The SAG1 Toxoplasma gondii Surface Protein Is Not Required for ...

INFECTION AND IMMUNITY, Apr. 2007, p. 2079–2083 0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01685-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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The SAG1 Toxoplasma gondii Surface Protein Is Not Required for Acute Ocular Toxoplasmosis in Mice䌤 Elizabeth Charles,1 Michelle C. Callegan,1,2 and Ira J. Blader1* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104,1 and Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731042 Received 20 October 2006/Returned for modification 4 January 2007/Accepted 28 January 2007

The SAG1 Toxoplasma gondii surface protein stimulates acute ileitis. To determine whether SAG1 is also important in the eye, wild-type or SAG1 knockout parasites were injected intravitreally into mice. No differences in retinal damage or parasite growth were observed, indicating that unlike the case for the intestine, factors besides SAG1 are important for retinal damage. Toxoplasma gondii is an obligate intracellular pathogen that infects humans congenitally or postnatally and causes a number of diseases, including ocular toxoplasmosis (OT) (18). In humans and other intermediate hosts, Toxoplasma organisms exist in two forms, disease-causing tachyzoites and non-disease-causing bradyzoites, which encyst in the eye, brain, and muscle. Cysts, which each contain approximately 1,000 bradyzoites, periodically reactivate, and the released parasites convert into tachyzoites and initiate an acute phase of the infection (10, 35). Thus, experiments with tachyzoites are necessary to examine how Toxoplasma causes disease. Assessing the function of tachyzoite-expressed proteins in the eye and other immune-privileged sites is difficult for several reasons. First, mice systemically infected with type I Toxoplasma strains, which are associated with postnatally acquired OT (8), die before the onset of ocular pathology (5). Second, mice infected with type II and III strains, which are more commonly associated with congenital OT (1), develop tissue cysts; however, the number of retinal cysts is highly variable and irreproducible (6, 19). Moreover, the animals display only minor retinal damage and develop acute OT only after becoming immunocompromised. Third, direct injection of tachyzoites into the anterior chamber of the mouse eye causes retinal damage (14). However, Toxoplasma is found in the anterior chamber only in severe cases of human OT. This suggests that disease in the anterior chamber is secondary to that in the retina, presumably because the integrity of the posterior segment of the eye is compromised since the two regions are physically separated. The parasite’s cell surface is covered by a family of developmentally regulated, glycosylphosphatidylinositol-linked surface proteins named SAGs (15). SAG1 is the most abundant tachyzoite surface protein and functions in host cell attachment and immune modulation (21, 22, 28). More recently, SAG1 was demonstrated to be important for intestinal tissue

damage after parasites were directly injected into the intestines of mice (28). However, it is not known if SAG1’s role in causing tissue damage is limited to the intestine or extends to other tissues. To examine this question, we directly tested whether SAG1 is responsible for Toxoplasma-induced retinal damage. Intravitreal injection of tachyzoites induces retinochoroiditis. To address the question, we first needed to develop an infection model that would allow us to directly assess tachyzoites in the retina without the need for them to cross anatomical borders. Thus, we adapted an intravitreal injection protocol previously used to induce OT in monkeys and rabbits (3, 12, 26). Here, a 0.5-␮l solution of parasites was injected by using a pneumatically controlled liquid dispenser (MDI, South Plainfield, NJ) into the vitreous of a mouse eye with a 35-␮m glass needle prepared with a pipette puller (Sutter Instruments, Novato, CA). C57BL/6 mice were intravitreally injected with buffer or 103 or 104 tachyzoites of the type I RH strain for 4 and 6 days. At each time point, retinal damage was assessed histologically and functionally. First, hematoxylin-and-eosinstained retinas were assigned clinical scores using the scoring system described by Hu et al. (13). The data indicated that whereas mock-injected eyes had no damage, significant retinal damage was observed 6 days after mice were injected with 104 parasites (mean score, 2.9) (Fig. 1A). This damage was dose and time dependent, since mice injected with 104 parasites for 4 days or with 103 parasites for 6 days had significantly less damage (mean scores of 1.95 and 1.3, respectively). We could not examine eyes more than 6 days after they were injected with 104 parasites because they were severely necrotic and could not be harvested. Eyes injected with 104 parasites for 4 days or 103 parasites for 6 days displayed leukocyte infiltration and disorganization of the retinal architecture (Fig. 1B to E). Six days after 104 parasites were injected, the retinitis was significantly more severe and focalized necrosis was evident in all retinal layers, with the inner layers of the retina appearing the most necrotic. The damage in these mice was similar to that in human OT patients or mice systemically infected with type II parasites and then immunosuppressed (11, 31). In contrast to mice injected with Toxoplasma, mice intravitreally injected with Staphylococcus aureus or Bacillus cereus develop severe vitreal inflamma-

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 1034, Oklahoma City, OK 73034. Phone: (405) 271-2133. Fax: (405) 271-3117. E-mail: iblader @ouhsc.edu. 䌤 Published ahead of print on 5 February 2007. 2079

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FIG. 1. Histopathology of intravitreally injected mice. (A) Clinical scores for mice intravitreally injected with buffer only (mock) or with the indicated number of parasites. Student’s t tests were used to determine statistically significant differences in results between each group and the mock-infected group. HK, heat-killed parasites; *, P value of ⬍0.05 relative to value for mock-injected eyes. (B to E) Representative sections of eyes injected with buffer (B), 104 tachyzoites for 6 days (C), 103 tachyzoites for 6 days (D), 104 tachyzoites for 4 days (E), and 104 heat-killed tachyzoites for 6 days (F). D, days. All images were taken with a 4⫻ objective.

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FIG. 2. ERG analysis of Toxoplasma-infected eyes. Mice were intravitreally injected with buffer or the indicated numbers of parasites in one eye and the other eye was left uninjected. Student’s t tests were used to determine statistically significant differences between results for each group of mice and mock-infected mice. D, days; Pun, an eye punctured with a parasite-loaded needle but not injected with parasites; *, P value of ⬍0.05 relative to value for mock-infected eyes.

tion and damage (4, 29). Thus, Toxoplasma-induced retinal damage appears to be pathogen specific and not a general feature of a pathogen growing in the posterior segment of the eye. The experiments described above were performed with type I parasites. Because humans infected with type II parasites can also develop OT, we tested whether retinal damage occurred in mice intravitreally injected with the ME49 type II strain. The data indicated that 6 days after 104 ME49 parasites were injected, mice also developed significant retinal damage, albeit not as severe as that caused by the RH strain (clinical score ⫽ 1.5 ⫾ 0.8) (data not shown). It is important to note that 6 days after mice were intravitreally injected with either dose, they appeared healthy, with no signs of systemic disease. In fact, mice intravitreally injected with 104 parasites died 10 to 11 days postinjection (not shown). This was in contrast to mice that died 7 or 9 days after they were intraperitoneally injected with 104 or 102 RH parasites, respectively (not shown). Thus, the RH tachyzoites we used were as virulent as previously described (5, 23), but for unknown reasons mice succumbed later to intravitreal infections than to intraperitoneal infections. No histopathological or functional damage was observed in mice injected with 104 heat-killed parasites (Fig. 1F and 2) or eyes in which a parasite-loaded needle was placed into the vitreous but without injection of parasites (Fig. 2). These results are in contrast to another report that intravitreal injections caused lens damage and retinal edema independently of the parasite (34). This was surprising since intravitreal injections are commonly used to study retinal development, degeneration, and inflammation in mice (2, 20, 33). However, Tedesco et al. (34) used 24-gauge needles that are at least 10 times larger than our needles. The large size of these needles would make it very difficult to use them without causing lens and/or retina damage. In addition, they injected 5 ␮l, or 10 times more than we did, which could significantly increase intraocular pressure and retinal edema. Thus, with proper

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technique and instrumentation, intravitreal injections are suitable for the study of the acute phase of OT. The intravitreal injection model does have some inherent limitations. First, it cannot be used to study parasite dissemination to the eye. In addition, immunological and pathological events may differ when cysts reactivate than when parasites are injected into the vitreous. However, the retinal damage seen in our mice is very similar to that seen in mice systemically infected with type II parasites and then immunosuppressed or in hamsters orally infected with type II parasites (6, 7, 27, 32). Intravitreal injection of tachyzoites causes defects in retinal function. Electroretinography (ERG) measures retinal responses to light and was previously used to assess retinal function in Toxoplasma-infected humans and mice (24, 30). To perform ERGs, mice were dark adapted for 18 h and then anesthetized and both pupils were dilated. A ground electrode was placed in the animal’s mouth, while bipolar electrodes were placed on each eye and then flashes of light were presented simultaneously to both eyes and the responses recorded. The percentage of retinal function was defined as the ratio of the b-wave amplitude of the infected eye to the b-wave amplitude of the uninjected eye. The data show that responses were reduced more than 85% 6 days after 104 tachyzoites were injected, while responses were reduced ⬃45% 4 days after 104 parasites were injected or 6 days after 103 parasites were injected (Fig. 2). Toxoplasma tachyzoites grow in the retina. Sections prepared from eyes 6 days after they were injected with buffer or 104 parasites were stained with SAG1 antisera to determine where tachyzoites were located in the eye. Parasites were detected only in damaged regions of the retina and were present in the optic nerve (data not shown), which has been previously reported to occur in humans (9). Only when eyes were extremely necrotic could parasites be detected in the anterior segment or in extraocular tissues, suggesting that parasites entered those areas only after the integrity of the retina was compromised (not shown). SAG1 is not required for retinal damage. To test if SAG1 is important for retinal damage, we compared the histopathologies of mice injected with 103 or 104 parental RH⌬ or SAG1 knockout (⌬sag1) parasites for 6 days or 102 parasites for 8 days, which was the same dose used by Lu et al. (17). We found that regardless of the size of the infectious dose, there was no significant difference in the tissue damage caused by RH⌬ or ⌬sag1 parasites (Fig. 3A). We next examined whether SAG1 was required for Toxoplasma-induced decreases in retinal function. Mice were therefore injected with 103 or 104 RH⌬ or ⌬sag1 parasites for 6 days, and light responses were measured by ERG. These two doses were used because they resulted in two distinct degrees of retinal damage. Similar to the histopathology data, no significant differences in ERG responses were observed after mice were intravitreally injected with RH⌬ or ⌬sag1 parasites (Fig. 3B). These data are in contrast to a previous report demonstrating that ⌬sag1 parasites caused less retinal pathology than RH⌬ parasites after they were injected into the anterior chamber (17). The basis for this difference is not clear but likely may be due to the different injection sites. Because the retina is physically separated from the anterior chamber and parasites would need to traffic to the retina, it is possible that loss of

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FIG. 3. SAG1 is not required for retinal damage. (A) Clinical scores for eyes injected with 103 or 104 RH⌬ or ⌬sag1 parasites for 6 days or with 102 RH⌬ or ⌬sag1 parasites for 8 days. No significant differences were observed. (B) Electroretinography of mice intravitreally injected with 103 or 104 RH⌬ or ⌬sag1 parasites for 6 days. (C and D) Representative hematoxylin-and-eosin-stained sections from mice intravitreally injected with 104 RH⌬ (C) and ⌬sag1 parasites (D) for 6 days.

SAG1 affects the ability of parasites to spread across anatomical barriers. In the intestine, ⌬sag1 parasites grow better than wild-type parasites (28). To determine if SAG1 affects parasite growth in the eye, parasites were enumerated with quantitative real-time PCR (16). Mice were injected with 104 RH⌬ or ⌬sag1 parasites for 6 days, and then the numbers of parasites were determined. No statistically significant difference in the numbers of ⌬sag1 or RH⌬ parasites was observed. Overall, an average of 2.5 ⫻ 108 parasites of either strain was present (Fig. 4). Other studies have also quantified parasites in the eyes of Toxoplasma-infected mice. Approximately 107 parasites were present in gamma interferon knockout mice perorally infected with five tissue cysts of a type II strain (25). Gamma interferon knockout mice were necessary because type II strains will encyst in wild-type mice and tachyzoites cannot be detected in their eyes. Thus, our data are consistent with the notion that high numbers of tachyzoites can grow in the eye. In summary, we have developed a new murine model to study the acute phase of OT. Using this model, we demonstrated with histopathological, functional, and parasite enumeration assays that the SAG1 surface protein does not appear

FIG. 4. Enumeration of wild-type and ⌬sag1 parasites in mouse eyes. Quantitative PCR was used to determine the numbers of parasites 6 days after eyes were injected with 104 RH⌬ or ⌬sag1 parasites. A Student’s t test determined no significant difference between the numbers of wild-type (n ⫽ 11) and ⌬SAG1 (n ⫽ 11) parasites (P ⫽ 0.67).

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to play a significant role in Toxoplasma-induced retinal damage. Given that the mouse eye is an experimentally manipulatable, immune-privileged organ and the large number of available mouse mutants and reagents, this model will likely help elucidate pathogenic mechanisms underlying Toxoplasma-associated diseases in the brain as well as the eye. In addition, the ability to grow parasites to high numbers (⬎108 parasites/eye) provides a unique opportunity to examine, in a physiologically relevant tissue, Toxoplasma gene and protein expression. We thank Mark Dittmar for animal care, Raniyah Ramadan for assistance with the intravitreal injection, and John Boothroyd for providing ⌬SAG1 parasites and anti-SAG1 antisera. We also thank Dan Carr, James Chodosh, and Justine Smith for helpful discussions and Kristina Wasson-Blader and Jimmy Ballard for reviewing the manuscript. This work was supported in part by grants to I.B. from the University of Oklahoma’s College of Medicine Alumni Association and the Oklahoma Center for the Advancement of Science and Technology (grant HR05-138S). REFERENCES 1. Ajzenberg, D., N. Cogne, L. Paris, M. H. Bessieres, P. Thulliez, D. Filisetti, H. Pelloux, P. Marty, and M. L. Darde. 2002. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J. Infect. Dis. 186:684–689. 2. Blanks, J. C., and R. H. Blanks. 1980. Autoradiographic pattern of 3Hfucose incorporation in the developing mouse retina. Investig. Ophthalmol. Vis. Sci. 19:457–467. 3. Culbertson, W. W., K. F. Tabbara, and R. O’Connor. 1982. Experimental ocular toxoplasmosis in primates. Arch. Ophthalmol. 100:321–323. 4. Engelbert, M., and M. S. Gilmore. 2005. Fas ligand but not complement is critical for control of experimental Staphylococcus aureus endophthalmitis. Investig. Ophthalmol. Vis. Sci. 46:2479–2486. 5. Eyles, D. E., and N. Coleman. 1956. Relationship of size of inoculum to time to death in mice infected with Toxoplasma gondii. J. Parasitol. 42:272–276. 6. Gazzinelli, R. T., A. Brezin, Q. Li, R. B. Nussenblatt, and C. C. Chan. 1994. Toxoplasma gondii: acquired ocular toxoplasmosis in the murine model, protective role of TNF-alpha and IFN-gamma. Exp. Parasitol. 78:217–229. 7. Gormley, P. D., C. E. Pavesio, P. Luthert, and S. Lightman. 1999. Retinochoroiditis is induced by oral administration of Toxoplasma gondii cysts in the hamster model. Exp. Eye Res. 68:657–661. 8. Grigg, M. E., J. Ganatra, J. C. Boothroyd, and T. P. Margolis. 2001. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184:633–639. 9. Grossniklaus, H. E., C. S. Specht, G. Allaire, and J. A. Leavitt. 1990. Toxoplasma gondii retinochoroiditis and optic neuritis in acquired immune deficiency syndrome. Report of a case. Ophthalmology 97:1342–1346. 10. Holland, G. N. 2003. Ocular toxoplasmosis: a global reassessment. Part I. Epidemiology and course of disease. Am. J. Ophthalmol. 136:973–988. 11. Holland, G. N., G. R. O’Connor, R. Belfort, and J. S. Remington. 1996. Toxoplasmosis, p. 1183–1223. In J. Pepose, G. N. Holland, and K. R. Wilhelmus (ed.), Ocular infection and immunity. Mosby, St. Louis, MO. 12. Holland, G. N., G. R. O’Connor, R. F. Diaz, P. Minasi, and W. M. Wara. 1988. Ocular toxoplasmosis in immunosuppressed nonhuman primates. Investig. Ophthalmol. Vis. Sci. 29:835–842. 13. Hu, M. S., J. D. Schwartzman, A. C. Lepage, I. A. Khan, and L. H. Kasper. 1999. Experimental ocular toxoplasmosis induced in naive and preinfected mice by intracameral inoculation. Ocul. Immunol. Inflamm. 7:17–26. 14. Hu, M. S., J. D. Schwartzman, G. R. Yeaman, J. Collins, R. Seguin, I. A.

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