Targeted Disruption of theGRA2Locus inToxoplasma gondiiDecreases Acute Virulence in Mice

Abstract

Following invasion into the host cell, the protozoanToxoplasma gondii secretes a variety of proteins that modify the parasitophorous vacuole. Within the vacuole, the 28-kDa dense granule protein known as GRA2 is specifically targeted to the tubulovesicular network which forms connections with the vacuolar membrane. To investigate the importance of GRA2, we derived from strain RH a mutant T. gondii line in which GRA2 was disrupted by replacement with the marker Ble (selecting for phleomycin resistance). The Δgra2 mutant invaded and grew normally in both fibroblasts and macrophages in vitro; however, it was less virulent during acute infection in mice. The survival rate of mice inoculated with Δgra2 was significantly higher; some infected mice survived the acute infection, whereas all mice infected with the wild-type strain RH succumbed to early death. Chronic infection by Δgra2 was detected by positive serology, immunohistochemical detection of parasites and cysts in the brain, and reisolation of parasites by bioassay at 6 weeks postinfection. Thus, absence of GRA2 partially attenuates the virulence of T. gondii during the acute phase of infection and allows for establishment of chronic infection by the otherwise highly virulent RH strain. These results establish that GRA2 plays an important role during in vivo infection and provide a potential model for examining acute pathogenesis by T. gondii.

Mechanisms ofToxoplasma gondiipersistence and latency

“…Isolated reports claim RH parasites can form cysts in mice, via treatment with atovaquone and pyrrolidine dithiocarbamate (Djurkovic‐Djakovic et al ., ) or prior vaccination with soluble tachyzoite antigen and IL‐12, although the bradyzoites formed by RH differ ultrastructurally and in their sensitivity to pepsin–HCl (Yap et al ., ). In addition, RH strain parasites attenuated in virulence owing to disruption of dense granule gene GRA2 are capable of establishing chronic infection in mice (Mercier et al ., ). Other type I strains that have not been subject to extensive in vitro culture, such as GT1, are able to form normal cyst walls in vitro during stress (Khan et al ., ).…”

Biogenesis of Nanotubular Network inToxoplasmaParasitophorous Vacuole Induced by Parasite Proteins

“…T. gondii tachyzoites of the RH wild-type; the ⌬gra2 mutant; the complemented ⌬gra2 (Mercier et al, 1998b), the wild-type expressing GRA2HA9 (Mercier et al, 1998a); and the RH hxgprt - (Donald et al, 1996) were propagated in human foreskin fibroblasts (HFFs) maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 25 g/ml gentamicin. Parasites were harvested after complete lysis of the monolayer, purified through 3.0-m filters, and washed in phosphate-buffered saline.…”

“…To construct the double knockout parasite ⌬gra6-⌬gra2, tachyzoites of the ⌬gra6 Toxoplasma mutant clone A11 (GRA6 Ϫ , CAT ϩ , hxgprt Ϫ ) were electroporated with 100 g of the circular plasmid GRA2/Ble/GRA2 (8.9) used previously to target the GRA2 locus in the RH strain (Mercier et al, 1998b). The phleomycin selection was applied as described previously (Messina et al, 1995), and surviving parasites were cloned by limiting dilution to obtain the clones A26 and A81.…”

“…To complement the ⌬gra2 mutant (Mercier et al, 1998b) with the mutated forms of GRA2-HA9, tachyzoites were cotransfected with 50 g of the circular plasmid expressing either the deleted form of GRA2-HA9, ⌬␣1, or GRA2-HA9 containing the scrambled form of ⌬␣1, the S␣1 construct, mixed with 5 g of the circular selectable plasmid TUB/CAT, and the CAT selection was carried out as described previously (Kim et al, 1993).…”

“…Therefore, we have undertaken to study the biological functions of the GRA proteins by constructing knockout mutants and examining their cellular phenotypes. Herein, we report a detailed analysis of the previously generated ⌬gra2 knockout mutant (Mercier et al, 1998b), showing for the first time that one of the functions of GRA2 is to organize the membranous structure of the network. Through the construction of newly derived ⌬gra6 and ⌬gra6-⌬gra2 mutants, we also demonstrate that GRA6 is involved in organization of the network.…”