Saeij lab:Research

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==Overview==
==Overview==
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The primary research interest of our laboratory is to understand how intracellular parasites exploit and manipulate the host cells in which they live to ensure their survival, replication and transmission and hence their success. Our experimental model is ''Toxoplasma gondii'', considered the most successful protozoan parasite of warm-blooded animals. The focus of my laboratory over the last years has been to identify genes of ''Toxoplasma'' that determine virulence, host genes and pathways that determine resistance/susceptibility, and to characterize their specific interactions. To achieve this we use a combination of genomics, biochemistry, genetics, microscopy, immunology and computational tools.
The primary research interest of our laboratory is to understand how intracellular parasites exploit and manipulate the host cells in which they live to ensure their survival, replication and transmission and hence their success. Our experimental model is ''Toxoplasma gondii'', considered the most successful protozoan parasite of warm-blooded animals. The focus of my laboratory over the last years has been to identify genes of ''Toxoplasma'' that determine virulence, host genes and pathways that determine resistance/susceptibility, and to characterize their specific interactions. To achieve this we use a combination of genomics, biochemistry, genetics, microscopy, immunology and computational tools.
==''Toxoplasma'' biology, life cycle and disease==
==''Toxoplasma'' biology, life cycle and disease==
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''Toxoplasma gondii'' is an obligate intracellular parasite capable of infecting virtually any warm-blooded animal. In humans, ''Toxoplasma'' infections are widespread (~between 20-85% of humans are chronically infected, depending on the region) and can lead to severe disease (toxoplasmosis) in individuals with an immature (fetus) or suppressed immune system (AIDS patients). The life cycle of ''Toxoplasma'' is complex and includes both sexual and asexual stages. While the sexual cycle is limited to the gut of felines, the asexual cycle can occur in a wide range of hosts and has two major forms: the rapidly growing tachyzoite and the slowly growing encysted bradyzoite. Bradyzoite cysts can persist within the infected tissue for the life of the host, hidden from the immune system and anti-parasitic drugs. In addition to studying ''Toxoplasma'' biology in order to develop anti-''Toxoplasma'' agents, researchers are studying ''Toxoplasma'' as an important model of the pathogenesis of other disease-causing Apicomplexan parasites such as ''Plasmodium'' (malaria), and ''Cryptosporidium parvum'', another opportunistic pathogen associated with AIDS.
''Toxoplasma gondii'' is an obligate intracellular parasite capable of infecting virtually any warm-blooded animal. In humans, ''Toxoplasma'' infections are widespread (~between 20-85% of humans are chronically infected, depending on the region) and can lead to severe disease (toxoplasmosis) in individuals with an immature (fetus) or suppressed immune system (AIDS patients). The life cycle of ''Toxoplasma'' is complex and includes both sexual and asexual stages. While the sexual cycle is limited to the gut of felines, the asexual cycle can occur in a wide range of hosts and has two major forms: the rapidly growing tachyzoite and the slowly growing encysted bradyzoite. Bradyzoite cysts can persist within the infected tissue for the life of the host, hidden from the immune system and anti-parasitic drugs. In addition to studying ''Toxoplasma'' biology in order to develop anti-''Toxoplasma'' agents, researchers are studying ''Toxoplasma'' as an important model of the pathogenesis of other disease-causing Apicomplexan parasites such as ''Plasmodium'' (malaria), and ''Cryptosporidium parvum'', another opportunistic pathogen associated with AIDS.
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We have also used linkage mapping to identify Toxoplasma loci involved in modulation of host gene expression. Our initial analysis of the data identified that most of the strain-specific differences in the modulation of host cell transcription are mediated by two genes ROP16 and GRA15. Upon invasion by the parasite, ROP16 is injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signaling pathways (Saeij et al. 2007). GRA15, encodes a dense granule protein, which affects activation of NF-kB, a transcription factor involved in the activation of the inflammatory response (Rosowski et al. 2011). Different Toxoplasma strains (e.g. type I, II and III) have different alleles of ROP16 and GRA15. Our results showed that the precise allelic combination of ROP16 and GRA15 determines how distinct Toxoplasma strains modulate the host immune response (Jensen et al. 2011). For example, type II strains can kill susceptible mice because they induce a hyper-inflammatory response that damages host tissue (Jensen et al. 2011). We are now combining genetic and biochemical approaches to identify host factors that interact with GRA15. Furthermore, we are continuing to investigate the host genes and pathways that mediate ROP16’s strong anti-inflammatory effects.  
We have also used linkage mapping to identify Toxoplasma loci involved in modulation of host gene expression. Our initial analysis of the data identified that most of the strain-specific differences in the modulation of host cell transcription are mediated by two genes ROP16 and GRA15. Upon invasion by the parasite, ROP16 is injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signaling pathways (Saeij et al. 2007). GRA15, encodes a dense granule protein, which affects activation of NF-kB, a transcription factor involved in the activation of the inflammatory response (Rosowski et al. 2011). Different Toxoplasma strains (e.g. type I, II and III) have different alleles of ROP16 and GRA15. Our results showed that the precise allelic combination of ROP16 and GRA15 determines how distinct Toxoplasma strains modulate the host immune response (Jensen et al. 2011). For example, type II strains can kill susceptible mice because they induce a hyper-inflammatory response that damages host tissue (Jensen et al. 2011). We are now combining genetic and biochemical approaches to identify host factors that interact with GRA15. Furthermore, we are continuing to investigate the host genes and pathways that mediate ROP16’s strong anti-inflammatory effects.  
Overall, these results suggest that analogous to bacterial pathogens and their secretion system, it seems that Toxoplasma can secrete effectors into host cells to subvert host-cell signaling pathways. ROP16, ROP18 and ROP5 are members of a large protein family suggesting that Toxoplasma has a wide arsenal of effectors to modulate diverse host cell signaling pathways. The detailed characterization of these effectors represents a major focus of the laboratory.
Overall, these results suggest that analogous to bacterial pathogens and their secretion system, it seems that Toxoplasma can secrete effectors into host cells to subvert host-cell signaling pathways. ROP16, ROP18 and ROP5 are members of a large protein family suggesting that Toxoplasma has a wide arsenal of effectors to modulate diverse host cell signaling pathways. The detailed characterization of these effectors represents a major focus of the laboratory.
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==Identification of host genes and pathways that influence resistance or susceptibility to ''Toxoplasma''==
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In addition to parasite strain differences, host genetic differences also determine the outcome of toxoplasmosis. We found that inflammatory pathways are differentially regulated in macrophages from susceptible and resistant mice. Using a genetic approach we have identified the mouse genomic regions that determine these differences and we are now in the position to identify the exact causative genes.
==Projects the lab is currently working on==
==Projects the lab is currently working on==
     1. Pathogenesis: what properties make certain strains more virulent than others and what proteins are involved?
     1. Pathogenesis: what properties make certain strains more virulent than others and what proteins are involved?
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     2. How does Toxoplasma co-opt host-cell gene expression and how does this differ between strains?
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     2. How does ''Toxoplasma'' co-opt host-cell gene expression and how does this differ between strains?
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     3. How does Toxoplasma modulate the NF-κB signaling pathway?
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     3. How does ''Toxoplasma'' modulate the NF-κB signaling pathway?
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    4. How can we use ''Toxoplasma'' whole genome data to understand its population biology?
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    5. What are the host genes and pathways that influence resistance or susceptibility to ''Toxoplasma''?
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Revision as of 12:20, 26 August 2011

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Contents

Overview

The primary research interest of our laboratory is to understand how intracellular parasites exploit and manipulate the host cells in which they live to ensure their survival, replication and transmission and hence their success. Our experimental model is Toxoplasma gondii, considered the most successful protozoan parasite of warm-blooded animals. The focus of my laboratory over the last years has been to identify genes of Toxoplasma that determine virulence, host genes and pathways that determine resistance/susceptibility, and to characterize their specific interactions. To achieve this we use a combination of genomics, biochemistry, genetics, microscopy, immunology and computational tools.

Toxoplasma biology, life cycle and disease

Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually any warm-blooded animal. In humans, Toxoplasma infections are widespread (~between 20-85% of humans are chronically infected, depending on the region) and can lead to severe disease (toxoplasmosis) in individuals with an immature (fetus) or suppressed immune system (AIDS patients). The life cycle of Toxoplasma is complex and includes both sexual and asexual stages. While the sexual cycle is limited to the gut of felines, the asexual cycle can occur in a wide range of hosts and has two major forms: the rapidly growing tachyzoite and the slowly growing encysted bradyzoite. Bradyzoite cysts can persist within the infected tissue for the life of the host, hidden from the immune system and anti-parasitic drugs. In addition to studying Toxoplasma biology in order to develop anti-Toxoplasma agents, researchers are studying Toxoplasma as an important model of the pathogenesis of other disease-causing Apicomplexan parasites such as Plasmodium (malaria), and Cryptosporidium parvum, another opportunistic pathogen associated with AIDS.

Toxoplasma population biology

The majority of Toxoplasma isolates from Europe and North America belong to three distinct clonal lines, referred to as types I, II and III. The three types have been shown to differ widely in a number of phenotypes in mice such as virulence, persistence, migratory capacity, attraction of different cell-types and induction of cytokine expression. Recent data suggest that such differences may also exist in human infection. In South America many other Toxoplasma strains exist, some of which can cause severe disease even in healthy individuals. One of our long-term goals is to understand how distinct Toxoplasma strains differ in their ability to cause disease in humans. Determining how particular Toxoplasma genotypes differ in their capacity to induce pathology in a particular animal species could enable prediction of the outcome of infection based on the genotype of the infecting organism. For example, not all seropositive AIDS patients develop toxoplasmic encephalitis; the ones that do might be infected with a particular subset of parasite strains. Similarly, seroconversion during pregnancy does not always lead to infection of the fetus; this might be a result of variability in the ability of different strains to cross the placental barrier. We have sequenced whole genomes of strains with different phenotypes and compared the differences and commonalities. This approach allowed us to correlate genotype with phenotype and have led to the identification of Toxoplasma loci and genes that affect fitness, clonality, virulence and modulation of host signaling pathways.

Identification of Toxoplasma virulence genes

Toxoplasma has a haploid genome with 14 chromosomes that total 65 Mbp in size, representing ~7900 genes (http://www.toxodb.org ). Classical genetic crosses can be performed in Toxoplasma and several experimental crosses have been used to generate genetic linkage maps. To identify the Toxoplasma loci involved in virulence, we mapped virulence in F1 progeny derived from crosses between type II and type III strains. Five virulence loci were thus identified, and for three of these, genetic complementation showed that protein kinases (ROP18 and ROP16, respectively) and pseudokinases (ROP5) are the key molecules (Saeij et al. 2006, Saeij et al. 2007, Reese et al. 2011). These are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. We have also used linkage mapping to identify Toxoplasma loci involved in modulation of host gene expression. Our initial analysis of the data identified that most of the strain-specific differences in the modulation of host cell transcription are mediated by two genes ROP16 and GRA15. Upon invasion by the parasite, ROP16 is injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signaling pathways (Saeij et al. 2007). GRA15, encodes a dense granule protein, which affects activation of NF-kB, a transcription factor involved in the activation of the inflammatory response (Rosowski et al. 2011). Different Toxoplasma strains (e.g. type I, II and III) have different alleles of ROP16 and GRA15. Our results showed that the precise allelic combination of ROP16 and GRA15 determines how distinct Toxoplasma strains modulate the host immune response (Jensen et al. 2011). For example, type II strains can kill susceptible mice because they induce a hyper-inflammatory response that damages host tissue (Jensen et al. 2011). We are now combining genetic and biochemical approaches to identify host factors that interact with GRA15. Furthermore, we are continuing to investigate the host genes and pathways that mediate ROP16’s strong anti-inflammatory effects. Overall, these results suggest that analogous to bacterial pathogens and their secretion system, it seems that Toxoplasma can secrete effectors into host cells to subvert host-cell signaling pathways. ROP16, ROP18 and ROP5 are members of a large protein family suggesting that Toxoplasma has a wide arsenal of effectors to modulate diverse host cell signaling pathways. The detailed characterization of these effectors represents a major focus of the laboratory.

Identification of host genes and pathways that influence resistance or susceptibility to Toxoplasma

In addition to parasite strain differences, host genetic differences also determine the outcome of toxoplasmosis. We found that inflammatory pathways are differentially regulated in macrophages from susceptible and resistant mice. Using a genetic approach we have identified the mouse genomic regions that determine these differences and we are now in the position to identify the exact causative genes.

Projects the lab is currently working on

   1. Pathogenesis: what properties make certain strains more virulent than others and what proteins are involved?
   2. How does Toxoplasma co-opt host-cell gene expression and how does this differ between strains?
   3. How does Toxoplasma modulate the NF-κB signaling pathway?
   4. How can we use Toxoplasma whole genome data to understand its population biology?
   5. What are the host genes and pathways that influence resistance or susceptibility to Toxoplasma?



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