TANDEMLY REPEATED GENES ENCODE NUCLEOSIDE TRIPHOSPHATE HYDROLASE ISOFORMS SECRETED INTO THE PARASITOPHOROUS VACUOLE OF TOXOPLASMA-GONDII

The obligate intracellular parasite Toxoplasma gondii produces a nucle oside triphosphate hydrolase (NTPase) (nucleoside-triphosphatase, EC 3 .6.1.15) activable by dithiol-containing compounds. We have isolated t he genomic DNA for the NTPase from the RH strain of Toxoplasma and det ermined the nucleotide sequence of three tandemly arranged open readin g frames termed NTP1, NTP2, and NTP3. We have also isolated and sequen ced cDNAs for NTP1 and NTP3; no cDNA for NTP2 was obtained. The two cD NA clones encode proteins that are more than 97% identical at the amin o acid level but significantly differ within two small domains, indica ting the presence of NTPase isoforms. Both possess N-terminal signal s equences and two regions with partial homology to certain known ATP bi nding motifs: the glycine-rich loop common to many ATP binding protein s and the beta-phosphate binding domain found in the hexokinase-actin- hsp70 family. Antiserum against a NTP1-fusion protein immunoprecipitat ed NTPase activity from extracellular parasites that was increased in activity by treatment with dithiothreitol, confirming the identity of the cloned genes. By immunofluorescence, the NTPase is located in vesi cular structures within the parasite, and in infected cells it is secr eted into the vacuolar space and becomes partially associated with the parasitophorous vacuolar membrane. Since the vacuolar membrane is fre ely permeable to small molecules of <1300 Da, host cell ATP may serve as a substrate for the NTPase and supply the energy for parasite-direc ted processes in the vacuolar space.