Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from<i>Toxoplasma gondii</i>

Grujic, Ognjen; Grigg, Michael E.; Boulanger, Martin J.

doi:10.1107/s1744309108010920

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Expression, Purification, and Crystallization-BSR4 from the type II clonal lineage of T. gondii was recombinantly produced in insect cells, purified, and crystallized as described previously (22). Briefly, insect cell medium containing secreted BSR4 was concentrated by tangential flow, and the recombinant protein was purified to homogeneity using nickel-nitrilotriacetic acid and Superdex 75 size exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Crawford

Grujic

Bruic

et al. 2009

Journal of Biological Chemistry

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Toxoplasma gondii is an obligate intracellular protozoan parasite that infects nearly one-third of the human population. The success of T. gondii is based on its complex life cycle; a lytic tachyzoite form disseminates infection, whereas an encysted bradyzoite form establishes a latent, chronic infection. Persistence and transmissibility is central to the survival of the parasite and is, in part, mediated by a family of antigenically distinct surface antigen glycoprotein (SAG)-related sequences (SRS) adhesins that play a dual role in host cell attachment and host immune evasion. More than 160 members of the SRS family have been identified with only the tachyzoite-expressed SAG1 structurally characterized. Here we report the first structural description of the bradyzoite adhesin BSR4 using x-ray crystallography and small angle x-ray scattering. The 1.90-Å crystal structure of BSR4 reveals an architecture comprised of tandem ␤ sandwich domains organized in a head to tail fashion with the N-terminal domain responsible for dimer formation. A restructured topology in BSR4 results in a ligand-binding site that is significantly reorganized in both structure and chemistry relative to SAG1, consistent with BSR4 binding a distinct physiological ligand. The small angle x-ray scattering solution structure of BSR4 highlights a potentially important structural role for the interdomain polymorphic linker that imparts significant flexibility that may promote structural adaptation during ligand binding. This study reveals an unexpected level of structural diversity within the SRS superfamily and provides important insight into the role of these virulence factors.The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is a serious global pathogen that infects nearly one-third of the human population (1-3). T. gondii infections can be lethal to a developing fetus and immunocompromised, cancer, AIDS, and organ transplant patients. Clinical features range from asymptomatic infection to lymphadenopathy, ileitis, encephalitis, and/or blinding ocular infections in both children and adults with the severity of symptoms tending to increase with age (1, 4 -9). During infection, T. gondii cycles between the rapidly growing, lytic tachyzoite stage and the slow growing, cyst-forming bradyzoite stage. Upon challenge by the immune system, the tachyzoites, which are responsible for rapid dissemination of the parasite, differentiate into encysted bradyzoites that promote chronic infection. The molecular switches that regulate interconversion of the two parasitic stages in the host remain largely undefined. Despite the complex life cycle of T. gondii, it exists as a single species and has been referred to as one of the most, if not the most, successful protozoan parasite of animals on this planet (3).The success of T. gondii is largely due to its ability to infect a broad range of host cells (10), which is, in part, mediated by a family of developmentally expressed, antigenically distinct surface antigen glycoprotein (SAG)...

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Section: Methodsmentioning

confidence: 99%

Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Crawford

Grujic

Bruic

et al. 2009

Journal of Biological Chemistry

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“…Initial crystallization trials produced a P6 crystal form that diffracted to 1.60 Å. Attempts to solve the SporoSAG structure using molecular replacement with the individual core domains of SAG1 (25) and BSR4 (28,46) as search models were unsuccessful. Additional rounds of crystallization produced a cadmium-derivatized P2 1 2 1 2 crystal form from which we were able to generate high quality phases using cadmium single-wavelength anomalous dispersion.…”

Section: Figure 2 T Gondii-infected Patients Fail To Mount Antibodymentioning

confidence: 99%

Structural and Functional Characterization of SporoSAG

Crawford

Lamb

Wasmuth

et al. 2010

Journal of Biological Chemistry

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Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG (surface antigen glycoprotein) is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG clusters with the SAG2 subfamily of the SAG1-related superfamily (SRS) and is non-polymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 Å resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS ␤-sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the ␤-sandwich fold and may have important implications for multimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.

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“…Os estudos na literatura com foco em antígenos de T. gondii exclusivamente expressos na forma bradizoíta são escassos. Pesquisas anteriores mostraram que a proteína BSR4 está provavelmente relacionada com a manutenção do parasita no hospedeiro ( VAN et al, 2007;BOULANGER, 2008). O membro SRS9 da família de proteínas de SRS desempenha um papel importante na persistência do parasita no tecido cerebral e no controle da replicação do parasita no intestino (KIM; KARASOV; BOOTHROYD, 2007).…”

Section: Imunoprofilaxiaunclassified

Avaliação da resposta imune de camundongos C57BL/6 imunizados com antígeno solúvel ou com as proteínas recombinantes SAG2A e BSR4 de Toxoplasma gondii formuladas em lipossomos

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Agradeço em especial ao Prof. Dr. José Roberto Mineo que iniciou esta trajetória de pesquisa antigênica de Toxoplasma gondii, e assim propiciou o desenvolvimento científico de inúmeros trabalhos, incluindo este.A meu orientador Jair Pereira da Cunha Júnior principalmente pelo ensinamento, mas também pela paciência, solidariedade e generosidade.Ao Silas Silva Santana meu eterno coorientador de suma importância na minha formação profissional.Ao Prof. Dr. Carlos Priminho Pirovani por ceder gentilmente as proteínas utilizadas nesta pesquisa, sem ele este trabalho não poderia ser realizado.

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Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 fromToxoplasma gondii

Cited by 4 publications

References 21 publications

Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Structural and Functional Characterization of SporoSAG

Avaliação da resposta imune de camundongos C57BL/6 imunizados com antígeno solúvel ou com as proteínas recombinantes SAG2A e BSR4 de Toxoplasma gondii formuladas em lipossomos

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