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Protozoan Parasites: Familiar Faces, New Directions
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Protozoan Parasites: Familiar Faces, New Directions K.C. Carter, R.S. Phillips and C.W. Roberts Glasgow, UK April 1998 In spite of our acquired knowledge of protozoan parasites and the diseases they cause, we still have a lot to learn, and many aspects of progress were discussed at the British Section of the Society of Protozoologists (BSSP) annual meeting. Vestigial Plastids in Apicomplexans Iain Wilson [National Institute for Medical Research (NIMR), London, UK] gave an overview of the vestigial plastid organelle of apicomplexans, which was originally identified in electron micrographs1. The origin of the apicomplexan plastid is not clear, but initial studies suggested a red algal ancestry, since an open reading frame (ORF) from the plastid genome of Plasmodium falciparum has considerable homology with a red algal plastid ORF2. However, more recent sequence analysis of tufA gene, which encodes a protein elongation factor, confirms the algal origin of the plastid, but is suggestive of a green algal ancestry3. Like Epifagus, a parasitic plant, the apicomplexan vestigial plastid genome has only a subset of the features common to chloroplasts of higher plants. The apparent similarity between two unrelated, divergent species perhaps represents convergent evolution, presumably due to the selective pressures generally operative on parasitic organisms. A possible scenario involving a series of endosymbiotic events was presented to explain the acquisition of the plastid by apicomplexans, the ‘Mr Gobbler Theory’. Firstly, a cyanobacterium endocytosed by a eukaryote formed the common ancestor of modern day algae and higher plants. This organism was subsequently endocytosed by a dinoflagellate-like progenitor of the Apicomplexa. In the course of such events the nuclear genome of the endosymbiotic organism would eventually be lost or selectively transferred to the host’s genome. These events would result in the plastid having multiple membranes instead of the normal two found in the chloroplasts of higher plants1. The issue of how many membranes the plastid Parasitology Today, vol. 14, no. 9, 1998
has in any one apicomplexan has been the subject of considerable controversy1,3. Transmission electron micrographs of serial sections through various stages of P. falciparum clearly showed the presence of at least three membranes. These were often ruffled and had convolutions – factors contributing to the difficult task of determining their exact number (L. Bannister, London, UK). The precise ancestry of the plastid organelle may remain a contentious issue for many years to come, but this will not detract from certain fundamental questions, such as: What functions does it perform and can these functions be disrupted to prevent parasite growth?
(University College London, UK) described the introduction of P. falciparum plastid genes into Chlamydomonas using gene particle bombardment. This technique will determine if the tufA gene from the plastid genome of P. falciparum will function in Chlamydomonas, and may provide a more easily manipulatable and in some ways more phylogenetically related system for the expression of apicomplexan plastid genes. The possibility that the plastid may be a site of action for nuclearencoded enzymes as occurs in algae and plants was discussed (C.W. Roberts, University of Strathclyde, Glasgow, UK). Cloning Novel Parasite Molecules
Plastid Function and Potential as a Drug Target On the question of plastid function, inhibitors of plastid protein synthesis were able to restrict the growth of P. falciparum in culture. Theoretical and supportive experimental evidence demonstrates that thiostrepton preferentially binds P. falciparum large subunit ribosome RNAs (LSU rRNAs) encoded by the plastid, rather than those encoded by nuclear or mitochondrial genes. Similar analyses of LSU rRNAs from Eimeria and Toxoplasma gondii were described. Further studies demonstrate that recombinant P. falciparum protein synthesis elongation factor (EF-Tu) binds antibiotics of the kirromycin family. Collectively, these studies highlight plastid protein synthesis as a potential chemotherapeutic target in apicomplexans (B. Clough, NIMR, London, UK). A novel method was outlined that used Synechocystis as a surrogate system for studying plastid genes such as ORF470. The ORF470 gene is highly conserved and has homologues in bacteria and algae (ycf 24), but its function remains unknown. The Synechocystis ycf 24 gene was cloned, inactivated by insertion of the kanamycin resistance gene and transformed into Synechocystis. These organisms had reduced growth rates and altered morphology compared with the wild type, indicating that this mutation is detrimental to Synechocystis (A. Law, NIMR, London, UK). Using a similar approach with the green unicellular alga Chlamydomonas, J. Bateman
A common methodology adopted for the cloning of genes encoding novel drug targets was PCR with degenerate primers. This method facilitated the cloning and characterization of the gene encoding DNA polymerase-␣ catalytic subunit of Leishmania donovani (K. Lyons, University of Technology, Sydney, Australia). A cysteine-rich region in the midst of the active site of the enzyme appears to be unique to Kinetoplastids. The DNA-binding zinc finger in the Cterminus of the protein is a potential drug target for blocking enzyme catalysis in the parasite. A similar PCR-based approach allowed cloning and sequencing of cytoplasmic HSP70 from T. gondii. Differences in the number of copies of a seven-amino-acid repeat sequence at the C-terminal of the protein correlated with parasite virulence (R. Lyons, University of Technology, Sydney, Australia). In addition, virulent strains also had a twofold increase in transcription rate for this gene. Overexpression of a cyclophilin (PfCy P19) from P. falciparum in Escherichia coli showed that, unlike two previously identified cyclophilins from P. falciparum, PfCy P19 had no signal peptide or N-terminal sequence (M. Berriman, Dundee University, UK). Host–Parasite Interplay Studies demonstrated that a bidirectional interplay between the host and parasite is an important factor determining disease outcome. Previous studies have shown that nitric oxide (NO)
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(98)01307-6
341
News production is associated with ocular inflammation4. However, inhibition of NO production in mice chronically infected with T. gondii resulted in reactivation of ocular disease and increased ocular inflammation (F. Roberts, Western Infirmary, Glasgow, UK). Mice with a disrupted iNOS gene were highly susceptible to L. major infection compared with their wild type counterparts (L. Proudfoot, Napier University, Edinburgh, UK). The importance of NO for parasite killing in human cells was debated, although there is now evidence that certain human cells produce NO, which may play a protective role during disease5,6. Interestingly, Leishmania parasites have molecules which inhibit NO production. Thus, highly conserved major surface glycolipids, glycoinositolphospholipids (GIPLs) (L. Proudfoot) and lipophosphoglycans (LPG) are potent inhibitors of NO. Moreover, LPG can also inhibit synthesis of interleukin 12 (IL-12). Further studies demonstrated that synthetic phosphoglycan (sPG) selectively inhibit the production by macrophages of IL-12, but not of tumour necrosis factor ␣ (TNF-␣), IL-6 or NO. This inhibition is at the transcription levels
for the IL-12 p40 subunit (D. Piedrafita, University of Glasgow, UK). However, surprisingly, mucin-like GPI (glycosylphosphatidylinositol)-anchored glycoproteins present on all life-stages of Trypanosoma cruzi are potent inducers of pro-inflammatory cytokines (IL-12, TNF-␣) and can stimulate NO production in primed macrophages. This implies that the parasite invokes immune responses which, on the basis of present knowledge, seem counterprotective. These activities reside in the fatty acid portion of the GPI-anchor (I. Almeida, Dundee University, UK). K.C. Carter (University of Strathclyde, Glasgow, UK) presented evidence to show that IL-4 had a beneficial effect on therapeutic efficacy of sodium stibogluconate treatment of L. donovani infected mice. These studies reveal the subtle interplay between parasites and their hosts, and emphasize that it is inappropriate to characterize particular types of immune responses as protective or exacerbative7. 100 Years of Research in Malaria Peter Billingsley (University of Aberdeen, UK) described the events that
led to Ronald Ross’s description of the life cycle of Plasmodium and highlighted major breakthroughs in malariology over the past 100 years. Acknowledgements The BSSP annual meeting was held at the University of Glasgow, UK, 6–8 April 1998. The BSSP warmly thanks the Universities of Glasgow and Strathclyde for their financial support. References 1 Wilson, R.J.M., Williamson, D.H. and Prieser, P. (1994) Infect. Agents Dis. 3, 29–37 2 Williamson, D.H. et al. (1994) Mol. Gen. Genet. 43, 249–252 3 Kohler, S. et al. (1997) Science 275, 1485–1489 4 Wang, Z.Y. and Hakanson, R. (1995) Br. J. Pharmacol. 116, 2447–2450 5 Albina, J.E. (1995) J. Leukocyte Biol. 58, 643–649 6 Vouldoukis, I. et al. (1997) Eur. J. Immunol. 27, 860–865 7 Allen, J.E. and Maizels, R.M. (1997) Immunol. Today 18, 387–392
K. Chris Carter and Craig W. Roberts are at the Department of Immunology, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow, UK G4 0NR. R. Stephen Phillips is at the Division of Infection and Immunity, Joseph Black Bldg, University of Glasgow, Glasgow, UK G12 8QQ. Tel: +44 141 552 4400 x 3748, Fax: +44 141 552 6674, e-mail: [email protected]
The Plasmodium falciparum Genome Project C. Fletcher Orlando, FL, USA December 1997 The completion of DNA sequences for over a dozen microbial genomes1 is focusing attention on genome sequencing of pathogens as never before. The genomic sequences of several major bacterial pathogens have already been completed2–4. Now sequencing of the much larger genome of the malaria parasite, Plasmodium falciparum, is progressing well within the framework of an international consortium. Two of the 14 chromosomes in the 30 Mb P. falciparum genome are almost completely sequenced, sequencing of two other chromosomes is well under way, and work on the remaining ten chromosomes has begun. The consortium is a coordinated effort by funding agencies, sequencing centres and malariologists to achieve the complete sequencing of the P. falciparum genome (clone 3D7) and to promote its use in developing new strategies to 342
control malaria, including diagnostics, vaccines and drugs. Building on the malaria genome mapping project5 and impressive advances in genomics, the consortium was initiated in 1996. To date, The Wellcome Trust (London, UK), Burroughs Wellcome Fund (Durham, NC, USA), the US National Institutes of Health (NIH) (Bethesda, MD, USA) and the US Department of Defense (Washington, DC, USA) have committed in total about US$ 25 million, although funds have not been pooled. Sequencing efforts began with pilot projects to overcome the technical difficulties of cloning and sequencing highly A–T rich P. falciparum DNA. The encouraging results led to expanded efforts at the three high-throughput sequencing centres: the Sanger Centre (Hinxton, UK), The Institute for Genomic Research (TIGR) (Rockville, MD, USA) in collaboration with the Naval Medical Research Institute (NMRI) (MD, USA), and Stanford University (Palo Alto, CA, USA).
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(98)01300-3
Progress – Nuts and Bolts The consortium is tackling the sequencing of the entire genome on a chromosome-by-chromosome basis. Because large fragments of P. falciparum DNA are unstable in Escherichia coli, large-insert (⬎20 kb) libraries in E. coli cannot be produced for use in sequencing. Moreover, although several libraries are available as yeast artificial chromosomes (YACs), these are poor substrates for high-throughput sequencing. Consequently, all three sequencing centres are sequencing the P. falciparum chromosomes by the ‘shotgun’ method. Shotgunning involves breaking up large (megabase) fragments of genomic DNA into small (1–2 kb) fragments by random shearing, and cloning the size-selected fragments into bacterial plasmids; these then serve as the templates for highthroughput sequencing. Sequencing of chromosomes 1, 3, 4, 5–9 (known as ‘the blob’ because of its Parasitology Today, vol. 14, no. 9, 1998
Protozoan Parasites: Familiar Faces, New Directions K.C. Carter, R.S. Phillips and C.W. Roberts Glasgow, UK April 1998 In spite of our acquired knowledge of protozoan parasites and the diseases they cause, we still have a lot to learn, and many aspects of progress were discussed at the British Section of the Society of Protozoologists (BSSP) annual meeting. Vestigial Plastids in Apicomplexans Iain Wilson [National Institute for Medical Research (NIMR), London, UK] gave an overview of the vestigial plastid organelle of apicomplexans, which was originally identified in electron micrographs1. The origin of the apicomplexan plastid is not clear, but initial studies suggested a red algal ancestry, since an open reading frame (ORF) from the plastid genome of Plasmodium falciparum has considerable homology with a red algal plastid ORF2. However, more recent sequence analysis of tufA gene, which encodes a protein elongation factor, confirms the algal origin of the plastid, but is suggestive of a green algal ancestry3. Like Epifagus, a parasitic plant, the apicomplexan vestigial plastid genome has only a subset of the features common to chloroplasts of higher plants. The apparent similarity between two unrelated, divergent species perhaps represents convergent evolution, presumably due to the selective pressures generally operative on parasitic organisms. A possible scenario involving a series of endosymbiotic events was presented to explain the acquisition of the plastid by apicomplexans, the ‘Mr Gobbler Theory’. Firstly, a cyanobacterium endocytosed by a eukaryote formed the common ancestor of modern day algae and higher plants. This organism was subsequently endocytosed by a dinoflagellate-like progenitor of the Apicomplexa. In the course of such events the nuclear genome of the endosymbiotic organism would eventually be lost or selectively transferred to the host’s genome. These events would result in the plastid having multiple membranes instead of the normal two found in the chloroplasts of higher plants1. The issue of how many membranes the plastid Parasitology Today, vol. 14, no. 9, 1998
has in any one apicomplexan has been the subject of considerable controversy1,3. Transmission electron micrographs of serial sections through various stages of P. falciparum clearly showed the presence of at least three membranes. These were often ruffled and had convolutions – factors contributing to the difficult task of determining their exact number (L. Bannister, London, UK). The precise ancestry of the plastid organelle may remain a contentious issue for many years to come, but this will not detract from certain fundamental questions, such as: What functions does it perform and can these functions be disrupted to prevent parasite growth?
(University College London, UK) described the introduction of P. falciparum plastid genes into Chlamydomonas using gene particle bombardment. This technique will determine if the tufA gene from the plastid genome of P. falciparum will function in Chlamydomonas, and may provide a more easily manipulatable and in some ways more phylogenetically related system for the expression of apicomplexan plastid genes. The possibility that the plastid may be a site of action for nuclearencoded enzymes as occurs in algae and plants was discussed (C.W. Roberts, University of Strathclyde, Glasgow, UK). Cloning Novel Parasite Molecules
Plastid Function and Potential as a Drug Target On the question of plastid function, inhibitors of plastid protein synthesis were able to restrict the growth of P. falciparum in culture. Theoretical and supportive experimental evidence demonstrates that thiostrepton preferentially binds P. falciparum large subunit ribosome RNAs (LSU rRNAs) encoded by the plastid, rather than those encoded by nuclear or mitochondrial genes. Similar analyses of LSU rRNAs from Eimeria and Toxoplasma gondii were described. Further studies demonstrate that recombinant P. falciparum protein synthesis elongation factor (EF-Tu) binds antibiotics of the kirromycin family. Collectively, these studies highlight plastid protein synthesis as a potential chemotherapeutic target in apicomplexans (B. Clough, NIMR, London, UK). A novel method was outlined that used Synechocystis as a surrogate system for studying plastid genes such as ORF470. The ORF470 gene is highly conserved and has homologues in bacteria and algae (ycf 24), but its function remains unknown. The Synechocystis ycf 24 gene was cloned, inactivated by insertion of the kanamycin resistance gene and transformed into Synechocystis. These organisms had reduced growth rates and altered morphology compared with the wild type, indicating that this mutation is detrimental to Synechocystis (A. Law, NIMR, London, UK). Using a similar approach with the green unicellular alga Chlamydomonas, J. Bateman
A common methodology adopted for the cloning of genes encoding novel drug targets was PCR with degenerate primers. This method facilitated the cloning and characterization of the gene encoding DNA polymerase-␣ catalytic subunit of Leishmania donovani (K. Lyons, University of Technology, Sydney, Australia). A cysteine-rich region in the midst of the active site of the enzyme appears to be unique to Kinetoplastids. The DNA-binding zinc finger in the Cterminus of the protein is a potential drug target for blocking enzyme catalysis in the parasite. A similar PCR-based approach allowed cloning and sequencing of cytoplasmic HSP70 from T. gondii. Differences in the number of copies of a seven-amino-acid repeat sequence at the C-terminal of the protein correlated with parasite virulence (R. Lyons, University of Technology, Sydney, Australia). In addition, virulent strains also had a twofold increase in transcription rate for this gene. Overexpression of a cyclophilin (PfCy P19) from P. falciparum in Escherichia coli showed that, unlike two previously identified cyclophilins from P. falciparum, PfCy P19 had no signal peptide or N-terminal sequence (M. Berriman, Dundee University, UK). Host–Parasite Interplay Studies demonstrated that a bidirectional interplay between the host and parasite is an important factor determining disease outcome. Previous studies have shown that nitric oxide (NO)
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(98)01307-6
341
News production is associated with ocular inflammation4. However, inhibition of NO production in mice chronically infected with T. gondii resulted in reactivation of ocular disease and increased ocular inflammation (F. Roberts, Western Infirmary, Glasgow, UK). Mice with a disrupted iNOS gene were highly susceptible to L. major infection compared with their wild type counterparts (L. Proudfoot, Napier University, Edinburgh, UK). The importance of NO for parasite killing in human cells was debated, although there is now evidence that certain human cells produce NO, which may play a protective role during disease5,6. Interestingly, Leishmania parasites have molecules which inhibit NO production. Thus, highly conserved major surface glycolipids, glycoinositolphospholipids (GIPLs) (L. Proudfoot) and lipophosphoglycans (LPG) are potent inhibitors of NO. Moreover, LPG can also inhibit synthesis of interleukin 12 (IL-12). Further studies demonstrated that synthetic phosphoglycan (sPG) selectively inhibit the production by macrophages of IL-12, but not of tumour necrosis factor ␣ (TNF-␣), IL-6 or NO. This inhibition is at the transcription levels
for the IL-12 p40 subunit (D. Piedrafita, University of Glasgow, UK). However, surprisingly, mucin-like GPI (glycosylphosphatidylinositol)-anchored glycoproteins present on all life-stages of Trypanosoma cruzi are potent inducers of pro-inflammatory cytokines (IL-12, TNF-␣) and can stimulate NO production in primed macrophages. This implies that the parasite invokes immune responses which, on the basis of present knowledge, seem counterprotective. These activities reside in the fatty acid portion of the GPI-anchor (I. Almeida, Dundee University, UK). K.C. Carter (University of Strathclyde, Glasgow, UK) presented evidence to show that IL-4 had a beneficial effect on therapeutic efficacy of sodium stibogluconate treatment of L. donovani infected mice. These studies reveal the subtle interplay between parasites and their hosts, and emphasize that it is inappropriate to characterize particular types of immune responses as protective or exacerbative7. 100 Years of Research in Malaria Peter Billingsley (University of Aberdeen, UK) described the events that
led to Ronald Ross’s description of the life cycle of Plasmodium and highlighted major breakthroughs in malariology over the past 100 years. Acknowledgements The BSSP annual meeting was held at the University of Glasgow, UK, 6–8 April 1998. The BSSP warmly thanks the Universities of Glasgow and Strathclyde for their financial support. References 1 Wilson, R.J.M., Williamson, D.H. and Prieser, P. (1994) Infect. Agents Dis. 3, 29–37 2 Williamson, D.H. et al. (1994) Mol. Gen. Genet. 43, 249–252 3 Kohler, S. et al. (1997) Science 275, 1485–1489 4 Wang, Z.Y. and Hakanson, R. (1995) Br. J. Pharmacol. 116, 2447–2450 5 Albina, J.E. (1995) J. Leukocyte Biol. 58, 643–649 6 Vouldoukis, I. et al. (1997) Eur. J. Immunol. 27, 860–865 7 Allen, J.E. and Maizels, R.M. (1997) Immunol. Today 18, 387–392
K. Chris Carter and Craig W. Roberts are at the Department of Immunology, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow, UK G4 0NR. R. Stephen Phillips is at the Division of Infection and Immunity, Joseph Black Bldg, University of Glasgow, Glasgow, UK G12 8QQ. Tel: +44 141 552 4400 x 3748, Fax: +44 141 552 6674, e-mail: [email protected]
The Plasmodium falciparum Genome Project C. Fletcher Orlando, FL, USA December 1997 The completion of DNA sequences for over a dozen microbial genomes1 is focusing attention on genome sequencing of pathogens as never before. The genomic sequences of several major bacterial pathogens have already been completed2–4. Now sequencing of the much larger genome of the malaria parasite, Plasmodium falciparum, is progressing well within the framework of an international consortium. Two of the 14 chromosomes in the 30 Mb P. falciparum genome are almost completely sequenced, sequencing of two other chromosomes is well under way, and work on the remaining ten chromosomes has begun. The consortium is a coordinated effort by funding agencies, sequencing centres and malariologists to achieve the complete sequencing of the P. falciparum genome (clone 3D7) and to promote its use in developing new strategies to 342
control malaria, including diagnostics, vaccines and drugs. Building on the malaria genome mapping project5 and impressive advances in genomics, the consortium was initiated in 1996. To date, The Wellcome Trust (London, UK), Burroughs Wellcome Fund (Durham, NC, USA), the US National Institutes of Health (NIH) (Bethesda, MD, USA) and the US Department of Defense (Washington, DC, USA) have committed in total about US$ 25 million, although funds have not been pooled. Sequencing efforts began with pilot projects to overcome the technical difficulties of cloning and sequencing highly A–T rich P. falciparum DNA. The encouraging results led to expanded efforts at the three high-throughput sequencing centres: the Sanger Centre (Hinxton, UK), The Institute for Genomic Research (TIGR) (Rockville, MD, USA) in collaboration with the Naval Medical Research Institute (NMRI) (MD, USA), and Stanford University (Palo Alto, CA, USA).
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(98)01300-3
Progress – Nuts and Bolts The consortium is tackling the sequencing of the entire genome on a chromosome-by-chromosome basis. Because large fragments of P. falciparum DNA are unstable in Escherichia coli, large-insert (⬎20 kb) libraries in E. coli cannot be produced for use in sequencing. Moreover, although several libraries are available as yeast artificial chromosomes (YACs), these are poor substrates for high-throughput sequencing. Consequently, all three sequencing centres are sequencing the P. falciparum chromosomes by the ‘shotgun’ method. Shotgunning involves breaking up large (megabase) fragments of genomic DNA into small (1–2 kb) fragments by random shearing, and cloning the size-selected fragments into bacterial plasmids; these then serve as the templates for highthroughput sequencing. Sequencing of chromosomes 1, 3, 4, 5–9 (known as ‘the blob’ because of its Parasitology Today, vol. 14, no. 9, 1998