The T. gondii tachyzoite cell cycle.   [MS97 PowerPoint]   Exploiting new molecular tools and synchronous growth, we have now characterized the major features of the tachyzoite cell cycle.  Tachyzoites divide using a cycle comprised of a G1 (60%), S phase (30%) and mitosis/cytokinesis (10%).  Cytokinesis is not a separate phase in these parasites as it overlaps late S and mitosis.  There is very little pause between S and mitosis, suggesting G2 may be absent.  S-phase is unusual in that the DNA distributions of parasites in S are distinctly bimodal (Radke, Striepen, Guerini et al., 2001).  As part of this effort, we discovered tachyzoites express two distinct DNA clamps (proliferating-cell-nuclear-antigens)--where normally there is one.  While it is too soon to suggest a connection to bimodal S-phase, two DNA clamps in one cell suggests the apicomplexan DNA synthetic machinery is different from other eukaryotes (Guerini, et. al., 2000).  This summer we successfully deleted the TgPCNA2 gene in RH tachyzoites (Guerini and White, 2001) and are now characterizing the phenotype of the knockout mutant.

Whereas G2 parasites are absent from tachyzoite populations, during differentiation we readily detect pre-mitotic parasites with a 2N DNA content demonstrating that a G2 period is associated with development.  FACS analysis of these populations shows that G2 parasites are intermediate in the pathway (positive for tachyzoite and bradyzoite markers) while mature bradyzoites are haploid (Guerini, Jerome, White, 2001).  These recent data, added to what we know about the tachyzoite cell cycle, have led to the following working model for tachyzoite-to-bradyzoite stage differentiation.

                We propose that differentiation is triggered by a change in the chromosome cycle which is manifest by a late-S/G2 pause.  Parasites emerging from this pause are committed to differentiate into bradyzoites.  This process is likely stochastic, as suggested for differentiation of Theileria schizonts, thus, the probability of differentation is dependent on a change in the steady-state level of a factor(s) above (or below) a threshold margin.  Consequently, differentiation is not synchronous as one or more cycles may be required to achieve the thereshold level.  Laboratory adapted strains, such as RH, have a fast cell cycle with a very short G2, and therefore, a lowered potential to differentiate, and differentation does not occur if growth is blocked because cell cycle progression is required.  A prediction of this model is that any condition that slows DNA synthesis appropriately has the potential to alter late-S/G2 and induce bradyzoite differentiation.  This would explain why a variety of drugs with different modes of action induce differentiation in T. gondii.  Understanding the mechanisms that underly this working model is a major focus of the lab.