Abstract
ABSTRACTToxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δgra12), no major defects in acute virulence were observed in Δgra12A, Δgra12B, or Δgra12D parasites, though Δgra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δgra12A parasites and were increased in mice infected with Δgra12B parasites. However, Δgra12B cysts were not efficiently maintained in vivo. Δgra12A, Δgra12B, and Δgra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δgra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts.IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection.
Highlights
Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system
It is remarkable that the majority of proteins that are found associated with the cyst wall are GRA proteins [30,31,32,33,34]
In view of the fact that parasites released from ruptured cysts are cleared by the immune system [52], it has been hypothesized that the cyst wall provides a barrier to cyst clearance
Summary
Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. Our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection. Toxoplasma efficiently invades a vast variety of nucleated host cells using an arsenal of secreted effectors stored in secretory organelles [8] These secreted effector proteins are released from secretory organelles called rhoptries, micronemes, and dense granules (the organelle containing GRA proteins) in a coordinated fashion to mediate the parasite’s attachment and invasion into host cells, as well as the establishment of the intracellular parasitophorous vacuole (PV) that supports parasite survival and replication inside the host cell [9,10,11]. GRA12 displays little homology to proteins that are not expressed by Coccidian species, which suggests a specificity to the functional role(s) of GRA12 in Coccidians
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