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1.  Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii *  
The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.
PMCID: PMC2704969  PMID: 19585669
DNA vaccine; Granule protein 4 (GRA4); Liposome; Toxoplasma gondii
2.  DNA Vaccination with Genes Encoding Toxoplasma gondii Antigens GRA1, GRA7, and ROP2 Induces Partially Protective Immunity against Lethal Challenge in Mice 
Infection and Immunity  2000;68(1):38-45.
C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-γ) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-γ. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.
PMCID: PMC97099  PMID: 10603366
3.  DNA prime and peptide boost immunization protocol encoding the Toxoplasma gondii GRA4 induces strong protective immunity in BALB/c mice 
BMC Infectious Diseases  2013;13:494.
Toxoplasma gondii is a widespread intracellular parasite, which infects most vertebrate animal hosts and causes zoonotic infection in humans. Vaccine strategy remains a promising method for the prevention and control of toxoplasmosis. T. gondii GRA4 protein has been identified as a potential candidate for vaccine development. In our study, we evaluated the immune response induced by four different immunization vaccination strategies encoding TgGRA4.
BALB/c mice were intramuscularly (i.m.) immunized four times according to specific immunization schedules. Generally, mice in experimental groups were immunized with polypeptide, pGRA4, peptide/DNA, or DNA/peptide, and mice in the control groups were injected with PBS or pEGFP. After immunization, the levels of IgG antibodies and cytokine productions were determined by enzyme-linked immunosorbent assays (ELISA). The survival time of mice was also evaluated after challenge infection with the highly virulent T. gondii RH strain.
The results showed that mice vaccinated with different immunization regimens (polypeptide, pGRA4, peptide/DNA, or DNA/peptide) elicited specific humoral and cellular responses, with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ), which suggested a specific Th1 immunity was activated. After lethal challenge, an increased survival time was observed in immunized mice (11.8 ± 4.8 days) compared to the control groups injected with PBS or pEGFP (P < 0.05). Mice injected with PBS or pEGFP died within 8 days, and there was no significant difference in the protection level in two groups (P > 0.05).
These results demonstrated that this DNA prime and peptide boost immunization protocol encoding the TgGRA4 can elicit the highest level of humoral and cellular immune responses compared to other immunized groups, which is a promising approach to increase the efficacy of DNA immunization.
PMCID: PMC3871000  PMID: 24148219
4.  Construction and In vitro Expression Analyses of a DNA Plasmid Encoding Dense Granule GRA5 Antigen of Toxoplasma gondii  
Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii (T.gondii) throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic loss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 antigen of T.gondii as a possible DNA vaccine. GRA5 gene fragment devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 (pGRA5) was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HEK 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst I enzyme showed correct insertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. immunofluorescence and Western blotting analyses of HEK 293-T cells transfected with pGRA5 showed specific expression of GRA5. Immunogenicity of pGRA5 will be evaluated in mice.
PMCID: PMC3558189  PMID: 23407179
Toxoplasma gondii; DNA vaccine; GRA5 protein
5.  A GRA1 DNA Vaccine Primes Cytolytic CD8+ T Cells To Control Acute Toxoplasma gondii Infection  
Infection and Immunity  2003;71(1):309-316.
Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-γ). The contribution of CD4+ and CD8+ T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4+ and CD8+ T-cell subsets produced IFN-γ upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8+ T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8+ T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4+ T cells led to an increase in brain cyst burden during the chronic phase of infection.
PMCID: PMC143232  PMID: 12496180
6.  Recombinant GRA4 or ROP2 Protein Combined with Alum or the gra4 Gene Provides Partial Protection in Chronic Murine Models of Toxoplasmosis 
The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant.
PMCID: PMC440599  PMID: 15242945
7.  Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis 
Bioengineered  2012;3(6):358-364.
Toxoplasmosis is a globally distributed foodborne zoonosis caused by a protozoan parasite Toxoplasma gondii. Usually asymptomatic in immunocompetent humans, toxoplasmosis is a serious clinical and veterinary problem often leading to lethal damage in an infected host. In order to overcome the exceptionally strong clinical and socio-economic impact of Toxoplasma infection, the construction of an effective vaccine inducing full immunoprotection against the parasite is an urgent issue. In the last two decades many live attenuated, subunit and DNA-based vaccines against toxoplasmosis have been studied, however only partial protection conferred by vaccination against chronic as well as acute infection has been achieved. Among various immunization strategies, no viable subunit vaccines based on recombinant secretory (ROP2, ROP4 and GRA4) and surface (SAG1) T. gondii proteins have been found as attractive tools for further studies. This is due to their high, but still partial, protective efficacy correlated with the induction of cellular and humoral immune responses.
PMCID: PMC3489714  PMID: 22892593
immunoprotection; live attenuated vectors; recombinant Toxoplasma gondii proteins; subunit vaccines; toxoplasmosis
8.  Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria 
Toxoplasmosis is a worldwide-distributed infection which is mostly asymptomatic but can cause serious health problems in congenitally-infected newborns and immunecompromised individuals. Research is undergoing both to improve Toxoplasma serological tests, which play the main role in laboratory diagnosis of the infection, and develop an effective vaccine to prevent the infection. Some studies showed usefulness of rhoptry protein 1 (ROP1) antigen of Toxoplasma gondii (T. gondii) in serodiagnosis of the infection and induction of protective immunity. The purpose of this study was to produce recombinant ROP1 and evaluate its antigenicity against human infected sera.
DNA encoding ROP1, amino acids 171 to 574, was obtained from T. gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-15b. rROP1 was expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography.
DNA sequencing showed 99% similarity between the cloned sequence and the corresponding sequence in Gene bank. Results indicated the proper antigenicity of rROP1. Sera from Toxoplasma infected individuals specifically recognized rROP1 in Western blotting.
rROP1 is antigenic toward human infected sera and can be used in studies for development of both a Toxoplasma serological test and a protective vaccine.
PMCID: PMC3838767  PMID: 24285997
Gene expression; Purification; ROP1 protein; Toxoplasma gondii
9.  Protective Immunity Induced by Toxoplasma gondii Rhoptry Protein 16 against Toxoplasmosis in Mice ▿  
Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly (P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.
PMCID: PMC3019779  PMID: 21106780
10.  DNA vaccination with a gene encoding Toxoplasma gondii GRA6 induces partial protection against toxoplasmosis in BALB/c mice 
Parasites & Vectors  2011;4:213.
Infection with the protozoan Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Protection from acute toxoplasmosis is known to be mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity have been poorly defined. The T. gondii dense granule protein 6 (GRA6), recently proved to be highly immunogenic and produces fully immune protection in T. gondii infected BALB/c mice with an H-2Ld gene. The CD8+ T cell response of H-2Ld mice infected by the T. gondii strain seemed to target entirely to a single GRA6 peptide HF10-H-2Ld complex.
To determine whether a GRA6-based DNA vaccine can elicit protective immune responses to T. gondii in BALB/c mice, we constructed a eukaryotic expression vector pcDNA3.1-HisGRA6 and tested its immunogenicity in a mouse model. BALB/c mice were vaccinated intramuscularly with three doses of GRA6 DNA and then challenged with a lethal dose of T. gondii RH strain tachyzoites. All immunized mice developed high levels of serum anti-GRA6 IgG antibodies, and in vitro splenocyte proliferation was strongly enhanced in mice adjuvanted with levamisole (LMS). Immunization with pcDNA3.1-HisGRA6 with LMS resulted in 53.3% survival of challenged BALB/c mice as compared to 40% survival of BALB/c without LMS. Additionally, immunized Kunming mice without an allele of H-2Ld failed to survive.
Our result supports the concept that the acquired immune response is MHC restricted. This study has a major implication for vaccine designs using a single antigen in a population with diverse MHC class I alleles.
PMCID: PMC3229464  PMID: 22070984
11.  Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice 
Parasitology Research  2012;111(5):2001-2009.
A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4 weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50 ± 0.22 to 7.60 ± 0.74 days); induced high levels of IgG antibody (from 0.252 ± 0.080 to 0.790 ± 0.083), IFN-gamma (from 598.74 ± 67.50 to 853.77 ± 66.74 pg/ml), and IL-2 (from 89.44 ± 10.66 to 192.24 ± 19.90 pg/ml); changed the CD4+/CD8+ lymphocyte ratio (from 1.81 ± 0.14 to 1.09 ± 0.19); and stimulated NK cell-killing activity (from 46.81 ± 3.96 to 64.15 ± 7.71 %). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.
PMCID: PMC3480591  PMID: 22167371
12.  Protective Efficacy of a Toxoplasma gondii Rhoptry Protein 13 Plasmid DNA Vaccine in Mice 
Clinical and Vaccine Immunology : CVI  2012;19(12):1916-1920.
Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-γ), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.
PMCID: PMC3535859  PMID: 23015648
13.  Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation 
PLoS ONE  2012;7(12):e51448.
Host defense against the parasite Toxoplasma gondii requires the cytokine interferon-gamma (IFNγ). However, Toxoplasma inhibits the host cell transcriptional response to IFNγ, which is thought to allow the parasite to establish a chronic infection. It is not known whether all strains of Toxoplasma block IFNγ-responsive transcription equally and whether this inhibition occurs solely through the modulation of STAT1 activity or whether other transcription factors are involved. We find that strains from three North American/European clonal lineages of Toxoplasma, types I, II, and III, can differentially modulate specific aspects of IFNγ signaling through the polymorphic effector proteins ROP16 and GRA15. STAT1 tyrosine phosphorylation is activated in the absence of IFNγ by the Toxoplasma kinase ROP16, but this ROP16-activated STAT1 is not transcriptionally active. Many genes induced by STAT1 can also be controlled by other transcription factors and therefore using these genes as specific readouts to determine Toxoplasma inhibition of STAT1 activity might be inappropriate. Indeed, GRA15 and ROP16 modulate the expression of subsets of IFNγ responsive genes through activation of the NF-κB/IRF1 and STAT3/6 transcription factors, respectively. However, using a stable STAT1-specific reporter cell line we show that strains from the type I, II, and III clonal lineages equally inhibit STAT1 transcriptional activity. Furthermore, all three of the clonal lineages significantly inhibit global IFNγ induced gene expression.
PMCID: PMC3519884  PMID: 23240025
14.  Protective Immune Response in BALB/c Mice Induced by DNA Vaccine of the ROP8 gene of Toxoplasma gondii 
Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.
PMCID: PMC3752752  PMID: 23509124
15.  Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite 
PLoS Pathogens  2013;9(6):e1003449.
CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood.
Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells.
Author Summary
Toxoplasma gondii is a widespread intracellular parasite that can cause severe disease in immunocompromised individuals and lead to fetal abnormalities if contracted during pregnancy. Establishment of protective immunity relies on CD8 T cells, which recognize antigenic peptides presented by MHC class I molecules on the surface of T. gondii-infected cells. Intriguingly, while the proteome of T. gondii is large, CD8 T cell responses target a very limited set of peptides. These peptides can be ranked according to the magnitude of the associated CD8 response (from immunodominant down to subdominant). Yet, little is known about the rules that define their immunogenicity and the hierarchy of the associated T cell responses. Using a panel of genetically modified T. gondii where the GRA6 dominant antigen was mutated, we show that the C-terminal location of the epitope within the source antigen is the critical parameter for immunodominance. Interestingly, when placed at the C-terminus of GRA6, the subdominant status of an epitope can be overturned. Our results unravel the mechanisms that make parasite antigens accessible for the MHC I presentation pathway. They may help to ameliorate natural immune responses and improve vaccine design against intravacuolar pathogens.
PMCID: PMC3688528  PMID: 23818852
16.  Toxoplasma gondii Rhoptry 16 Kinase Promotes Host Resistance to Oral Infection and Intestinal Inflammation Only in the Context of the Dense Granule Protein GRA15 
Infection and Immunity  2013;81(6):2156-2167.
Toxoplasma gondii transmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (strain II+ROP16I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by a lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16I strain occurred independently of STAT6 and the T cell coinhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, coexpression of ROP16 and GRA15 enhanced parasite susceptibility within tumor necrosis factor alpha/gamma interferon-stimulated macrophages in a STAT3/6-independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from mice orally challenged with strain II+ROP16I suggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 in Toxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.
PMCID: PMC3676013  PMID: 23545295
17.  A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins 
PLoS Biology  2012;10(7):e1001358.
A secreted kinase from the parasitic protozoan, Toxoplasma gondii, is shown to cooperate with a phylogenetically related pseudokinase to phosphorylate and inactivate a mouse resistance protein of the IRG system.
The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.
Author Summary
Toxoplasma gondii is an intracellular parasitic protozoan infecting about a third of humankind. Humans, however, are unlikely to be important hosts in terms of the evolution of the parasite because, for completion of the parasite sexual cycle, the infected animal must be eaten by a cat. Therefore, important intermediate hosts for Toxoplasma are species like mice that are often preyed on by cats. Mice use an intracellular resistance mechanism, the IRG proteins, against Toxoplasma. In turn, Toxoplasma appears to have evolved a virulence factor, a protein kinase called ROP18, that inactivates IRG proteins. We show that ROP18 does not act alone. It needs help from the ROP5 pseudokinases, proteins related to ROP18 but without enzymatic activity. ROP5 pseudokinases assist ROP18 by binding to the IRG proteins, holding them inactive, and laying them open to enzymatic attack and likely permanent inactivation by the ROP18 kinase. This mechanism illustrates the principle that members of an enzyme family can lose their enzymatic activity and evolve into regulators or co-factors for the active members.
PMCID: PMC3393671  PMID: 22802726
18.  Recombinant Antigens To Detect Toxoplasma gondii-Specific Immunoglobulin G and Immunoglobulin M in Human Sera by Enzyme Immunoassay 
Journal of Clinical Microbiology  2000;38(3):1144-1150.
We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94.5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.
PMCID: PMC86359  PMID: 10699010
19.  GRA2 and ROP1 Recombinant Antigens as Potential Markers for Detection of Toxoplasma gondii-Specific Immunoglobulin G in Humans with Acute Toxoplasmosis▿  
A goal of the current study was to evaluate serological applications of Toxoplasma gondii GRA2 and rhoptry protein 1 (ROP1) antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at the N and C terminals were obtained using an Escherichia coli expression system. Purification by one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per liter of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 serum samples from patients with acute toxoplasmosis was compared to a panel of 90 serum samples from individuals with past infection. The results indicated that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently in samples from individuals with acute infections (100% and 94.6%, respectively) than in samples from individuals with chronic infections (22.5% and 15.5%, respectively). These results suggest that immunoglobulin G antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.
PMCID: PMC2668274  PMID: 19225074
20.  Effects of specific monoclonal antibodies to dense granular proteins on the invasion of Toxoplasma gondii in vitro and in vivo 
Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins). Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAG1, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoites pretreated with anti-GRA6 or anti-SAG1 mAb were significantly increased. Mice that received tachyzoites pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.
PMCID: PMC2721072  PMID: 11590913
Toxoplasma gondii; monoclonal antibody; dense-granule molecules; complement; major surface protein; host cell invasion
21.  Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It 
PLoS ONE  2011;6(9):e24434.
IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.
PMCID: PMC3169597  PMID: 21931713
22.  Seroreactivity to and Avidity for Recombinant Antigens in Toxoplasmosis 
To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinant Toxoplasma gondii antigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned in Escherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points.
PMCID: PMC1182181  PMID: 16085916
23.  Role of Cytokines and Major Histocompatibility Complex Restriction in Mouse Resistance to Infection with a Natural Recombinant Strain (Type I-III) of Toxoplasma gondii  
Infection and Immunity  2003;71(11):6392-6401.
Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype “d” were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.
PMCID: PMC219541  PMID: 14573660
24.  Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection ▿ † 
Eukaryotic Cell  2011;10(9):1193-1206.
Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
PMCID: PMC3187049  PMID: 21531875
25.  The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4▿  
Infection and Immunity  2008;76(12):5853-5861.
The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.
PMCID: PMC2583583  PMID: 18809661

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