New hope for the neglected diseases Paul McKean

As we enter the 21st century it is a sobering Clearly there is little financial incentive to develop There is an urgent thought that many of the parasitic protozoan drugs against diseases afflicting human populations need to develop diseases that were thought to be under control, in some of the world’s poorest countries. However, new drugs against and indeed eradicated from some areas during the 1960s the dearth of anti-parasite drugs is not just a financial protozoan are again resurgent in many tropical countries. Diseases issue. Our academic understanding of parasite biology such as , trypanosomiasis and leishmaniasis has often not been at a level adequate for target parasites. Paul are once again exerting an enormous toll in terms identification. McKean explains of mortality and morbidity on the populations of how greater developing countries (see Table 1). New opportunities from the distinct understanding of Although there are reasons for this degenerating biology of parasites parasite biology situation, including complex political and socio- Despite this rather dismal picture of tropical disease and and genome economic factors (such as military conflicts, collapsing drug development there is now hope that progress in sequencing health programmes and population displacement) it is parasite molecular genetics, genome sequencing information clear that the parasites themselves are also fighting back. projects, as well as fundamental studies into the may lead to a There is widespread development of drug resistance biology of parasites, will provide scope for new drug breakthrough. in many parasitic , a worrying situation for development. diseases where the treatments are often unsatisfactory Within the next 2–3 years, the genomes of four in the first place. major protozoan pathogens ( falciparum, Although modern medicine has made enormous Leishmania major, Trypanosoma brucei and Trypanosoma technological advances over the past 40 years, this has cruzi) will be completely sequenced. Careful inter- not been reflected in the field of tropical medicine. For rogation of this wealth of information, allied to a greater instance, Melarsoprol, the most commonly used understanding of parasite biology is creating exciting drug for the treatment of African trypanosomiasis or new opportunities for the development of novel sleeping sickness, was introduced over 50 years ago. This chemotherapeutics. Nowhere is this approach better arsenic derivative causes severe pain upon intravenous exemplified than by the recent advances in the injection and actually kills up to 10 % of patients identification of new targets for anti-malarial drug due to severe side effects. therapy, based in part on the realization that during its evolutionary history the malarial parasite Plasmodium The neglected diseases ‘ate a plant’. Although there is an urgent requirement for the development of new drugs against protozoan parasites, Plasmodium – a green parasite? these organisms have been largely neglected by Plasmodium, along with all other parasites from the pharmaceutical companies in the developed world. phylum , contains three classes of DNA; At a recent meeting in New York organized by the a nuclear genome and two circular DNA elements of French charity Médecins Sans Frontières (MSF) it 6 and 35 kb in size. It was originally suggested that was emphasized that whilst nearly 1,400 drugs the 35 kb element could represent the mitochon- have been introduced over the last 25 years, only 13 drial genome of apicomplexan parasites. However, of these were for the treatment of tropical infectious the subsequent identification of the 6 kb DNA diseases. element as the true mitochondrial genome presented Table 1. Disease statistics associated with several important protozoan parasites

Disease Parasite Cases At risk Malaria 300–500 million 2,400 million Plasmodium malariae Leishmaniasis Leishmania major 12 million 350 million Leishmania donovani Leishmania mexicana Leishmania brasiliensis American trypanosomiasis Trypanosoma cruzi 18 million 100 million Chagas disease African trypanosomiasis Trypanosoma brucei gambiense 300,000–500,000 60 million Sleeping sickness Trypanosoma brucei rhodesiense

Source: World Health Organization

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Nu M

RIGHT: a conundrum. Exactly where within the cell did this G ** Fig. 1. Transmission electron 35 kb DNA element reside and what possible function micrograph of the apicomplexan parasite . The could it serve? Sequencing of the 35 kb DNA element organellar structures containing clearly indicated that it resembled the plastid genome M the three genomes of this parasite; of non-photosynthetic algae. Research on a related Nu the nucleus (Nu), mitochondria (M) apicomplexan parasite, Toxoplasma gondii (an important and apicoplast (**) are indicated. opportunistic pathogen in AIDS patients), localized The golgi (G), and the anterior (A) and posterior (P) ends of the cells the 35 kb DNA element to a small membrane-bound are also indicated. organelle adjacent to the apical end of the nucleus PHOTO COURTESY PROFESSOR DAVID (Fig. 1). This structure, a relic chloroplast-like plastid P ROOS, DEPARTMENT OF BIOLOGY, termed the apicoplast, appears to have been acquired UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, USA by an early progenitor of the apicomplexan phyla through a process of secondary endosymbiosis. It is OPPOSITE PAGE: proposed that a progenitor algal cell initially engulfed bipartite apicoplast targeting signal is well conserved Fig. 2. Schematic showing a cyanobacterial-like prokaryotic cell in a primary phylogenetically, enabling nuclear-encoded apicoplast apicoplast metabolic pathways. endosymbiotic event (see Saul Purton’s article on pp. proteins to be identified from the P. falciparum genome Biosynthetic pathways within the apicoplast are dependent upon 126–128 for the development of chloroplasts in algae). sequencing project solely on the basis of primary pyruvate. This is acquired through The subsequent engulfment of this algal cell by the sequence information (see below). the import of sugars and/or apicomplexan ancestor (secondary endosymbiosis) and phosphoenol pyruvate and the the subsequent retention of the plastid gave rise to The apicoplast – the enemy within! subsequent activity of the enzyme pyruvate kinase. Isoprenoids are the apicoplast. Whilst the unusual evolutionary history and cell synthesized via the DOXP- It is important to appreciate that the apicoplast (and biology of the apicoplast is clearly a fascinating area dependent pathway and are indeed all plastids) retain a considerable degree of of academic research, the functional relevance of the required for the isoprenylation of autonomy despite their intracellular existence within apicoplast was initially obscure. However, the fact tRNAs and possibly other metabolic eukaryotic cells. This autonomy extends not only to that apicomplexan cells had retained this organelle functions within the cell. Acetyl CoA is produced from pyruvate retaining their own reduced genome, but also to the over millions of years of evolution strongly argued that by the action of a pyruvate retention of transcription and translation systems it may have a vital cellular role. The precise nature of dehydrogenase complex and used that are fundamentally prokaryotic in nature. this is at last beginning to be unravelled, partly as a to produce fatty acids via acetyl result of information derived from genome sequencing CoA carboxylase and a type II FAS Apicoplast genes and proteins complex. Although not discussed projects. Significantly, apicoplast metabolic pathways in the text it is also likely that Sequencing of the plastid genome from Plasmodium are more characteristic of bacteria and plants than the biosynthesis of haem is a and Toxoplasma revealed that many apicoplast genes animal cells. Obviously the cyanobacterial origin of metabolic function of the were exclusively involved in gene expression, including the apicoplast now takes on added importance, as apicoplast. large and small subunit rRNAs, 25 species of tRNA, pathways fundamentally different from mammalian DIAGRAM COURTESY P. MCKEAN (BASED ON A FIGURE BY PROFESSOR subunits of eubacterial RNA polymerases and the cells provide excellent targets for the design of new DAVID ROOS, DEPARTMENT OF translation elongation factor Tu. The presence of parasiticidal drugs. BIOLOGY, UNIVERSITY OF prokaryotic-like proteins explains the susceptibility PENNSYLVANIA, PHILADELPHIA, USA) of apicomplexan parasites to antibiotics that interfere Apicoplast metabolic pathways – the with bacterial transcription and translation, such as Achilles heel of apicomplexan parasites? rifampicin, erythromycin and doxycycline. Isoprenoid biosynthesis. Isoprenoids represent a However many apicoplast-specific genes (probably remarkably diverse group of essential lipids that include more than 100) have also been transferred laterally to cholesterol and the respiratory chain electron carrier, the nuclear genome of the apicomplexan cell. The coenzyme Q (ubiquinone). Isoprenoid synthesis depends targeting of nuclear-encoded proteins to the apicoplast upon the production of a critical subunit, isopentyl presents a complex problem for cells as the apicoplast diphosphate (See Fig. 2). Synthesis of isopentyl is surrounded by four membranes, reflecting the diphosphate can proceed via two distinct routes, the two endosymbiotic events leading to its formation. melavonate-dependent pathway and the melavonate- Toxoplasma has been instrumental in the elucidation of independent pathway. apicoplast-targeting sequences due to the exceptional In mammals and fungi, isopentyl diphosphate is ultrastructure of these cells. These studies demonstrate synthesized by the melavonate pathway, whereas in that nuclear-encoded apicoplast proteins encode an eubacteria, plants and algae this critical subunit is amino-terminal bipartite targeting signal. This consists synthesized by the melavonate-independent or 1-deoxy- of a typical amino-terminal secretory signal sequence D-xylulose 5-phosphate (DOXP) pathway. Significantly, immediately followed by a plastid-targeting . inhibitors of the melavonate pathway are ineffective These two domains together are sufficient to target as antimalarials, indicating that this pathway is absent proteins with great efficiency to the apicoplast. The in Plasmodium. However, two key enzymes in the

130 MICROBIOLOGYTODAY VOL 29/AUG 02 not surprising therefore that the type II FAS pathway Further reading Triose phosphate has recently been identified in Plasmodium. and/or phosphoenol Website of Médecins Sans pyruvate Crucially the two FAS pathways differ in Frontières, the independent susceptibility to inhibition by the drug triclosan (a humanitarian medical charity: broad-spectrum antibiotic); the type I FAS pathway is http://www.msf.org/ refractory, but the type II FAS pathway is inhibited. Ralph, S.A., D’Ombrain, Administration of triclosan to malaria-infected mice M.C. & McFadden, G.I. results in the complete clearance of malarial parasites (2001). The apicoplast as with no adverse effect on the mammalian host. an antimalarial drug target. Pyruvate kinase One of the major attractions of the apicoplast as a Drug Resist Updates 4, Haem drug target is its prokaryotic character. This is not solely 145–151. biosynthesis because apicoplast biochemistry is fundamentally Roos, D.S., Crawford, different from that of the mammalian host, but also M.J., Donald, R.G.K., Pyruvate Acetyl CoA because considerable investment in antibacterial Pyruvate DH Fraunholz, M., Harb, O.S., complex drug development now provides a pre-existing source He, C.Y., Kissinger, J.C., of inhibitors that may be effective as anti-malarial Shaw, M.K. & Striepen, B. drugs. (2002). Mining the Isoprenoid Fatty acid Plasmodium genome database biosynthesis biosynthesis Unfortunately, trypanosomes and Leishmania do not harbour an apicoplast-like organelle to target for to define organellar function: chemotherapy. However, with these pathogens too what does the apicoplast do? DOXP Acetyl CoA Philos Trans R Soc Lond B 357, pathway carboxylase there is renewed optimism that the wealth of 35–46. information from genome sequencing projects, allied Type II Singh, D. & Habib, S. Isoprenyl tRNA FAS to greater understanding of parasite biology will lead to better target identification and ultimately to the (2000). The plastid in Apicomplexa: genome, development of new and desperately needed drugs. function and evolution. J Parasit Dis 24, 18. Dr Paul G. McKean is a Lecturer in the Wilson, R.J.M. & ??? ??? Department of Biological Sciences, Lancaster Williamson, D.H. (1997). University, Lancaster, Lancashire LA1 4YQ, UK. Extrachromosomal DNA in DOXP pathway have been identified in the Plasmodium Tel. 01524 594717 the Apicomplexa. Microbiol Mol genome; DOXP synthase and DOXP reducto- email [email protected] Biol Rev 61, 1–16. isomerase. The apicoplast localization of these two enzymes was strongly indicated by the presence of the bipartite apicoplast targeting sequence at the amino terminus. Although the exact role of isoprenoid biosynthesis is still under investigation (as it is likely that Plasmodium salvages cholesterol from the host cell), this path- way is unquestionably important to Plasmodium. For instance, mice infected with the rodent malarial parasite Plasmodium vinckei can be completely cured of their infection by the administration of the antibacterial drug fosidomycin (an inhibitor of DOXP reducto- isomerase.

Fatty acid synthesis. The synthesis of fatty acids, which are important for cell growth, differentiation and general cellular homeostasis, represents one of the most critical biochemical pathways within cells. Fatty acids are synthesized de novo by most organisms, with two distinct types of fatty acid synthase (FAS) being described to date. Type I FAS is typically found in the cytosol of most eukaryotic cells, including man. Although the type II FAS pathway is widespread in bacteria, in eukaryotic cells it is almost exclusively limited to those organisms containing plastids. It is

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