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 (~50% of all people are infected) 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 bradyzoite. Tachyzoites can invade virtually any nucleated cell, replicate and exit quickly, thereby disseminating throughout the infected host. In most cases the infection is controlled by the immune system, although some tachyzoites can evade elimination by switching to the dormant bradyzoite form contained within a walled cyst. Bradyzoite cysts can persist within the infected tissue for the life of the host, hidden from the immune system and anti-parasitic drugs. Ingesting cysts in undercooked meat is thought to be one of the main sources of human infection.
In immunocompromised individuals such as AIDS, leukemia and lymphoma patients, new infections or rupture of pre-existing cysts can lead to toxoplasmic encephalitis. Additionally, in congenital infections, the disease can lead to severe neurological problems or even death. The most effective drugs are poorly tolerated and do not affect the cysts therefore new drugs and therapies are needed.
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, and Cryptosporidium parvum, another opportunistic pathogen associated with AIDS. Many questions that have proven difficult to study in other Apicomplexans such as gene regulation and drug resistance are attainable in Toxoplasma. In addition, because of its transmission to humans through contaminated food and water sources, Toxoplasma is considered a potential bioterrorism agent by the CDC.
Toxoplasma population biology
The majority of Toxoplasma gondii isolates identified to date within 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. 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.
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). The whole genome of a type I, II and III strain has been sequenced and this information has been used to construct Toxoplasma microarrays. Classical genetic crosses can be performed in Toxoplasma and several experimental crosses have been used to generate genetic linkage maps for the chromosomes.
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 two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule (Saeij et al. 2006) Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.
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 some of the strain-specific differences in the modulation of host cell transcription are mediated by ROP16. Upon invasion by the parasite, this polymorphic protein 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).
These results suggest that analogous to bacterial pathogens and their secretion system, it seems that Toxoplasma can secrete protein kinases into host cells to subvert host-cell signaling pathways. ROP16 and ROP18 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.
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?