back to main

 

 

 

 

 

 

 

 

 

 

Research interest
     
 

 

Pathogenesis caused by Toxoplasma gondii coincides with going through the lytic cycle, either in a primary infection, or as the result of a relapsing chronic infection. This stage is characterized by repeated host-cell invasion, establishment of the parasitophorous vacuole, and replication within the host cell. As such parasite proliferation can potentially be inhibited by intervention at each of these steps. Our goal is to enhance the knowledge about the specific biology of parasite invasion as well as its replication. These processes are at the core of parasitic behavior and are interesting from a biological perspective as well. Both problems are being approached with the power of genetics. The advantage of this approach is the un-biased identification of genes involved in these processes.

 
     
Toxoplasma as a genetic model parasite
 
 
  In contrast to most other apicomplexan parasites, Toxoplasma is easily cultured in the lab; it grows in virtually any cell type and has a short generation time. Transient and stable transfections are easily established. To enable genetic experiments an extensive set of tools has been developed over the past decade. Among these are multiple selectable markers (positive and negative), promoters of differential strength, expression of green fluorescent protein (GFP) and color variants as well a other reporters (B-galactosidase, Luciferase, B-lactamase). Random genomic tagging is highly efficient since plasmid DNA integrates at high frequency at random in the genome. Homologous recombination to create gene knock-outs or allelelic replacement is aslo possible. Most genes are present as single copy genes in a haploid genome. The genome sequencing project is in the late stages of annotation (see www.ToxoDB.org). Many other Apicomplexa have been, or are being sequenced supplying an extensive additional data set. One aspect shared by these organisms is that 60% of their predicted open reading frames have no homologs outside the phylum Apicomplexa, likely reflecting the parastic life-style requiring dedicated machinery.  
     
Growth mutants
     
 

The genes that play essential roles in the parasite's biology are hard to identify through biochemical studies. However, making random genetic mutants and screen them for specific biological defects overcomes this problem. Subsequently the mutated gene can be identified and studied in further detail. One problem with making mutants in essential genes is that it results in death parasites. To circumvent this we made a pool of conditional growth mutants through chemical mutagenesis; all mutants grow at low temperature (35°C) but display a growth defect at high temperature (40°C).

In order to make this growth screen high throughput, parasite lines were cloned in 384-well plates and subsequently duplicated. Growth could be screened on a fluorescent plate reader because a transgenic parasite expressing cytoplasmic YFP was used to generate mutants. The Figure on the left shows the increase in fluorescence signal upon increasing infection dose in a single well of a 384-well plate. Images show the distribution of parasite plaques in the read wells. See paper abstract.

 
     
Invasion mutants
     
 

The temperature-senstive mutants were screened for defects in host-cell invasion. A number of these mutants was identified. One of the mutants had an in addition to an invasion defect also an egress defect; the parasited divided fine in the vacuole but when it was time to come out and invade neighboring cells they remained within the vacuole. Egress is considered the first step in invasion since mechanistically it looks like reverse invasion and the parasite likely uses (part of) the same machinery. Also, egress means activation of the parasite's motility machinery and apparently our mutant is not motile anymore.

To delineate this defect, parasite egress was artifically stimulated by addition of a calcium ionophore, forcing even small vacuoles to egress. As shown in the picture on the right, mutants do not egress anymore (parasites shown in green). This indicates the problem likely lies in the motility machinery rather than in the signal transduction pathway activating motility. We developed a powerful genetic screen to isolate mutants in the egress/invasion pathway, described in the following abstracts: Eidell et al and Coleman et al

 
 
 
Identification of the mutated gene
     
  To identify the gene mutated, we employ genetic complementation. Various DNA libraries from wild-type parasites were made and transfected into the mutant parasite. Successul complementation is screened by growth restoration at 40°C; only if the mutant picks up an intact copy of the mutated gene from the library will it be able to grow. Subsequently, the library insert supplying complemtentation is identified, isolated and re-confirmed. To this end we have generated of a cosmid library with ~40 kb insert length to cover the large open reading frames present in the genome. This library successfully complemented several TS-mutants. The latest development is the application of whole genome sequencing to identify point mutations in mutants (see abstract of our sequencing paper).  
     
Parasite replication
     
  Our second interest is parasite replication, as this process is significantly different from the host cell. The parasite replicates by an internal budding process, where two new daughter parasites are assembled within the mother. The complex cytoskeleton, which also anchors the motility machinery, serves as a scaffold for daughter assembly. The cytoskeleton is compsed of three basic elements: flattened membrane vesicles (cisternae) in complex with a set of microtubuli and a meshwork of intermediate filaments. This structure is known as the inner membrane complex. Tagging of one of the IMC filaments with YFP allowed the study of parasite assembly in real time by time-lapse video microscopy as shown below.  
     
 
 
  The image panel above shows a set of time-lapse frames of a newly identified inner membrane complex (IMC3) protein fused to YFP throughout cell division. At t=0 two parasites with the first signs of forming daughters are visible, which progressively enlarge over time to emerge as new parasitess towards the last frame.  
 
     
 

In the past we identified a protein highlighting several cytoskleletal structure involved in division. This protein, TgMORN1, is localizing to two distinct structures: the spindle pole and the apical and posterior extremities of the IMC. Although these appear to be quite different at first sight, both structures are at a crossroad of membrane and cytoskelelton (as are MORN proteins in other systems). The spindle pole composed of microtubuli is in T. gondii embedded in the nuclear envelope, which is maintained during mitosis . The IMC extremties is where the membrane meets the intermediate filaments. Check here for an abstract of our MORN1 and conditional MORN1 knock-down paper.

 
 
  In addition we have chararcterized the whole IMC protein family, which highlighted a highly dynamic, sequential assembly process of the IMC proteins into the daughter parasite cytoskeleton scaffold. In addition, this study identified the unexpected complesity of the basal complex of the parasite, which is a current reserach focus in our lab. Check here for an abstract of our IMC paper.  
     
  A 3D deconvolution of two dividing parasites is shown below. The DNA is visualized with an H2B-RFP protein; TgMORN-YFP is shown in green. The green band halfway across the red dividing nucleus is the posterior end of the newly forming IMC. The green spot sitting on top of each red lobe is the spindle pole. The spot halfway the spindle pole and the curve shaped anterior location of TgMORN1 at the anterior side is thought to be the centrosome. Furthermore there is an uncharacterized green TgMORN1 spot at the posterior end of the nuclear envelope.  
     
 

 

Time lapse fluorescent microscopy through division.

TgMORN-YFP in green, nucleus in red (H2B-mRFP). Note the ring formed by TgMORN1 though which the nucleus is split.

 

   
top scanning electron microscopy image of parasites with courtesy from David Morrisson.
     
 
back to main