Toxoplasma gondii: Characterization of Temperature-Sensitive Tachyzoite Cell Cycle Mutants

Abstract

Radke, J. R., Guerini, M. N., and White, M. W. 2000. Toxoplasma gondii: Characterization of temperature-sensitive tachyzoite cell cycle mutants. Experimental Parasitology96, 168–177. We mutagenized RHδhxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40°C. These isolates grew normally at 34°C, but showed variable growth at temperatures between 34 and 39°C. The inability to grow at 40°C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (≅ 85%) than that observed for RHδhxgprt (≅ 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40°C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40°C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters—indicating that the initiation of daughter formation can occur in the absence of nuclear division.