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Host–microbe interactions: parasites
Host–microbe interactions: parasites Insights from parasite genomes Editorial overview Ken Stuart Current Opinion in Microbiology 2004, 7:359–361 This review comes from a themed issue on Host–microbe interactions: parasites Edited by Ken Stuart Available online 20th July 2004 1369-5274/$ – see front matter ß 2004 Published by Elsevier Ltd. DOI 10.1016/j.mib.2004.06.015
Ken Stuart Seattle Biomedical Research Institute and Department of Pathobiology, University of Washington, 307 Westlake Ave N, Suite 500, Seattle, WA 98109-5219, USA e-mail: [email protected]
Ken’s research focuses on Trypanosomatids: Trypanosoma brucei, T. cruzi and pathogenic Leishmania species. He studies molecular process and gene function in these organisms and plays a leading role in the genome projects. His lab also studies RNA editing with an emphasis on molecular mechanisms in addition to editing complex composition, structure and specific functions with an eye toward drug development.
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Now that the sequencing of the genomes of key Apicomplexan and Trypanosomatid pathogens have been completed, attention is turning to using this information for insights into the roles of these genes and how this information can be used to develop strategies to combat diseases. The diploid genomes of three Trypanosomatid pathogens Trypanosoma brucei, T. cruzi and Leishmania major have substantial similarity, as illustrated by conservation of extensive synteny, despite their ancient divergence. However, these genomes have characteristic differences including minichromosomes and variant surface glycoprotein expression sites in T. brucei, distinct families of repeated sequences in T. cruzi, and high allele similarity in L. major. Trypanosomatid genomes are about 20% larger than those of the Apicomplexan genomes and there are more similarities between Trypanosomatid genomes than between Apicomplexan genomes, such as P. falciparum and Toxoplasma gondii, although synteny cannot be analyzed in detail as the T. gondii genome sequence that has been determined currently exists as numerous sizeable subchromosomal sequence segments (contigs). The above five pathogens are transmitted by different vectors (typically insects), cause different diseases, reside in different host tissues, and have different immune evasion strategies. Trypanosoma brucei (group) trypanosomes are transmitted by Tsetse flies, proliferate extracellularly in the blood and cerebral spinal fluid, and evade immune elimination by antigenic variation. They cause African sleeping sickness a neural pathological disease that is invariably fatal if untreated. T. cruzi is transmitted by Reduviid bugs, and proliferates intracellularly, primarily in the cytoplasm of muscle cells, which provides some immune defense, but they also undergo antigenic variation. T. cruzi causes Chagas’ disease with severe cardiac and alimentary pathology that is debilitating and ultimately fatal. Leishmania are transmitted by sand flies and proliferate in the phagolysosomes of macrophages where they avoid immune elimination as a result of interaction with the immune system. Leishmania cause a spectrum of pathologies that range from localized to disseminated cutaneous lesions, to horribly disfiguring muco-cutaneous erosion, to severe liver and spleen pathology associated with visceral disease. Chagas’ disease and Leishmaniasis are chronic diseases with an apparent immune basis for the resultant pathology. African Sleeping Sickness is a more acute disease with an uncertain basis for its pathology. Plasmodium falciparum is transmitted by mosquitoes and multiplies intracellularly, first in the liver and then in erythrocytes. The intracellular environment and antigenic variation evade immune elimination and adhesion-based Current Opinion in Microbiology 2004, 7:359–361
360 Host–microbe interactions: parasites
sequestration evades elimination of infected erythrocytes by the spleen. Malaria is a spectral disease ranging from debilitating to lethal with pathologies including placental inflammation, severe anemia and cerebral disease. Toxoplasma is transmitted by domestic and wild cats by fecal contamination and proliferates intracellularly. While infection is typically asymptomatic and chronic it causes neuropathology especially in immune compromised persons and fetuses. Hence, related pathogenic protozoa have adapted to varied environments and cause different diseases. The availability of their genome sequences presents the opportunity to learn how these pathogens have adapted and cause disease and for devising strategies to prevent or treat these diseases. A key challenge presented by the completed genome sequences is the use or development of methods to determine the functions of the genes, as the function of most are not known. Motyka and Englund describe how RNA interference (RNAi) is used in T. brucei for specific gene silencing in order to identify the functions of genes, to elucidate complex biological processes, and to identify those that are essential for pathogen survival and hence are potential drug targets. They describe its use in combination with tetracycline-regulated systems, which allows analysis of essential genes. RNAi is more convenient and efficient than previous methods for determining gene function and Motyka and Englund describe how RNAi libraries may provide an approach for forward genetic studies in Trypanosomatids. However, RNAi is not a gene function panacea. Motyka and Englund indicate some of the pitfalls with RNAi. RNAi has not been achieved in T. cruzi or Leishmania despite numerous attempts, perhaps reflecting differences in RNA degradation systems and RNA metabolism among Trypanosomatids. Adaptation of RNAi and regulatable promoter systems is at an earlier stage in Plasmodium and Toxoplasma and not yet routine, perhaps reflecting the difficulty of DNA transfection, especially in Plasmodium. Overall, there is a substantial need for high throughput tools for the study of gene function in pathogenic protozoa. Pays et al. describe antigenic variation in T. brucei, a key phenomenon for this pathogen and one with a rich history in molecular parasitology. Their summary of how these parasites evade immune elimination by a combination of transcriptional control and DNA recombination parallels insights stemming from the organization of the T. brucei genome and the need to elucidate molecular processes in this organism. The multiple telomeric expression sites for variant surface glycoprotein (VSG) genes with their associated genes (ESAGs) and the numerous additional VSG genes that are non-telomeric or on the numerous minichromosomes illustrates the adaptation of the T. brucei genome to the process of antigenic variation. Pays et al. describe the expression of only one VSG gene at a time, Current Opinion in Microbiology 2004, 7:359–361
periodic switching of expression to an alternate VSG expression site, and inactivation of VSG expression concomitant with activation of procyclic specific genes, which reveals the early stage of understanding of control of transcription and RNA turnover in all Trypanosomatids. It also implies that there are features specific to antigenic variation. Their descriptions of the periodic recombination that switches and remodels the VSG gene in the expression site reveals the importance of the fundamental process of recombination. In this case it will not only accomplish an antigenic switch but also provide for substantial antigenic diversification and hence expansion of the potential antigenic repertoire. The variation in gene family size and sequence, allelic variation or lack thereof among the different Trypanosomatids implies that recombination may be adapted to different ends in these organisms. The availability of the complete T. brucei genome sequence may provide for advancing understanding of the mechanisms and factors that control and mediate antigenic variation, which has resisted elucidation despite intensive analysis. The roles of cysteine peptidases of Leishmania, which were discovered some time ago, in parasite–host interactions and virulence in Leishmanial pathogens is the topic of the article by Mottram et al. These enzymes are important for parasite growth and survival in the animal host and have been extensively studied as virulence factors, potential drug targets and vaccine candidates. Mottram et al. describe the great diversity of Leishmanial cysteine peptidases that have been identified from analysis of the genome sequence of Leishmania. These are clearly complex families of proteins with critical functions. Indeed, the detailed analysis of one cysteine peptidase, CPB, reveals how it influences the parasite– mammalian host interaction. The interaction between Leishmania and its mammalian target cell, the macrophage, is an intimate one and one with critical consequences that influence the immune response to infection, which can lead to disease or to control of the pathogen. Understanding these interactions provides hope for preventing disease. The genome wide expression analysis of asexual intraerythrocytic developmental cycle of P. falciparum described by Llina´ s and DeRisi provides a comprehensive overview of the program of gene expression in the parasite stage that is responsible for disease in humans. The study was made possible by elucidation of the complete P. falciparum genome sequence. The review reveals a striking pattern of temporal regularity of gene expression, which stimulates new research directions. These directions include the relationship between the temporal order of expression and the functions of the genes in order to elucidate the physiological processes, which appear to be occurring in an orderly fashion, especially those related to disease. They also include elucidating the fundamental www.sciencedirect.com
Editorial overview Stuart 361
mechanisms by which the gene expression is regulated at the level of RNA abundance. The availability of the completed P. falciparum genome sequence and expression data will aid the needed studies. Llina´ s and DeRisi indicate that studies into the basic mechanisms of Plasmodium gene regulation have begun and are helping to define gene functions. The very early stage of understanding the transcription apparatus and its regulatory elements reveals that there is a long way to go. Given the rapid spread of resistance to anti-malarial drugs these studies are encouraging as they can help identify novel drug targets and aid in the development of a malaria vaccine. Four of the five pathogens discussed in this issue have an intracellular location in the mammalian host (T. brucei is extracellular), apparently providing for immune evasion. The Apicomplexa have obvious organelles that function in invasion, namely the cell surface, micronemes and rhoptries. While both Plasmodium and Toxoplasma have these organelles there must be differences in the invasion process given the life cycle differences and different target tissues. Until recently, knowledge of the invasion process was essentially limited to microscopic observations entailing cellular reorientation and the participation of actin and myosin. However, substantial progress has been made on the identification of the molecules and processes that function in cellular invasion and the roles of the molecules are being characterized. Dowse and Soldati describe the process of invasion, which is active
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and distinct from phagocytosis. They describe the gliding motility associated with invasion and the actin polymerization, which along with the action of at least one myosin motor protein accompanies invasion. They describe the large repertoire of micronemal proteins, their contributions to and precise roles in invasion as well as their functional redundancy. The focus of the paper is on the calcium regulated microneme proteins that are secreted during the active penetration. Interestingly, the resultant parasitophorous vacuole lacks host cell transmembrane proteins rendering it resistant to acidification and degradative fusion. The micronemal proteins undergo proteolytic processing. The processing events and the responsible proteases are at an early stage of investigation but may be targets for drugs against Toxoplasma and perhaps other Apicomplexan pathogens. As the genomes of these important pathogens are explored, we should anticipate surprises as well as new insights that will be useful for the development of interventions to avert disease. Insights gained on the adaptations that these organisms evolved to cope with the host environment may well form the bases for the development of the interventions.
Acknowledgments Research in the KS lab received support from NIH grants AI14102 and GM42188 for RNA editing and AI45039 and AI40599 for Trypanosomatid genome projects.
Current Opinion in Microbiology 2004, 7:359–361
Ken Stuart Seattle Biomedical Research Institute and Department of Pathobiology, University of Washington, 307 Westlake Ave N, Suite 500, Seattle, WA 98109-5219, USA e-mail: [email protected]
Ken’s research focuses on Trypanosomatids: Trypanosoma brucei, T. cruzi and pathogenic Leishmania species. He studies molecular process and gene function in these organisms and plays a leading role in the genome projects. His lab also studies RNA editing with an emphasis on molecular mechanisms in addition to editing complex composition, structure and specific functions with an eye toward drug development.
www.sciencedirect.com
Now that the sequencing of the genomes of key Apicomplexan and Trypanosomatid pathogens have been completed, attention is turning to using this information for insights into the roles of these genes and how this information can be used to develop strategies to combat diseases. The diploid genomes of three Trypanosomatid pathogens Trypanosoma brucei, T. cruzi and Leishmania major have substantial similarity, as illustrated by conservation of extensive synteny, despite their ancient divergence. However, these genomes have characteristic differences including minichromosomes and variant surface glycoprotein expression sites in T. brucei, distinct families of repeated sequences in T. cruzi, and high allele similarity in L. major. Trypanosomatid genomes are about 20% larger than those of the Apicomplexan genomes and there are more similarities between Trypanosomatid genomes than between Apicomplexan genomes, such as P. falciparum and Toxoplasma gondii, although synteny cannot be analyzed in detail as the T. gondii genome sequence that has been determined currently exists as numerous sizeable subchromosomal sequence segments (contigs). The above five pathogens are transmitted by different vectors (typically insects), cause different diseases, reside in different host tissues, and have different immune evasion strategies. Trypanosoma brucei (group) trypanosomes are transmitted by Tsetse flies, proliferate extracellularly in the blood and cerebral spinal fluid, and evade immune elimination by antigenic variation. They cause African sleeping sickness a neural pathological disease that is invariably fatal if untreated. T. cruzi is transmitted by Reduviid bugs, and proliferates intracellularly, primarily in the cytoplasm of muscle cells, which provides some immune defense, but they also undergo antigenic variation. T. cruzi causes Chagas’ disease with severe cardiac and alimentary pathology that is debilitating and ultimately fatal. Leishmania are transmitted by sand flies and proliferate in the phagolysosomes of macrophages where they avoid immune elimination as a result of interaction with the immune system. Leishmania cause a spectrum of pathologies that range from localized to disseminated cutaneous lesions, to horribly disfiguring muco-cutaneous erosion, to severe liver and spleen pathology associated with visceral disease. Chagas’ disease and Leishmaniasis are chronic diseases with an apparent immune basis for the resultant pathology. African Sleeping Sickness is a more acute disease with an uncertain basis for its pathology. Plasmodium falciparum is transmitted by mosquitoes and multiplies intracellularly, first in the liver and then in erythrocytes. The intracellular environment and antigenic variation evade immune elimination and adhesion-based Current Opinion in Microbiology 2004, 7:359–361
360 Host–microbe interactions: parasites
sequestration evades elimination of infected erythrocytes by the spleen. Malaria is a spectral disease ranging from debilitating to lethal with pathologies including placental inflammation, severe anemia and cerebral disease. Toxoplasma is transmitted by domestic and wild cats by fecal contamination and proliferates intracellularly. While infection is typically asymptomatic and chronic it causes neuropathology especially in immune compromised persons and fetuses. Hence, related pathogenic protozoa have adapted to varied environments and cause different diseases. The availability of their genome sequences presents the opportunity to learn how these pathogens have adapted and cause disease and for devising strategies to prevent or treat these diseases. A key challenge presented by the completed genome sequences is the use or development of methods to determine the functions of the genes, as the function of most are not known. Motyka and Englund describe how RNA interference (RNAi) is used in T. brucei for specific gene silencing in order to identify the functions of genes, to elucidate complex biological processes, and to identify those that are essential for pathogen survival and hence are potential drug targets. They describe its use in combination with tetracycline-regulated systems, which allows analysis of essential genes. RNAi is more convenient and efficient than previous methods for determining gene function and Motyka and Englund describe how RNAi libraries may provide an approach for forward genetic studies in Trypanosomatids. However, RNAi is not a gene function panacea. Motyka and Englund indicate some of the pitfalls with RNAi. RNAi has not been achieved in T. cruzi or Leishmania despite numerous attempts, perhaps reflecting differences in RNA degradation systems and RNA metabolism among Trypanosomatids. Adaptation of RNAi and regulatable promoter systems is at an earlier stage in Plasmodium and Toxoplasma and not yet routine, perhaps reflecting the difficulty of DNA transfection, especially in Plasmodium. Overall, there is a substantial need for high throughput tools for the study of gene function in pathogenic protozoa. Pays et al. describe antigenic variation in T. brucei, a key phenomenon for this pathogen and one with a rich history in molecular parasitology. Their summary of how these parasites evade immune elimination by a combination of transcriptional control and DNA recombination parallels insights stemming from the organization of the T. brucei genome and the need to elucidate molecular processes in this organism. The multiple telomeric expression sites for variant surface glycoprotein (VSG) genes with their associated genes (ESAGs) and the numerous additional VSG genes that are non-telomeric or on the numerous minichromosomes illustrates the adaptation of the T. brucei genome to the process of antigenic variation. Pays et al. describe the expression of only one VSG gene at a time, Current Opinion in Microbiology 2004, 7:359–361
periodic switching of expression to an alternate VSG expression site, and inactivation of VSG expression concomitant with activation of procyclic specific genes, which reveals the early stage of understanding of control of transcription and RNA turnover in all Trypanosomatids. It also implies that there are features specific to antigenic variation. Their descriptions of the periodic recombination that switches and remodels the VSG gene in the expression site reveals the importance of the fundamental process of recombination. In this case it will not only accomplish an antigenic switch but also provide for substantial antigenic diversification and hence expansion of the potential antigenic repertoire. The variation in gene family size and sequence, allelic variation or lack thereof among the different Trypanosomatids implies that recombination may be adapted to different ends in these organisms. The availability of the complete T. brucei genome sequence may provide for advancing understanding of the mechanisms and factors that control and mediate antigenic variation, which has resisted elucidation despite intensive analysis. The roles of cysteine peptidases of Leishmania, which were discovered some time ago, in parasite–host interactions and virulence in Leishmanial pathogens is the topic of the article by Mottram et al. These enzymes are important for parasite growth and survival in the animal host and have been extensively studied as virulence factors, potential drug targets and vaccine candidates. Mottram et al. describe the great diversity of Leishmanial cysteine peptidases that have been identified from analysis of the genome sequence of Leishmania. These are clearly complex families of proteins with critical functions. Indeed, the detailed analysis of one cysteine peptidase, CPB, reveals how it influences the parasite– mammalian host interaction. The interaction between Leishmania and its mammalian target cell, the macrophage, is an intimate one and one with critical consequences that influence the immune response to infection, which can lead to disease or to control of the pathogen. Understanding these interactions provides hope for preventing disease. The genome wide expression analysis of asexual intraerythrocytic developmental cycle of P. falciparum described by Llina´ s and DeRisi provides a comprehensive overview of the program of gene expression in the parasite stage that is responsible for disease in humans. The study was made possible by elucidation of the complete P. falciparum genome sequence. The review reveals a striking pattern of temporal regularity of gene expression, which stimulates new research directions. These directions include the relationship between the temporal order of expression and the functions of the genes in order to elucidate the physiological processes, which appear to be occurring in an orderly fashion, especially those related to disease. They also include elucidating the fundamental www.sciencedirect.com
Editorial overview Stuart 361
mechanisms by which the gene expression is regulated at the level of RNA abundance. The availability of the completed P. falciparum genome sequence and expression data will aid the needed studies. Llina´ s and DeRisi indicate that studies into the basic mechanisms of Plasmodium gene regulation have begun and are helping to define gene functions. The very early stage of understanding the transcription apparatus and its regulatory elements reveals that there is a long way to go. Given the rapid spread of resistance to anti-malarial drugs these studies are encouraging as they can help identify novel drug targets and aid in the development of a malaria vaccine. Four of the five pathogens discussed in this issue have an intracellular location in the mammalian host (T. brucei is extracellular), apparently providing for immune evasion. The Apicomplexa have obvious organelles that function in invasion, namely the cell surface, micronemes and rhoptries. While both Plasmodium and Toxoplasma have these organelles there must be differences in the invasion process given the life cycle differences and different target tissues. Until recently, knowledge of the invasion process was essentially limited to microscopic observations entailing cellular reorientation and the participation of actin and myosin. However, substantial progress has been made on the identification of the molecules and processes that function in cellular invasion and the roles of the molecules are being characterized. Dowse and Soldati describe the process of invasion, which is active
www.sciencedirect.com
and distinct from phagocytosis. They describe the gliding motility associated with invasion and the actin polymerization, which along with the action of at least one myosin motor protein accompanies invasion. They describe the large repertoire of micronemal proteins, their contributions to and precise roles in invasion as well as their functional redundancy. The focus of the paper is on the calcium regulated microneme proteins that are secreted during the active penetration. Interestingly, the resultant parasitophorous vacuole lacks host cell transmembrane proteins rendering it resistant to acidification and degradative fusion. The micronemal proteins undergo proteolytic processing. The processing events and the responsible proteases are at an early stage of investigation but may be targets for drugs against Toxoplasma and perhaps other Apicomplexan pathogens. As the genomes of these important pathogens are explored, we should anticipate surprises as well as new insights that will be useful for the development of interventions to avert disease. Insights gained on the adaptations that these organisms evolved to cope with the host environment may well form the bases for the development of the interventions.
Acknowledgments Research in the KS lab received support from NIH grants AI14102 and GM42188 for RNA editing and AI45039 and AI40599 for Trypanosomatid genome projects.
Current Opinion in Microbiology 2004, 7:359–361