Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins

Abstract

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5 nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being 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 masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.

Identification of a sporozoite‐specific member of the Toxoplasma SAG superfamily via genetic complementation

“…1C1*). As we have shown previously, replicating parasites in sporozoite‐infected cells reside in a vacuole that is structurally distinct from the primary vacuole formed during sporozoite invasion (Tilley et al ., 1997). It is intriguing that sporozoite‐specific proteins are absent from the parasitophorous vacuole space and membrane in Fig.…”

“…Note staining of sporozoite surface, cytoplasm, and parasitophorous vacuole 2 h post invasion which is lost at later times. CPA cells are shown here as the relatively larger vacuole size formed (Tilley et al ., 1997) in these cells readily demonstrate secreted antigen(s), similar results are observed using HFF monolayers (data not shown). Molecular mass markers (kDa) are indicated to the left of (A,B).…”

“…It is intriguing that sporozoite‐specific proteins are absent from the parasitophorous vacuole space and membrane in Fig. 1C2* (24 h post sporozoite infection) indicating that sporozoite proteins are secreted only into the primary invasion vacuole (PV1) and not into secondary vacuoles (PV2) (Tilley et al ., 1997). The bulk of sporozoite antigens were downregulated in CPA cells (Fig.…”

“…2A; lane a versus b). The presence of SAG1 (band 2) in VEG sporozoite protein extracts was surprising as we and others had reported sporozoites lacked this antigen (Kasper et al ., 1984; Tilley et al ., 1997). The sensitivity of this antigen to fixation (M. E. Jerome and M. W. White, unpubl.)…”

Hammondia hammondihas a developmental programin vitrothat mirrors its stringent two host life cycle

“…While T. gondii oocysts and sporozoites have been studied extensively for their ability to cause disease in intermediate hosts (13, 19, 20), very little is known about the biology of H. hammondi oocysts and sporozoites. We compared sporozoite yield and infectivity between T. gondii and H. hammondi using oocyst preparations derived from multiple cat infections and consistently found that the yield of sporozoites from HhCatEth1 was lower than that from TgVEG oocysts.…”