SPOROZOITES OF TOXOPLASMA-GONDII LACK DENSE-GRANULE PROTEIN GRA3 AND FORM A UNIQUE PARASITOPHOROUS VACUOLE

The invasion of host cells by sporozoites of Toxoplasma gondii leads t o the formation of parasitophorous vacuoles that are distinctly differ ent from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. C onsistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of t achyzoites, was not detected by indirect immunofluorescence in sporozo ite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts, Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule prot eins, revealed numerous other antigenic differences between sporozoite s and tachyzoites. Cell cultures inoculated with sporozoites were moni tored at various intervals for the expression of GRA3 and the developm entally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoit e-infected cells that were positive for GRA3 and SAG1, Thus, these dat a indicate that sporozoites and their interaction with host cells diff er substantially from tachyzoites and the expression of tachyzoite-spe cific proteins is likely required for parasite replication.