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First published online 23 April 2003
doi: 10.1242/jcs.00382

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Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii

Valerian Nakaar*, Huân M. Ngô*, Emily P. Aaronson, Isabelle Coppens, Timothy T. Stedman and Keith A. Joiner{ddagger}

Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St, New Haven, CT, 06520-8022, USA



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  		Fig. 1. Antisense RNA specifically inhibits ROP2 expression. Analysis of the steady-state levels of protein of ROP2AS clones was done by immunoblot with equal numbers of parasites using a monoclonal antibody T34A7 (Sadak et al., 1988Go). (A) Because the antibody also recognizes ROP3 and ROP4, each clone conveniently serves as its own internal control for loading and specificity of targeting by antisense RNA. (A) The autoradiogram was over-exposed to demonstrate the faint ROP2 band in the ROP2AS-7 lane. Densitometric scanning of a less intensely exposed autoradiogram demonstrated that ROP2 expression was lowered by 87-92% for ROP2AS-1, ROP2AS-7, ROP2AS-10 and ROP2AS-20, whereas ROP2 expression in the ROP2AS-8 clone was normal, probably reflecting recovery (see Fig. 6). The limited variation among the four repressed clones may be partly fortuitous, but in addition probably reflects the fact that the majority of clones (>80%) were not recovered at all, thereby skewing the results in favor of parasites expressing basal levels of ROP2. (B) ROP2 and NTPase levels are unaffected by expression of antisense HXGPRT construct and irrelevant vector control pminCAT.

 


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  		Fig. 2. Depletion of ROP2 produces aborted parasite vacuoles and alters rhoptry shape. (A-G) Transmission electron microscopy of ROP2AS-7 and ROP2AS-20 revealed that a small proportion of ROP2-deficient parasites were able to invade Vero cells and establish a vacuole with a delimiting membrane. The majority of the vacuoles contained large multinucleated (N) parasites (A) and the network membrane (NM) was significantly increased (A,B). Altered parasite morphology was because of an arrest in the formation of the cytokinetic furrow during cell division (endodyogeny) (arrows in A,B). Although dense granules (DG) and micronemes (M) appeared to be unaffected, the formation of mature rhoptries (R) was blocked in the majority of parasites (C-F). Defective rhoptries were most often no longer polarized to the apical half of the cell (A) and their categorical flask shape was neither formed nor maintained (C-F). Whereas the formation of the honeycombed basal portion containing packaged lumenal membranes was disrupted (arrowhead in F) in some rhoptries, the condensation of lumenal contents to form the electron-dense rhoptry distal tip (arrowheads in D,E) was severely disrupted in the majority of aberrant organelles. Bars: 1.0 µM (A-B,G), 0.2 µM (C-F,H-J). (H-J) Ultrathin cryoimmuno electron microscopy of ROP2AS-1. Serial cryosections of an unsegregated rhoptry cluster in ROP2AS-1 contains ROP2/3/4 (arrows) as immunolabelled with T34A7 monoclonal antibody. (K-R) Immunofluorescent microscopy of extracellular ROP2AS-7 immunostained with an antiserum specific to ROP2, as reported previously (Sadak et al., 1988Go). In up to one-third of parasites, rhoptries appeared normal, even when rhoptries in adjacent parasites in the same vacuole were distorted. This was evident both at the electron microscopic level (G, normal rhoptries, arrows; abnormal rhoptries, arrowheads) and by immunofluorescence (K-R, see text for description).

 


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  		Fig. 2. Depletion of ROP2 produces aborted parasite vacuoles and alters rhoptry shape. (A-G) Transmission electron microscopy of ROP2AS-7 and ROP2AS-20 revealed that a small proportion of ROP2-deficient parasites were able to invade Vero cells and establish a vacuole with a delimiting membrane. The majority of the vacuoles contained large multinucleated (N) parasites (A) and the network membrane (NM) was significantly increased (A,B). Altered parasite morphology was because of an arrest in the formation of the cytokinetic furrow during cell division (endodyogeny) (arrows in A,B). Although dense granules (DG) and micronemes (M) appeared to be unaffected, the formation of mature rhoptries (R) was blocked in the majority of parasites (C-F). Defective rhoptries were most often no longer polarized to the apical half of the cell (A) and their categorical flask shape was neither formed nor maintained (C-F). Whereas the formation of the honeycombed basal portion containing packaged lumenal membranes was disrupted (arrowhead in F) in some rhoptries, the condensation of lumenal contents to form the electron-dense rhoptry distal tip (arrowheads in D,E) was severely disrupted in the majority of aberrant organelles. Bars: 1.0 µM (A-B,G), 0.2 µM (C-F,H-J). (H-J) Ultrathin cryoimmuno electron microscopy of ROP2AS-1. Serial cryosections of an unsegregated rhoptry cluster in ROP2AS-1 contains ROP2/3/4 (arrows) as immunolabelled with T34A7 monoclonal antibody. (K-R) Immunofluorescent microscopy of extracellular ROP2AS-7 immunostained with an antiserum specific to ROP2, as reported previously (Sadak et al., 1988Go). In up to one-third of parasites, rhoptries appeared normal, even when rhoptries in adjacent parasites in the same vacuole were distorted. This was evident both at the electron microscopic level (G, normal rhoptries, arrows; abnormal rhoptries, arrowheads) and by immunofluorescence (K-R, see text for description).

 


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  		Fig. 3. The association of host mitochondria with the parasitophorous vacuole membrane (PVM) is inhibited in ROP2-deficient parasites. (A-B) Mitotracker red staining of wild-type (WT), ROP2AS-7 and ROP2AS-20 parasites. The morphology of parasites in B', illustrating both distention and occasional vacuolization, was observed in only a portion of antisense-expressing parasites. In comparison to WT, mitotracker labeling indicates that the host mitochondria are not localized to the parasite vacuole containing ROP2-deficient parasites, in which only the intracellular mitochondria are detected (A-A', arrows). (C-D) Transmission electron micrographs illustrate abrogation of mitochondrial association with the PVM (arrows) in ROP2AS (D) compared to WT (C). (E) Linear density of parasitophorous membrane that are associated with mitochondria in WT, ROP2AS-7 and ROP2AS-7 (recovered) as determined by EM morphometric analysis similar to that described in Sinai et al. (Sinai et al., 1977). ROP2AS-7 (recovered) is parasites in which ROP2 expression is recovered with continued passage. Random EM sections of parasite vacuoles containing WT (n=25), ROP2AS-7 (n=26) and ROP2AS-7 (recovered) (n=31) were examined by the double square overlay test system to calculate the percentage of PVM that is associated with host mitochondria. The percentage of PVM associated with mitochondria is reduced by ten-fold in ROP2AS-7 in comparison to WT (P<0.0001), whereas ROP2AS-7 (recovered) showed an increase by four-fold (P<0.001). Values shown in E are mean±s.d. Bar, 1 µM.

 


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  		Fig. 4. Reduction of rhoptry cholesterol content and [NBD-C]-LDL acquisition by antisense-expressing parasites. Fibroblasts infected with Toxoplasma gondii, wild-type (A-A') or ROP2AS (B-B') were cultivated for 24 hours in medium containing 10% FCS, fixed, cytochemically stained with filipin for cholesterol detection and observed by fluorescence microscopy as described (Coppens et al., 2000Go). Twenty-four hours post-infection with T. gondii, wild type (C-C') or ROP2AS (D-D') in medium containing 10% LPDS, fibroblasts were pulse-labeled at 37°C with 0.1 mg/ml of [NBD-C]-LDL for 60 minutes. Cells were washed and processed for fluorescence observation.

 


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  		Fig. 5. Expression of ROP2 antisense RNA compromises parasite invasion, replication and virulence in mice. ROP2AS clones and control RH parasites were inoculated into fresh human foreskin fibroblast (HFF) cells. After 2 hours, parasites that did not infect were washed off, and the infection was continued for 24 hours. (A) Invasion was monitored by counting the number of vacuoles for control (wild type) and antisense clones (ROP2AS-1, ROP2AS-7, ROP2AS-20). Data are derived by counting vacuoles in at least 50-100 randomly selected microscopic fields. Differences between values of the control and all experimental groups were statistically significant (P<0.005). (B) Parasite replication was monitored by [3H]uracil incorporation assays using HFF cells infected for 24 hours. Data are expressed as mean±s.e.m. from 3-6 independent experiments repeated at least four times. All ROP2AS clones over a 24-hour period post-infection reproducibly replicated more slowly than control (P<0.005). (C) Replication of intracellular parasites was determined at 24 hours after infection by counting the number of parasites within each of 130 randomly selected vacuoles. The median vacuole in the control contained 16 parasites (corresponding to four parasite doublings), whereas in the antisense clones this was between 4-8 parasites (2-3 doublings), reflecting a delay in replication. Wild type (•); ROP2AS-7 ({blacksquare}); ROP2AS-20 ({square}). (D) Survival curves for mice (10 mice in each group) infected with 105 parasites of control or ROP2AS clones. Wild type ({circ}); ROP2AS-1 ({triangleup}); ROP2AS-7 ({square}); ROP2AS-20 ({blacksquare}).

 


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  		Fig. 6. Recovery of ROP2 expression restores rhoptry shape, mitochondrial association and virulence in mice. After prolonged passage of ROP2AS clones in VERO cells, ROP2 protein expression recovered to nearly wild-type levels (A). A random sample of 31 vacuoles from `recovered' ROP2AS-7 clones was examined by transmission electron microscopy. Parasite vacuoles and individual organisms appeared more normal (B,C). An increasing number of vacuoles are associated with host mitochondria (B,C), as confirmed by morphometric analysis (see Fig. 3E). Most rhoptries are restored to the original club shape with an electron dense distal portion (D,E). Survival curves of mice infected with recovered ROP2AS clones are similar to those for wild-type parasites (Fig. 6F). Wild type ({circ}); ROP2AS-1 ({triangleup}); ROP2AS-7 ({square}); ROP2AS-20 ({blacksquare}). Bar, 1.0 µM (B-C), 0.2 µM (D), 0.4 µM (E).

 

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© The Company of Biologists Ltd 2003