| Home | E-Submission | Sitemap | Contact us |  
top_img
Korean J Parasitol > Volume 38(2):2000 > Article
Mun, Norose, Aosai, Chen, and Yano: A role of carboxy-terminal region of Toxoplasma gondii-heat shock protein 70 in enhancement of T. gondii infection in mice

Abstract

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g.HSP70-NH2-terminal region, or rT.g.HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T. gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T.gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NH2-terminal region did not. These results suggest that T.g.HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

Heat shock protein (HSP) 70 apparently stabilizes non-native polypeptides through the binding of hydrophobic peptide segments, which are exposed during protein synthesis, protein translocation, and protein degradation (Demand et al., 1998). Functional and structural studies of HSP70 have suggested that the molecule contains two major domains: a highly conserved NH2-terminal region, which contains the ATPase activity, and the more divergent carboxy-terminal region, which is thought to be important in substrate recognition (Demand et al., 1998).
An HSP70 has been shown to be a major immunogen in infections caused by a number of pathogens (Hedstrom et al., 1988; Behr et al., 1992; Wallace et al., 1992; Rico et al., 1998; Mun et al., 1999). It has been proposed that parasite-derived HSP70 may have potential as an antigen for vaccines (Behr et al., 1992; Yang et al., 1997; Rico et al., 1998). Behr et al. (1992) reported that Plasmodium falciparum HSP70 induced interferon-gamma (IFN-γ) production by patient's peripheral blood lymphocytes (PBL), and Rico et al. (1998) described that Leishmania infantum HSP70 induced IFN-γ production of lymph node cells from immunized mice. We have reported the existence of CD4+ helper T lymphocytes and CD4+CD8- and CD4-CD8+ cytotoxic T lymphocytes in peripheral blood lymphocytes of patients with toxoplasmosis (Yang et al., 1995) and demonstrated that human heat shock cognate protein 71 (HSC71) has an important role in antigen processing and antigen presentation by Toxoplasma gondii-infected melanoma cells to a CD4+CD8- cytotoxic T lymphocytes line specific for T. gondii-infected melanoma cells (Yang et al., 1997). We recently reported that the immunization of recombinant T. gondii (rT.g.) HSP70-full length inhibited host protective immunity in T. gondii-infected mice (Mun et al., 1999).
In order to localize the region of T.g.HSP70 involved in deterioration of T. gondii infection, we utilized T.g.HSP70-full length, T.g.HSP70-NH2-terminal region, and T.g.HSP70-carboxy-terminal region. The cloning and expression of rT.g.HSP70-full length, rT.g.HSP70-NH2-terminal region (1212 bp; from 1 to 1212), rT.g.HSP70-carboxy-terminal region (774 bp; from 1168 to 1941), and rT.g.HSP30/bag1, a bradyzoite-specific molecule, were done as described previously (Fig. 1) (Yano et al., 1998; Mun et al., 1999). Age/sex-matched C57BL/6 mice were immunized by intraperitoneal injection of 100 µg of rT.g.Hsp70-full length, rT.g.HSP70-NH2-terminal region, rT.g.HSP70-carboxy-terminal region, or rT.g.HSP30/bag1 that had been suspended in 100 µl of phosphate-buffered saline (PBS) and emulsified with 100 µl of complete Freund's adjuvant (Hoechst, La Jolla, CA, USA). Control groups were injected with an emulsified mixture of 100 µl of PBS combined with 100 µl complete Freund's adjuvant. Ten days after immunization, the mice were infected with five cysts of the Fukaya strain of T. gondii via the oral route. The infected mice were sacrificed at 3, 4, 5, and 6 weeks after infection and the number of T. gondii was then measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene, as reported previously (Luo et al., 1997). In brief, genomic DNA (1 µg) extracted from the brain tissue of each mouse was coamplified with a constant amount of the competitor DNA using a set of SAG1-specific primers. The amplified products were separated by agarose gel electrophoresis and stained with ethidium bromide. The ratio of the staining intensities of the amplified target and competitor sequences were determined using an IPLab Gel Densitometer (Signal Analytical Corp., Vienna, VA, USA). By comparing the ratio obtained to a standard curve, the T. gondii number in the brain was estimated.
The number of T. gondii in the brain tissues of mice immunized with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region was markedly greater 4 to 6 weeks after the challenge infection with Fukaya cysts than that in the brains of control mice (Fig. 2). Interestingly, the number of T. gondii in the brain tissues of mice immunized with rT.g.HSP70-carboxy-terminal region was enhanced compared to that of mice immunized with rT.g.HSP70-full length. In contrast, the number of T. gondii in the brain tissues of mice immunized with rT.g.HSP70-NH2-terminal region was not significantly increased compared with that in control mice. Additionally, when mice were immunized with rT.g.HSP30/bag1, the number of T. gondii in the brain tissues of C57BL/6 mice was lower than that in control mice 4 to 6 weeks after the challenge infection (Fig. 2).
The antigenicities of these recombinant proteins in antibody formation were examined (Fig. 3). Formation of IgG antibodies against T.g.HSP70-full length, T.g.HSP70-NH2-terminal region, T.g.HSP70-carboxy-terminal region, and T.g.HSP30/bag1 was examined in T. gondii-infected mice pre-immunized with or without the recombinant antigens. The sera were collected from each mouse and tested by ELISA (Mun et al., 1999).
Higher levels of IgG antibodies to T.g.HSP70-NH2-terminal region and T.g.HSP30/bag1 as well as T.g.HSP70-full length and T.g.HSP70-carboxy-terminal region were observed in T. gondii-infected mice pretreated with recombinant antigens than in T. gondii-infected mice without pretreatment (Fig. 3). The data indicate that the immunogenicity of T.g.HSP70-NH2-terminal region (and T.g.HSP30/bag1) in IgG antibody formation was equivalent to those of T.g.HSP70-full length and T.g.HSP70-carboxy-terminal region, and suggest that antibodies to T.g.HSP70 recombinant proteins were not involved in T.g.HSP70 mediated-enhancement of the number of T. gondii in the brain.
The deleterious effects induced by immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region with respect to the mortality of infected hosts may be due to several reasons. The first possibility is the activation of immunosuppression by down-regulating nitric oxide release of macrophages. The second possibility is that T.g.HSP70-full length and T.g.HSP70-carboxy-terminal region may function as a source of autoimmunity. In our hands, preliminary data suggest these two possible mechanisms.
We demonstrated here that T.g.HSP70-NH2-terminal region did not inhibit the host protective immunity (Fig. 2) as described above. These data indicate that the ATPase activity of T.g.HSP70-full length, which is critical for its role in protein refolding, is dispensable for T.g.HSP70-full length-mediated inhibition of the host protective immunity. Furthermore, since T.g.HSP70-carboxy-terminal region induces the deleterious effects on protective immunity as well as T.g.HSP70-full length (Fig. 2), T.g.HSP70-carboxy-terminal region may play a crucial role in T. gondii infection.
Wallace et al. (1992) reported that in visceral leishmaniasis infections, the carboxy-terminal region of Leishmania donovani HSP70 is a major target of the humoral immune response. Similarly, Hedstrom et al. (1988) reported that the immunodominant region of schistosome HSP70 is also located in the carboxy-terminal region, and the responses against S. mansoni and S. japonicum have been reported to be immunologically distinct and non-cross-reactive, although the epitopes involved were identified. James et al. (1997) also demonstrated that the carboxy-terminal region is a potential source of functional differences because the carboxy-terminal regions of the Ssa1 and Ssb1 HSP70 families of Saccharomyces cerevisiae are only 14 % identical.
Experimental murine toxoplasmosis provides a suitable tool for evaluating immune reactions to T. gondii because it closely mimics human disease (Choi et al., 1995; Chai et al., 1997; Park and Nam, 1999). Our present data suggest that the immunization with T.g.HSP70-carboxy-terminal region induces the deleterious effects on protective immunity in mice infected with T. gondii. Further studies are currently underway to analyze the mechanisms of T.g.HSP70-carboxy-terminal region-induced deterioration of toxoplasmosis in the brains of T. gondii-infected mice.

Notes

This work was supported in part by Grant-in-Aid 11670239 and 10670225 from the Ministries of Education, Science, and Culture, National Health of Japan, and Japan Science Promotion Society.

REFERENCES

1. Behr C, Sarthou JL, Rogier C, et al. Antibodies and reactive T cells against the malaria heat-shock protein Pf72/hsp70-1 and derived peptides in individuals continuously exposed to Plasmodium falciparum. J Immunol 1992;149: 3321-3330. PMID: 1431109.
pmid
2. Chai JY, Kook J, Guk SM, Chang YP, Yun CK. Experimental infection of murine splenic lymphocytes and granulocytes with Toxoplasma gondii RH tachyzoites. Korean J Parasitol 1997;35: 79-85. PMID: 9241981.
crossref pmid
3. Choi WY, Nam HW, Baek EJ, Cho SY. Lymphadenitis in experimental murine toxoplasmosis induced by intramuscular injection of tachyzoites. Korean J Parasitol 1995;33: 131-134. PMID: 7551804.
crossref pmid
4. Demand J, Luders J, Hohfeld J. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol Cell Biol 1998;18: 2023-2028. PMID: 9528774.
pmid pmc
5. Hedstrom R, Culpepper J, Schinski V, Agabian N, Newport G. Schistosome heat shock proteins are immunologically distinct host-like molecules. Mol Biol Parasitol 1988;29: 275-282.
crossref
6. James P, Pfund C, Craig LA. Functional specificity among HSP70 molecular chaperones. Science 1997;275: 387-389. PMID: 8994035.
crossref
7. Luo W, Aosai F, Ueda M, et al. Kinetics in parasite abundance in susceptible and resistant mice infected with an avirulent strain of Toxoplasma gondii by using quantitative competitive PCR. J Parasitol 1997;83: 1070-1074. PMID: 9406781.
crossref pmid
8. Mun HS, Aosai F, Yano A. Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii. Microbiol Immunol 1999;43: 471-479. PMID: 10449253.
crossref pmid
9. Park YK, Nam HW. Early recognized antigen (p34) of Toxoplasma gondii after peroral ingestion of tissue cyst forming strain (Me49 strain) in mice. Korean J Parasitol 1999;37: 157-162. PMID: 10507223.
crossref pmid pmc
10. Rico AI, Real GD, Soto M, et al. Characterization of the immunostimulatory properties of Leishmania infantum Hsp70 by fusion to the Escherichia coli maltose-binding protein in normal and nu/nu BALB/c mice. Infect Immun 1998;66: 347-352. PMID: 9423878.
pmid pmc
11. Wallace GR, Ball AE, MacFarlane J, El Safi SH, Miles MA, Kelly JM. Mapping of a visceral leishmaniasis-specific immuno-dominant B-cell epitope of Leishmania donovani HSP70. Infect Immun 1992;60: 2688-2693. PMID: 1377180.
pmid pmc
12. Yang TH, Aosai F, Norose K, Mun HS, Yano A. Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL. Microbiol Immunol 1997;41: 553-561. PMID: 9272701.
crossref pmid
13. Yang TH, Aosai F, Norose K, Ueda M, Yano A. Enhanced cytotoxicity of IFN-γ-producing CD4+ cytotoxic T lymphocytes specific for T. gondii-infected human melanoma cells. J Immunol 1995;154: 290-298. PMID: 7995948.
pmid
14. Yano A, Mun HS, Yang TH, et al. In ICOPA IX, Tada I, Kojima S, Tsuji M eds, Roles of IFN-γ in effector mechanisms and pathogenicity of HSPs in mice and human infected with Toxoplasma gondii. 1998, Chiba, Japan. International Proceedings Division. pp 457-466.

Fig. 1
Constructs of plasmids for recombinant protein product for T.g.HSP70-full length, T.g.HSP70-NH2-terminal region, and T.g.HSP70-carboxy-terminal region. The cDNA of T.g.HSP70-full length (1941 bp; from 1 to 1941), T.g.HSP70-NH2-terminal region (1212 bp; from 1 to 1212), and T.g.HSP70-carboxy-terminal region (774 bp; from 1168 to 1941) was cloned into the pET-15b (Novagen, Madison, USA) plasmid.
kjp-38-107-g001.jpg
Fig. 2
Effects of immunization with rT.g.HSP70-full length, rT.g.HSP70-NH2-terminal region, rT.g.HSP70-carboxy-terminal region, or rT.g.HSP30/bag1 on C57BL/6 mice infected perorally with the Fukaya strain of Toxoplasma gondii. At 3, 4, 5, and 6 weeks after infection, the infected mice were sacrificed and the number of T. gondii was then measured by QC-PCR targeting the SAG1 gene. The significance of differences between groups was determined by Student's t-test. P<0.05 was taken as significant.
*P<0.05 compared with control mice.
**P<0.005 compared with control mice.
kjp-38-107-g002.jpg
Fig. 3
IgG antibody production against T.g.HSP70-full length, T.g.HSP70-NH2-terminal region, T.g.HSP70-carboxy-terminal region, and T.g.HSP30/bag1 in Toxoplasma gondii-infected mice pre-immunized with or without the recombinant antigens. The sera were collected from each mouse and analyzed by ELISA. The solid line represents mice pre-immunized with recombinant antigens and then infected with T. gondii cysts. The dotted line represents without pre-immunization of the recombinant antigens. ELISA titers of T.g.HSP70-full length, T.g.HSP70-NH2-terminal region, T.g.HSP70-carboxy-terminal region, and T.g.HSP30/bag1 of control (non-infected, non-immunized) mouse sera were 0.045, 0.04, 0.05, and 0.04 respectively. The significance of differences between groups was determined by Student's t-test. P<0.05 was taken as significant.
*P<0.05 compared with non-immunized mice.
**P<0.005 compared with non-immunized mice.
kjp-38-107-g003.jpg
Editorial Office
c/o Department of Medical Environmental Biology
Chung-AngUniversity College of Medicine, Dongjak-gu, Seoul 06974, Korea
Tel: +82-2-820-5683   Fax: +82-2-826-1123   E-mail: kjp.editor@gmail.com
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Copyright © 2019 by The Korean Society for Parasitology and Tropical Medicine. All rights reserved.     powerd by m2community