identical to that found on the apicoplast genome [33,41], and crossreactivity limits conclusions regarding the impacts of antibiotics on native apicoplast proteins. Kiatfuengfoo et al. examined the effect of 1 mM tetracycline on the incorporation of [35S] methionine into parasite proteins, which were resolved by twodimensional SDS PAGE analysis. The two tetracyclinesensitive, cycloheximide-resistant proteins that were found were proposed to be mitochondrial in origin, although the proteins were not identified and this study preceded identification of the apicoplast [7]. Thus, although there is strong evidence that antibiotics interact with apicoplast ribosomes, a direct disruption of apicoplast translation by antibiotics remains to be demonstrated. Most of the prokaryotic translation inhibitors – including the tetracyclines, macrolides and lincosamides (see Table S1 in the Supplementary Data for a comprehensive list) – cause a delayed-death effect in cultured P. falciparum. A notable exception is thiostrepton [22], which is equally efficacious after one or two life cycles. Thiostrepton-treated parasites appeared unable to progress past the trophozoite stage, and the elongation of apicoplasts and mitochondria that occurs normally was not seen [22]. In bacterial ribosomes thiostrepton interferes with energy generation necessary to drive the ribosome [28,29], so it is possible that the drug might interfere with other targets in addition to the apicoplast, as has been hypothesized previously [22,42]. One possible second target is the mitochondrion; thiostrepton and tetracycline, but not macrolides or clindamycin, have been reported to inhibit translation in reconstituted bovine mitochondria in vivo [43]. This could explain the fast action of thiostrepton against malaria parasites and might also explain why the difference in IC50s at 48 and 96 h is less pronounced 281
Review for tetracyclines than for clindamycin and macrolides (Supplementary Data). Further elucidation of these mechanisms will require reliable methods to assess translation in both apicoplasts and mitochondria. Transcription The genes encoded by the apicoplast genome are all transcribed [44,45], and expression is highly coordinated, peaking during the schizont stage [21,46] and coinciding with apicoplast genome replication [47] and the beginning of apicoplast elongation and branching in preparation for segregation [48]. The apicoplast genome encodes three subunits (RpoB, RpoC1 and RpoC2) of a prokaryotic-like RNA polymerase, similar to the RNA polymerases encoded by chloroplast genomes [45]. These subunits, combined with a putative rpoA homolog (Pf13_0040) [33] encoded in the nucleus, could compose a bacterial-type RNA polymerase homologous to those in chloroplasts and predicted to be sensitive to rifampin [49]. Disrupting the plastidencoded RNA polymerase (PEP) genes in tobacco plastids revealed a second nuclear-encoded, phage-like RNA polymerase (NEP) [50,51] that transcribed a subset of plastid genes independently of the PEP. A search of the P. falciparum genome revealed a homolog (Pf11_0264) that had been identified previously as a putative mitochondrial RNA polymerase [33,52] and contains a targeting sequence suggestive of either mitochondrion or apicoplast localization [26,33,53,54]. Presumably, blocking translation of apicoplastencoded RNA polymerase subunits would lead to a loss of some transcription, and many researchers have used apicoplast transcription as an indirect measure of translation. Thiostrepton reduced expression of RpoB and RpoC transcripts relative to nuclear-encoded genes [31], as did doxycycline, which specifically reduced expression of most apicoplast-encoded genes, beginning during the first cycle of treatment [21]. Rifampin also reduced expression of RpoB and RpoC transcripts relative to nuclear-encoded genes [31] and, like thiostrepton, did not cause a delayed-death effect [22,25]. Rifampin-treated parasites appeared similar to thiostreptontreated parasites and had apicoplasts and mitochondria that never began elongation [22]. It was hypothesized recently that the RNA products of the plastid genome might be critical for maintaining the mitochondrion [42]; specifically, formylated methionine tRNA produced solely in the apicoplast might be needed to initiate translation in both organelles. If this hypothesis is true, inhibiting apicolplast transcription might cause an immediate effect on the parasite, whereas inhibiting apicoplast translation alone might cause a delayed-death effect, assuming the RNA polymerases are preformed in the cycle before they are used. Genome replication The apicoplast genome is replicated at the same time as the nuclear genome, at the beginning of schizogeny [47]. Replication initiates via replication bubbles that form between the genes for the large and small rRNAs in the inverted repeat region [47,55]. A rolling-circle mechanism, initiating at sites outside the inverted repeat, also 282
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contributes to replication [47]. The apicoplast genome itself does not encode any proteins known to be involved in DNA replication. However, a gene encoding a multifunctional protein, PfPrex, encompasses DNA helicase, primase and polymerase activities and is targeted to the apicoplast [56]. Other nuclear-encoded, apicoplast-targeted proteins probably involved in plastid replication include DNA gyrase subunits A and B [57,58], a DNA ligase and two hypothetical proteins with similarities to DNA-repair proteins [33]. The presence of bacterial-type DNA gyrases in Plasmodium was predicted after the observation that ciprofloxacin inhibits parasite growth [59]. DNA gyrases break doublestranded DNA to regulate topology and either introduce or remove supercoils as needed. Ciprofloxacin stabilizes the binding of the gyrases to DNA, which increases the number of double-strand breaks. Ciprofloxacin specifically increases the number of double-strand breaks in apicoplast, but not nuclear, DNA in P. falciparum [60]. Replication via replication bubbles is more sensitive to ciprofloxacin than replication by the rolling-circle mechanism, and ciprofloxacin treatment increases the proportion of linear apicoplast DNA [47]. Ciprofloxacin can cause a delayed-death effect in P. falciparum [22,25,47], although the difference in IC50 between effects measured after one or two life cycles is modest compared to that of the protein-synthesis inhibitors. In P. falciparum, the ciprofloxacin IC50 at 48 h appears to vary considerably by strain (see Table S1 in the Supplementary Data). This might indicate that although ciprofloxacin interferes with the apicoplast to cause a delayed effect, it also interferes with a second non-apicoplast target that has strain-specific variations in its susceptibility to the drug. This might explain sometimes contradictory data [22,25] regarding whether ciprofloxacin causes a delayed-death effect in P. falciparum. Ciprofloxacin activity could interfere with the apicoplast in two ways. First, it could interfere with transcription or replication because gyrase activity is required to unwind DNA as polymerases progress along the strand [61]. Alternatively, ciprofloxacin could block topoisomerase IV activity, which is required for unlinking concatamerized circular DNA after replication. This would result in replicated DNA that is unable to segregate into new apicoplasts. Though GFP-labeled apicoplasts appear to segregate normally into parasites treated with ciprofloxacin at a dose that induces a delayed-death effect [25], localization of the genome into each apicoplast inherited by the progeny of treated parasites has not been demonstrated. Topoisomerase IV typically unlinks concatamerized DNA in E. coli [61], and a putative homolog (PF10_0412) is present in the P. falciparum genome [33]. Thus, a second explanation for the narrow dose range at which a delayed-death effect is observed might be that DNA gyrase and topoisomerase IV are differentially sensitive to ciprofloxacin; inhibiting the gyrase necessary for transcription and replication might be lethal in the first cycle, whereas inhibiting topoisomerase IV might cause a delayed-death effect by interfering with separation of the replicated apicoplast genome.
Review A model for delayed death caused by antibiotics that inhibit plasmodial translation Available data indicate that antibiotics causing delayed death in P. falciparum do not interefere with apicoplast biosynthetic functions in the cycle during which the drug is applied. Because most of these functions are performed by nuclear-encoded proteins, blocking the production of apicoplast-encoded proteins would not be expected to affect them. However, apicoplasts generated in the presence of antibiotics and distributed into the progeny of treated parasites might lack apicoplast-encoded proteins required for importing and processing nuclear-encoded proteins. The progeny of antibiotic-treated parasites might, thus, die as they attempt to initiate cell division. Although their nuclei divide, the schizonts lack distinct membrane-bound merozoites and remain arrested at this stage [21]. In T. gondii, parasites initiate cell division earlier than in P. falciparum, and arrest occurs shortly after parasites invade new host cells. Ultrastructural examination showed that the progeny of clindamycintreated T. gondii were successively less able to complete cell division after each round of nuclear replication and formed multinucleated daughter cells that arrested after several rounds of nuclear division [38]. These phenotypes in both parasites might be expected if they were unable to synthesize new fatty acids required for generating new membranes. Enzymes involved in the type II fatty acid biosynthetic pathway typical of prokaryotes have been localized to the apicoplast of P. falciparum [27,62], as have enzymes important for synthesizing iron–sulfur clusters, heme and isoprenoids [27] needed for maintaining mitochondrial functions, which might explain the loss of mitochondrial rhodamine 123 staining [7] and subtle mitochondrial ultrastructural changes observed after two cycles of tetracycline treatment [22]. Antimalarial drugs that directly inhibit the type II fatty acid biosynthesis pathway found in P. falciparum, such as fosmidomycin [63], or collapse the mitochondrial membrane potential, such as atovaquone [64], do not cause delayed death. Although the antimalarial effects of antibiotic translation inhibitors are slow, they are also potent; one of these, doxycycline, remains a key drug for the treatment and prevention of falciparum malaria. Many of the antibiotics listed in Table 1 and Table S1 in the Supplementary Data are already used clinically to treat bacterial infections. They have, thus, already gone through rigorous approval processes and could be incorporated rapidly into malaria treatment or prevention regimes. Although too slow acting to be used as monotherapy, antibiotics targeting apicoplast housekeeping functions could be ideal partners for combination therapy with artemisinins or other rapidacting drugs. Acknowledgements Work by the authors on this subject was supported by the National Institutes of Health and the Medicines for Malaria Venture. P.J.R. is a Doris Duke Charitable Foundation Distinguished Clinical Scientist.
Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.pt.2008.03.007.
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Trends in Parasitology Vol.24 No.6 49 Gardner, M.J. et al. (1991) A circular DNA in malaria parasites encodes an RNA polymerase like that of prokaryotes and chloroplasts. Mol. Biochem. Parasitol. 44, 115–123 50 Allison, L.A. et al. (1996) Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. EMBO J. 15, 2802– 2809 51 Hajdukiewicz, P.T. et al. (1997) The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids. EMBO J. 16, 4041–4048 52 Li, J. et al. (2001) Identification and characterization of a Plasmodium falciparum RNA polymerase gene with similarity to mitochondrial RNA polymerases. Mol. Biochem. Parasitol. 113, 261–269 53 Bender, A. et al. (2003) Properties and prediction of mitochondrial transit peptides from Plasmodium falciparum. Mol. Biochem. Parasitol. 132, 59–66 54 Zuegge, J. et al. (2001) Deciphering apicoplast targeting signals– feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins. Gene 280, 19–26 55 Singh, D. et al. (2005) Multiple replication origins within the inverted repeat region of the Plasmodium falciparum apicoplast genome are differentially activated. Mol. Biochem. Parasitol. 139, 99–106 56 Seow, F. et al. (2005) The plastidic DNA replication enzyme complex of Plasmodium falciparum. Mol. Biochem. Parasitol. 141, 145–153 57 Raghu Ram, E.V. et al. (2007) Nuclear gyrB encodes a functional subunit of the Plasmodium falciparum gyrase that is involved in apicoplast DNA replication. Mol. Biochem. Parasitol. 154, 30–39 58 Dar, M.A. et al. (2007) Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit. Eukaryot. Cell 6, 398–412 59 Krishna, S. et al. (1988) Ciprofloxacin and malaria. Lancet 1, 1231–1232 60 Weissig, V. et al. (1997) Topoisomerase II inhibitors induce cleavage of nuclear and 35-kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol. 16, 1483–1492 61 Hawkey, P.M. (2003) Mechanisms of quinolone action and microbial response. J. Antimicrob. Chemother. 51 (Suppl 1), 29–35 62 Waller, R.F. et al. (2003) A type II pathway for fatty acid biosynthesis presents drug targets in Plasmodium falciparum. Antimicrob. Agents Chemother. 47, 297–301 63 Jomaa, H. et al. (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285, 1573–1576 64 Srivastava, I.K. et al. (1997) Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite. J. Biol. Chem. 272, 3961–3966 65 Pradines, B. et al. (2001) In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron. Antimicrob. Agents Chemother. 45, 1746–1750 66 Yeo, A.E. and Rieckmann, K.H. (1995) Increased antimalarial activity of azithromycin during prolonged exposure of Plasmodium falciparum in vitro. Int. J. Parasitol. 25, 531–532 67 Divo, A.A. et al. (1985) Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture. Antimicrob. Agents Chemother. 27, 21–27 68 Rogers, M.J. et al. (1998) The antibiotic micrococcin is a potent inhibitor of growth and protein synthesis in the malaria parasite. Antimicrob. Agents Chemother. 42, 715–716 69 Divo, A.A. et al. (1988) Activity of fluoroquinolone antibiotics against Plasmodium falciparum in vitro. Antimicrob. Agents Chemother. 32, 1182–1186
Apicoplast translation, transcription and genome replication: targets for antimalarial antibiotics Erica L. Dahl1,2 and Philip J. Rosenthal1 1
Department of Medicine, Division of Infectious Disease, Box 0811, University of California, San Francisco, San Francisco, CA 94143, USA 2 Institute for In Vitro Sciences, Inc., 30 West Watkins Mill Road, Suite 100, Gaithersburg, MD 20878, USA
Several antibiotics possess antimalarial properties, although the mechanisms by which they kill malaria parasites have been poorly understood. Recent data suggest that the target for multiple antimalarial antibiotics is the apicoplast, a chloroplast-like organelle of uncertain function. Translation inhibitors (such as tetracyclines, clindamycin and macrolides) and gyrase inhibitors (such as ciprofloxacin) cause modest antimalarial effects initially but are much more potent against the progeny of treated parasites. These progeny inherit nonfunctional apicoplasts, suggesting that blocking production of apicoplast proteins causes the ‘delayeddeath effect’. Interestingly, the antibiotics thiostrepton and rifampin are fast acting and might target additional processes outside the apicoplast. Antibiotics are effective antimalarial drugs The antimalarial activities of antibiotics were observed first in the late 1940s when animal studies and clinical trials demonstrated that chloramphenocol and tetracyclines had antimalarial properties, although they were slow acting and required up to a week of treatment to clear parasites. A review by Coatney and Greenberg in 1952 [1] concluded that these drugs were too slow to be useful clinically. The worldwide spread of chloroquine-resistant Plasmodium falciparum led to a re-evaluation of the use of antibiotics to treat malaria in the 1970s [2,3]. Though the term ‘antibiotic’ originally referred to microbial metabolites with antibacterial activity, it is used here to include naturally occurring antibacterial compounds and their synthetic derivatives and novel synthetic antibacterial drugs. This article focuses on antibiotics that inhibit the prokaryotic housekeeping functions of translation, transcription and genome replication. Compounds targeting folate metabolism (such as proguanil, pyrimethamine, sulfas and dapsone) [4] and fatty acid synthesis (such as fosmidomycin and triclosan) [5] will not be included. Because Plasmodium spp. are eukaryotes, it is unlikely that inhibitors of prokaryotic translation, transcription or genome maintenance disrupt these processes in the cytosol or nucleus. Indeed, several recent studies suggest that these antibiotics exert antimalarial effects by disrupting these processes in the apicoplast, a unique organelle of Corresponding author: Rosenthal, P.J. ([email protected]).
apicomplexan parasites. This review will summarize these studies and discuss how their findings have expanded our knowledge of apicoplast biology. In vitro studies: delayed death, the mitochondrion and the apicoplast In 1983 Geary and Jensen observed that antibiotics that inhibit protein or nucleic acid synthesis possessed antimalarial properties, whereas those that disrupted cellwall synthesis (penicillins) did not [6]. Moreover, they found that tetracycline and erythromycin were effective at clinically achievable concentrations only after cultured parasites were treated for 96 h, or two full erythrocytic cycles, consistent with the slow antimalarial activities observed clinically. It was thought that these antibiotics inhibited mitochondrial protein synthesis because of the prokaryotic ancestry of this organelle. Supporting this, prolonged exposure to tetracycline caused abnormal rhodamine 123 staining of mitochondria [7] and depressed Glossary Antibiotics: compounds having antibacterial activity, including naturally occurring antibacterial compounds and their synthetic derivatives and novel synthetic antibacterial drugs. Apicoplast: a nonphotosynthetic plastid organelle unique to apicomplexan parasites. Delayed-death effect: this specifically refers to a phenomenon observed in Toxoplasma and Plasmodium in which treatment with antiparasitic drugs does not kill the treated parasites directly but cause them to produce progeny that are unable to complete a replicative life cycle. The death of the progeny occurs even if the drugs are removed before the completion of the parents’ life cycle. Ef-Tu: a GTP/GDP-binding protein required for correct placement of the aatRNA into the acceptor site of the ribosome during elongation of the peptide strand. Gyrase: a topoisomerase enzyme that can introduce or relax supercoils in a DNA molecule by temporarily introducing double-strand breaks to wind or unwind the strand. Merozoites: free-swimming stage of the parasite asexual life cycle. Nuclear-encoded polymerase (NEP): a viral-like, nuclear-encoded RNA polymerase commonly found in plant chloroplasts and/or mitochondria that is encoded on the nuclear genome. Plastid-encoded polymerase (PEP): a bacterial-like RNA polymerase commonly found in plant chloroplasts that is often encoded on the plastid genome. Polysomes: ribosomes bound to an mRNA molecule. Schizont: intracellular stage of the parasite asexual life cycle, during nuclear and cellular division, before the release of merozoites. Topoisomerase IV: a topoisomerase enzyme that can introduce double-strand breaks to relax supercoils but also is responsible for unlinking circular DNA molecules after replication. Trophozoite: intracellular stage of the parasite asexual life cycle, before nuclear and cellular division begins.
1471-4922/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.03.007 Available online 29 April 2008
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Review activity of the mitochondrial enzyme dihydrooritate dehydrogenase [8]. Understanding the antimalarial mechanisms of antibiotics has been facilitated by studies in the related apicomplexan parasite Toxoplasma gondii. Several antibiotics are effective against cultured T. gondii, but as with P. falciparum, activity at clinically achievable concentrations was seen only after prolonged treatment [9]. This delayed action of antibiotics against T. gondii was observed to occur even after a short course of treatment [10]; parasites treated for as little as 2 h replicated normally and reinvaded host cells but died soon after reinvasion. This action against the progeny of treated parasites rather than the parasites exposed to the drugs was termed ‘delayed death’. In the early 1990s it became clear that Plasmodium and Toxoplasma species had a second genome-containing organelle in addition to the mitochondrion. In P. falciparum, sequencing demonstrated that a 35 kb extrachromosomal genome previously presumed to be mitochondrial had features of chloroplast genomes [11,12], whereas a smaller 6 kb genome carried genes encoding mitochondrial cytochromes [13,14]. The 35 kb genome of T. gondii was localized to a distinct organelle by in situ hybridization [15], confirming that these parasites contain a nonphotosynthetic, plastid-like organelle, named the ‘apicoplast’, which appears to have descended from an algal plastid after a secondary endosymbiotic event [16–18]. Because both the mitochondrion and the apicoplast are of prokaryotic origin, either was a logical target for antimalarial antibiotics. Sequencing of both genomes revealed putative antibiotic targets [12,14]. The apicoplast genome encodes components of prokaryotic 70 S ribosomes and subunits for a bacterial-type RNA polymerase; the ribosomes and RNA polymerases are likely targets of antibiotic inhibitors of translation or transcription, respectively. The mitochondrial genome also encodes fragments of 70 S ribosomes. In 1997 Fichera and Roos demonstrated that the plastid genome of T. gondii began to degrade in the cycles after antibiotic treatment [19], linking the effects of antibiotics to the apicoplast. In a transgenic T. gondii line possessing an apicoplast segregation defect [20], offspring that inherited an apicoplast reinvaded host cells and replicated normally, whereas those that did not inherit an apicoplast died shortly after invasion. This observation led to the hypothesis that antibiotics exert their effects against T. gondii and P. falciparum by blocking apicoplast segregation. However, a specific effect of antibiotics against apicoplast segregation was not demonstrated, and the nature of the apicoplast defect elicited by antibiotics in P. falciparum remained elusive. A delayed-death effect of antibiotics in P. falciparum, similar to that observed previously in T. gondii, was recently described by several groups. Parasites treated with clinically relevant concentrations of tetracyclines [21–23], clindamycin [22,23] or azithromycin [24,25] progressed through the erythrocytic life cycle and ruptured erythrocytes to release merozoites; these merozoites invaded new erythrocytes and completed most of a second cycle of development, forming multinucleated schizonts. However, these schizonts were unable to form functional 280
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merozoites capable of rupturing the host cell and appeared grossly abnormal [21]. These effects were observed even if antibiotics were present only during the first cycle and removed before merozoite rupture and reinvasion of host cells. Interestingly, during the first cycle of incubation with antibiotics, both the apicoplast and the mitochondrion, visualized by using targeted fluorescent proteins in transgenic parasites, appeared morphologically normal. Both organelles elongated, branched and segregated normally into developing progeny [21,22,25], demonstrating that the delayed-death effect was not due to blocking segregation of either the apicoplast or mitochondrion. How can the paradox of antibiotics affecting only the life cycle after incubation with malaria parasites be explained? Characterization of parasite transcription by microarray identified a specific decrease in apicoplast gene expression in doxycycline-treated parasites [21]. Apicoplasts inherited by the progeny of doxycycline-treated parasites were nonfunctional because they could not replicate their genomes, were unable to process apicoplast-targeted transgenic proteins and were unable to elongate into the branching pattern typically observed in schizont-stage parasites. By contrast, the mitochondrion displayed normal gene expression, genome replication and branching morphology [21,22]. Results similar to those obtained with doxycycline have been seen with tetracycline, azithromycin and clindamycin [22,24,25]. It appears likely that all the antibiotics that cause a delayed-death effect in P. falciparum target the apicoplast. However, rather than blocking apicoplast segregation, these antibiotics block apicoplast housekeeping functions, resulting in the distribution of nonfunctional apicoplasts into developing progeny. Multiple antibiotics with different structures and mechanisms of action display antimalarial effects (Table 1, and see Supplementary Data online). They can be grouped into three functional categories: inhibitors of translation, inhibitors of transcription and inhibitors of genome replication. The apicoplast genome The full sequence of the apicoplast genome, reported in 1996, provided surprisingly little information regarding the function of this organelle [12]. Phylogenetic analysis suggested the plastid descended from either a red [16] or green [17] algal symbiont. Most of the genes apparently are involved in organelle transcription and translation, and there are just nine exceptions: a putative component of protein import machinery (ClpC), a protein involved in iron-sulfur cluster biosynthesis (SufB) and seven open reading frames with products of unknown function. The sequencing of the full P. falciparum genome and the identification of an apicoplast-targeting sequence [26] allowed identification of several hundred nuclear-encoded proteins that are probably targeted to this organelle, leading to the assignment of several biochemical pathways including the biosynthesis of fatty acids, isoprenoids and heme [27] to the apicoplast. Translation Many of the antibiotics that cause a delayed-death effect in P. falciparum interact with prokaryotic 70 S ribosomes to
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Table 1. Antibiotics displaying antimalarial activity Antibiotic (class) Bacterial target Location of putative target in P. falciparum 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Doxycycline (peptidyl tRNA binding) mitochondrial genomes; nuclear-encoded components contain (Tetracycline) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Tetracycline (peptidyl tRNA binding) mitochondrial genomes; nuclear-encoded components contain (Tetracycline) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Clindamycin (peptidyl tRNA transfer) mitochondrial genomes; nuclear-encoded components contain (Lincosamide) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Azithromycin (peptide exit tunnel) mitochondrial genomes; nuclear-encoded components contain (Macrolide) either apicoplast- or mitochondrial-targeting signals 70 S ribosome 70 S ribosomes partially encoded on apicoplast and Thiostrepton (GTPase turnover) mitochondrial genomes; nuclear-encoded components contain (Cyclic either apicoplast- or mitochondrial-targeting signals thiopeptide) RNA polymerase Partially encoded on apicoplast genome; putative nuclearRifampin encoded subunit contains an apicoplast-targeting signal (Rifampin) DNA gyrase Nuclear encoded but contains apicoplast-targeting signals Ciprofloxacin (Fluoroquinoline)
inhibit translation. Macrolides block the peptide exit tunnel, preventing elongation of nascent peptides [28]. Clindamycin and chloramphenocol both interfere with peptidyl-transferase activity by blocking translocation of the peptidyl tRNAs [28]. Tetracyclines primarily interrupt translation by blocking the binding of the peptidyl tRNA to the acceptor site on the small subunit [28]. Thiopeptides, such as thiostrepton, interfere with the GTPase turnover that provides energy for translation [28,29]. The cytosolic ribosomes of Plasmodium spp. are the 80 S type typically found in eukaryotes and are unaffected by antibiotics targeting prokaryotic 70 S ribosomes. Chloramphenocol, doxycycline, tetracycline and thiostrepton, as well as the eukaryotic ribosomal inhibitor cycloheximide, all block incorporation of radiolabeled amino acids into cytosolic proteins after relatively brief incubations but only at concentrations well above the reported IC50s for growth inhibition [30,31]. Thus, the delayed effects observed at pharmacologically relevant concentrations probably involve targets other than the cytosolic ribosomes. The apicoplast genome encodes RNA for the large and small ribosomal subunits, 17 ribosomal proteins and the elongation factor Tu (ef-Tu). Several ribosomal proteins and elongation factors are encoded in the nucleus [32] and are likely targeted to the apicoplast [33]. The apicoplast genome also encodes 25 tRNAs, which is sufficient for a minimal translation system for apicoplast proteins [34]. Studies have suggested the presence of distinct 70 S polysomes that include rRNA enocoded by the apicoplast [12,35]. Much of the evidence linking antibiotic effects to the apicoplast ribosome has involved identifying mutations that would be expected to disrupt their interactions. Thiostrepton bound to a fragment of isolated apicoplast rRNA in vitro, and this interaction was abolished by inserting a base change found in thiostrepton-resistant ribosomes [36,37]. Clindamycin-resistant T. gondii [38] and azithromycin-resistant P. falciparum [24] generated in the laboratory accumulated mutations in ribosomal rRNA sequences and/or proteins, although additional mutations in nuclear-encoded genes could not be ruled out. These studies link the antibiotic actions to the
Delayed death Yes
Refs [21,25,65,66]
Yes
[6,21–23,65]
Yes
[6,22,23,25,67]
Yes
[24,25,66]
No
[22,24,31,68]
No
[6,22,23,25,65,67]
Strain dependent [22,25,59,69]
apicoplast ribosome, but a direct effect on apicoplast translation has been difficult to prove. Efforts to assess apicoplast translation have been problematic because reliable antibodies directed against apicoplast-encoded proteins are not available. Three studies have reported experiments using antibodies against the apicoplast-encoded ef-Tu [35,39,40]. However, P. falciparum encodes a copy of the gene encoding ef-Tu (tufA) on the nuclear genome that is 48% identical to that found on the apicoplast genome [33,41], and crossreactivity limits conclusions regarding the impacts of antibiotics on native apicoplast proteins. Kiatfuengfoo et al. examined the effect of 1 mM tetracycline on the incorporation of [35S] methionine into parasite proteins, which were resolved by twodimensional SDS PAGE analysis. The two tetracyclinesensitive, cycloheximide-resistant proteins that were found were proposed to be mitochondrial in origin, although the proteins were not identified and this study preceded identification of the apicoplast [7]. Thus, although there is strong evidence that antibiotics interact with apicoplast ribosomes, a direct disruption of apicoplast translation by antibiotics remains to be demonstrated. Most of the prokaryotic translation inhibitors – including the tetracyclines, macrolides and lincosamides (see Table S1 in the Supplementary Data for a comprehensive list) – cause a delayed-death effect in cultured P. falciparum. A notable exception is thiostrepton [22], which is equally efficacious after one or two life cycles. Thiostrepton-treated parasites appeared unable to progress past the trophozoite stage, and the elongation of apicoplasts and mitochondria that occurs normally was not seen [22]. In bacterial ribosomes thiostrepton interferes with energy generation necessary to drive the ribosome [28,29], so it is possible that the drug might interfere with other targets in addition to the apicoplast, as has been hypothesized previously [22,42]. One possible second target is the mitochondrion; thiostrepton and tetracycline, but not macrolides or clindamycin, have been reported to inhibit translation in reconstituted bovine mitochondria in vivo [43]. This could explain the fast action of thiostrepton against malaria parasites and might also explain why the difference in IC50s at 48 and 96 h is less pronounced 281
Review for tetracyclines than for clindamycin and macrolides (Supplementary Data). Further elucidation of these mechanisms will require reliable methods to assess translation in both apicoplasts and mitochondria. Transcription The genes encoded by the apicoplast genome are all transcribed [44,45], and expression is highly coordinated, peaking during the schizont stage [21,46] and coinciding with apicoplast genome replication [47] and the beginning of apicoplast elongation and branching in preparation for segregation [48]. The apicoplast genome encodes three subunits (RpoB, RpoC1 and RpoC2) of a prokaryotic-like RNA polymerase, similar to the RNA polymerases encoded by chloroplast genomes [45]. These subunits, combined with a putative rpoA homolog (Pf13_0040) [33] encoded in the nucleus, could compose a bacterial-type RNA polymerase homologous to those in chloroplasts and predicted to be sensitive to rifampin [49]. Disrupting the plastidencoded RNA polymerase (PEP) genes in tobacco plastids revealed a second nuclear-encoded, phage-like RNA polymerase (NEP) [50,51] that transcribed a subset of plastid genes independently of the PEP. A search of the P. falciparum genome revealed a homolog (Pf11_0264) that had been identified previously as a putative mitochondrial RNA polymerase [33,52] and contains a targeting sequence suggestive of either mitochondrion or apicoplast localization [26,33,53,54]. Presumably, blocking translation of apicoplastencoded RNA polymerase subunits would lead to a loss of some transcription, and many researchers have used apicoplast transcription as an indirect measure of translation. Thiostrepton reduced expression of RpoB and RpoC transcripts relative to nuclear-encoded genes [31], as did doxycycline, which specifically reduced expression of most apicoplast-encoded genes, beginning during the first cycle of treatment [21]. Rifampin also reduced expression of RpoB and RpoC transcripts relative to nuclear-encoded genes [31] and, like thiostrepton, did not cause a delayed-death effect [22,25]. Rifampin-treated parasites appeared similar to thiostreptontreated parasites and had apicoplasts and mitochondria that never began elongation [22]. It was hypothesized recently that the RNA products of the plastid genome might be critical for maintaining the mitochondrion [42]; specifically, formylated methionine tRNA produced solely in the apicoplast might be needed to initiate translation in both organelles. If this hypothesis is true, inhibiting apicolplast transcription might cause an immediate effect on the parasite, whereas inhibiting apicoplast translation alone might cause a delayed-death effect, assuming the RNA polymerases are preformed in the cycle before they are used. Genome replication The apicoplast genome is replicated at the same time as the nuclear genome, at the beginning of schizogeny [47]. Replication initiates via replication bubbles that form between the genes for the large and small rRNAs in the inverted repeat region [47,55]. A rolling-circle mechanism, initiating at sites outside the inverted repeat, also 282
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contributes to replication [47]. The apicoplast genome itself does not encode any proteins known to be involved in DNA replication. However, a gene encoding a multifunctional protein, PfPrex, encompasses DNA helicase, primase and polymerase activities and is targeted to the apicoplast [56]. Other nuclear-encoded, apicoplast-targeted proteins probably involved in plastid replication include DNA gyrase subunits A and B [57,58], a DNA ligase and two hypothetical proteins with similarities to DNA-repair proteins [33]. The presence of bacterial-type DNA gyrases in Plasmodium was predicted after the observation that ciprofloxacin inhibits parasite growth [59]. DNA gyrases break doublestranded DNA to regulate topology and either introduce or remove supercoils as needed. Ciprofloxacin stabilizes the binding of the gyrases to DNA, which increases the number of double-strand breaks. Ciprofloxacin specifically increases the number of double-strand breaks in apicoplast, but not nuclear, DNA in P. falciparum [60]. Replication via replication bubbles is more sensitive to ciprofloxacin than replication by the rolling-circle mechanism, and ciprofloxacin treatment increases the proportion of linear apicoplast DNA [47]. Ciprofloxacin can cause a delayed-death effect in P. falciparum [22,25,47], although the difference in IC50 between effects measured after one or two life cycles is modest compared to that of the protein-synthesis inhibitors. In P. falciparum, the ciprofloxacin IC50 at 48 h appears to vary considerably by strain (see Table S1 in the Supplementary Data). This might indicate that although ciprofloxacin interferes with the apicoplast to cause a delayed effect, it also interferes with a second non-apicoplast target that has strain-specific variations in its susceptibility to the drug. This might explain sometimes contradictory data [22,25] regarding whether ciprofloxacin causes a delayed-death effect in P. falciparum. Ciprofloxacin activity could interfere with the apicoplast in two ways. First, it could interfere with transcription or replication because gyrase activity is required to unwind DNA as polymerases progress along the strand [61]. Alternatively, ciprofloxacin could block topoisomerase IV activity, which is required for unlinking concatamerized circular DNA after replication. This would result in replicated DNA that is unable to segregate into new apicoplasts. Though GFP-labeled apicoplasts appear to segregate normally into parasites treated with ciprofloxacin at a dose that induces a delayed-death effect [25], localization of the genome into each apicoplast inherited by the progeny of treated parasites has not been demonstrated. Topoisomerase IV typically unlinks concatamerized DNA in E. coli [61], and a putative homolog (PF10_0412) is present in the P. falciparum genome [33]. Thus, a second explanation for the narrow dose range at which a delayed-death effect is observed might be that DNA gyrase and topoisomerase IV are differentially sensitive to ciprofloxacin; inhibiting the gyrase necessary for transcription and replication might be lethal in the first cycle, whereas inhibiting topoisomerase IV might cause a delayed-death effect by interfering with separation of the replicated apicoplast genome.
Review A model for delayed death caused by antibiotics that inhibit plasmodial translation Available data indicate that antibiotics causing delayed death in P. falciparum do not interefere with apicoplast biosynthetic functions in the cycle during which the drug is applied. Because most of these functions are performed by nuclear-encoded proteins, blocking the production of apicoplast-encoded proteins would not be expected to affect them. However, apicoplasts generated in the presence of antibiotics and distributed into the progeny of treated parasites might lack apicoplast-encoded proteins required for importing and processing nuclear-encoded proteins. The progeny of antibiotic-treated parasites might, thus, die as they attempt to initiate cell division. Although their nuclei divide, the schizonts lack distinct membrane-bound merozoites and remain arrested at this stage [21]. In T. gondii, parasites initiate cell division earlier than in P. falciparum, and arrest occurs shortly after parasites invade new host cells. Ultrastructural examination showed that the progeny of clindamycintreated T. gondii were successively less able to complete cell division after each round of nuclear replication and formed multinucleated daughter cells that arrested after several rounds of nuclear division [38]. These phenotypes in both parasites might be expected if they were unable to synthesize new fatty acids required for generating new membranes. Enzymes involved in the type II fatty acid biosynthetic pathway typical of prokaryotes have been localized to the apicoplast of P. falciparum [27,62], as have enzymes important for synthesizing iron–sulfur clusters, heme and isoprenoids [27] needed for maintaining mitochondrial functions, which might explain the loss of mitochondrial rhodamine 123 staining [7] and subtle mitochondrial ultrastructural changes observed after two cycles of tetracycline treatment [22]. Antimalarial drugs that directly inhibit the type II fatty acid biosynthesis pathway found in P. falciparum, such as fosmidomycin [63], or collapse the mitochondrial membrane potential, such as atovaquone [64], do not cause delayed death. Although the antimalarial effects of antibiotic translation inhibitors are slow, they are also potent; one of these, doxycycline, remains a key drug for the treatment and prevention of falciparum malaria. Many of the antibiotics listed in Table 1 and Table S1 in the Supplementary Data are already used clinically to treat bacterial infections. They have, thus, already gone through rigorous approval processes and could be incorporated rapidly into malaria treatment or prevention regimes. Although too slow acting to be used as monotherapy, antibiotics targeting apicoplast housekeeping functions could be ideal partners for combination therapy with artemisinins or other rapidacting drugs. Acknowledgements Work by the authors on this subject was supported by the National Institutes of Health and the Medicines for Malaria Venture. P.J.R. is a Doris Duke Charitable Foundation Distinguished Clinical Scientist.
Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.pt.2008.03.007.
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