Developmental Biology

The developmental biology of Toxoplasma has been well described in morphological terms but relatively little is understood about the detailed processes involved. We and others have focused on the asexual developmental cycle, i.e., the interconversion between the tachyzoite and bradyzoite stages. We focus on this rather than the sexual cycle because of the lack of an in vitro system for the latter (to date, the sexual cycle has only been observed in the intestine of cats).

The questions we are asking regarding Toxoplasma development are:

  1. What are the triggers that stimulate the developmental switch between tachyzoites and bradyzoites?
  2. What signaling events then ensue and what changes in the parasite result?
  3. And, finally, what is the biological or biochemical role of those changes (e.g., in promoting a difference in structure, metabolism or ability to persist and be transmitted)?

One of the first approaches we took was, in collaboration with many investigators but especially David Sibley, to look at a large library of ESTs and compare their frequency in bradyzoites and tachyzoites (Manger et al, 1998). This was a brute force approach but it yielded a large number of interesting, new genes that we are pursuing to this day using gene knock-outs and the like (e.g. some novel bradyzoite-specific surface antigens like SRS9; Kim et al., 2007).

The second major approach we took was to devise and use genetic screens and selections for mutants that are disrupted in development (e.g., Knoll et al., 1998; Vanchinathan et al., 2005). One of the genes we identified by this approach (twice, independently!) looks to be a RNA modifying enzyme. The RNA target of this enzyme and how its modification drives differentiation are current mysteries.

The third major strategy to study differentiation has been microarray analysis. We have exploited our proximity to some of the labs that have pioneered this technique to produce microarrays for Toxoplasma. For the first generation arrays, we used the set of ~4000 bradyzoite ESTs described above. We have used these to study changes in transcript abundance during development from tachyzoites to bradyzoites under different conditions and using different parasite lines and mutants (Cleary, et al. 2001). The results revealed several genes that we did not previously know to be developmentally regulated as well as information on which are affected early vs. late during the differentiation process. This has helped us to build a pathway or cascade of events that go on during differentiation.

One of the limitations of conventional microarray analysis is that it measures changes in abundance but without giving clues to whether these are a result of changes in the rate of synthesis or decay. To overcome this, Mike Cleary developed a protocol that makes good use of a pyrimidine salvage pathway in Toxoplasma to biosynthetically tag RNA in a way that it can be efficiently harvested from a pool of conventional RNA. In this way, he could do pulse/chase experiments and analyze synthesis and decay rates across the transcriptome on microarrays (Cleary et al., 2005). We are anxious to repeat these analyses with the Affymetrix arrays engineered by the Roos lab.

A further method that we have been using to ask questions about bradyzoite development again exploits the technical wizardry of some of our Stanford colleagues, in this case Chris Contag and his in vivo imaging. This approach required us to first engineer the parasite to express firefly luciferase. The resulting parasites, when exposed to luciferin, emit light. This occurs in sufficient quantity that foci of infection can be detected using sensitive cameras and photographing anesthetized animals in the dark (Saeij et al., 2005). Since the animals do not need to be sacrificed in order to be examined the method has two substantial advantages. First, we generate a time-course of data for each animal, avoiding the pitfalls of averaging data from multiple mice that are different for each time-point. Second, and in some ways even more importantly, it also allows us to significantly reduce the number of animals we must use to obtain conclusive results.

Recently, we have refined this analysis by engineering expression of the luciferase to be under control of a bradyzoite-specific promoter and seen that, as expected, the light signal does not come up until the infection has passed from the acute to chronic stages (Saeij et al., 2008). We are now using this to investigate infection in the brain.

Bradyzoites are difficult to study in vivo, because they are not abundant (a typical mouse might have only ~2000 tissue cysts (each containing maybe 1000 bradyzoites) per brain. We are testing the hypothesis that the bradyzoites are not totally passive occupiers of the infected host cell; instead, we propose that they maintain sufficient dialogue with the infected cell to allow persistence. We began by looking at bradyzoite-infected cells in tissue culture and saw that this dialogue is at the level of a whisper (especially compared to the shouting that goes on between a tachyzoite and its host cell; Fouts and Boothroyd, 2007). We are now working on ways to achieve the sensitivity necessary to examine this same process in the infected animal.