TOXOPLASMA-GONDII SPOROZOITES FORM A TRANSIENT PARASITOPHOROUS VACUOLE THAT IS IMPERMEABLE AND CONTAINS ONLY A SUBSET OF DENSE-GRANULE PROTEINS

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invas ion and the second (PV2) 18 to 24 h postinoculation. PV1 is structural ly distinctive due to its large size, yet it lacks a tubulovesicular n etwork (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, a nd M. W. White, Mel. Biochem. Parasitol. 75:75-86, 1995), Confirming t he finding that sporozoites have a different electron-dense-granule co mposition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTP ase first appears 12 h postinfection. Other tachyzoite dense-granule p roteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst ext racts, and antibodies against these proteins stained granules In the s porozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA 1, GRA2, or GRA4 during PV1 formation. Even after NTPase Induction, th ese proteins were retained within cytoplasmic granules rather than bei ng secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular mas ses as small as 330 Da, indicating that PV1 lacks channels through whi ch molecules can pass from the host cytoplasm into the vacuole. By con trast, lucifer yellow rapidly diffused into PV2, demonstrating the pre sence of molecular channels. These studies indicate that PV1 and PV2 a re morphologically, immunologically, and functionally distinct, and th at PV2 appears to be identical to the tachyzoite vacuole. The inaccess ibility of PV1 to host cell nutrients may explain why parasite replica tion does not occur in this vacuole.