Expression of herpes simplex virus thymidine kinase in Toxoplasma gondii attenuates tachyzoite virulence in mice

We tested the virulence in mice of Toxaplasma gondii RH strain tachyzoites containing various copies of the chloramphenicol acetyl transferase-herpes simplex virus thymidine kinase fusion sequence (CAT-HSTK). Tachyzoite isola tes containing greater than or equal to five copies of the fusion sequence were not lethal to female CD-1 outbred or BALB/c inbred mice, at doses up t o 10(6) parasites, while the parental RH strain caused 100% mortality withi n 2 weeks at doses as low as 10 parasites, Mice infected with CTK11, an iso late containing five copies of Be fusion sequence, showed no overt symptoms of disease and were protected from lethal challenge with the parental RH s train. The CTK11 isolate showed no difference in growth fate, the rate of h ost cell invasion, or extracellular viability in cell culture compared with parental RH parasites, demonstrating that the CAT-HSTK fusion protein does not affect the normal viability of this isolate. B11, B11C, and D1 isolate s contained one or two copies of the CAT-HSTK: coding sequence, were not se nsitive to thymidine in cell culture, and caused 100% mortality in CD-1 out bred mice in <12 days. A fourth isolate, D1C, contained seven copies of the CAT-HSTK fusion sequence and was sensitive to exogenous thymidine (50% inh ibitory concentration = 5.5 mu M). Mice infected with D1C showed no symptom s of disease and survived beyond 90 days, thus correlating increased CAT-HS TK gene copies with thymidine sensitivity in cell culture and attenuated vi rulence in mice. BALB/c mice containing a targeted disruption of the gamma interferon gene (gko) were also susceptible to infection with CTK11 parasit es but could be rescued by administration of subcutaneous thymidine once ea ch day for 5 or 10 days following infection. These results suggest that the attenuation of CAT-HSTK+ isolates in mice is directly due to active thymid ine kinase that likely alters the pyrimidine biosynthetic pathway in these parasites.