SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts

Particle and Fibre Toxicology

Tiago WP Mineo²  José R Mineo²  Deise AO Silva²  Carlos P Pirovani¹  João S Silva³  Fernanda M Santiago²  Murilo V Silva²  Thyago HS Cardoso¹  Jair P Cunha²  Arlindo G Macêdo² 

[1] Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, Rodovia Ilhéus/Itabuna km 16, Ilhéus, Bahia 45.662-900, Brazil;Laboratório de Imunoparasitologia “Dr. Mário Endsfeldz Camargo”, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Pará 1720 – Bloco 4C, Campus Umuarama, Uberlândia, Minas Gerais 38.400-902, Brazil;Laboratório de Imunoparasitologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo 14.015-000, Brazil

关键词: Recombinant protein;    Monoclonal antibodies;    SAG2A;    Protein modeling;    Toxoplasmosis;   

Others  :  1227018
DOI  :  10.1186/1756-3305-6-163

 received in 2013-04-20, accepted in 2013-05-26,  发布年份 2013

【 摘 要 】

Background

Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts.

Methods

To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays.

Results

Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells.

Conclusions

Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.

【 授权许可】

   
2013 Macêdo et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Blader IJ, Saeij JP: Communication between Toxoplasma gondii and its host: impact on parasite growth, development, immune evasion, and virulence. APMIS 2009, 117:458-476.
• [2]Weiss LM, Dubey JP: Toxoplasmosis: A history of clinical observations. Int J Parasitol 2009, 39:895-901.
• [3]Tenter AM, Heckeroth AR, Weiss LM: Toxoplasma gondii: from animals to humans. Int J Parasitol 2000, 30:1217-1258.
• [4]Montoya JG, Rosso F: Diagnosis and management of toxoplasmosis. Clin Perinatol 2005, 32:705-726.
• [5]Reese ML, Boothroyd JC: A helical membrane-binding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic 2009, 10:1458-1470.
• [6]Gong H, Kobayashi K, Sugi T, Takemae H, Kurokawa H, Horimoto T, Akashi H, Kato K: A Novel PAN/Apple Domain-Containing Protein from Toxoplasma gondii: Characterization and Receptor Identification. PLoS One 2012, 7:e30169.
• [7]Ong YC, Boyle JP, Boothroyd JC: Strain-dependent host transcriptional responses to Toxoplasma infection are largely conserved in mammalian and avian hosts. PLoS One 2011, 6:e26369.
• [8]Lekutis C, Ferguson DJ, Grigg ME, Camps M, Boothroyd JC: Surface antigens of Toxoplasma gondii: variations on a theme. Int J Parasitol 2001, 31:1285-1292.
• [9]Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM: Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii. Mol Cell Proteomics 2011, 10:M110.000745.
• [10]Fauquenoy S, Hovasse A, Sloves PJ, Morelle W, Dilezitoko Alayi T, Slomianny C, Werkmeister E, Schaeffer C, Van Dorsselaer A, Tomavo S: Unusual N-glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility. Mol Cell Proteomics 2011, 10:M111.008953.
• [11]Fritz HM, Bowyer PW, Bogyo M, Conrad PA, Boothroyd JC: Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance. PLoS One 2012, 7:e29955.
• [12]Hajagos BE, Turetzky JM, Peng ED, Cheng SJ, Ryan CM, Souda P, Whitelegge JP, Lebrun M, Dubremetz JF, Bradley PJ: Molecular dissection of novel trafficking and processing of the Toxoplasma gondii rhoptry metalloprotease toxolysin-1. Traffic 2012, 13:292-304.
• [13]Bai Y, He S, Zhao G, Chen L, Shi N, Zhou H, Cong H, Zhao Q, Zhu XQ: Toxoplasma gondii: Bioinformatics analysis, cloning and expression of a novel protein TgIMP1. Exp Parasitol 2012, 132:458-464.
• [14]Crawford J, Tonkin ML, Grujic O, Boulanger MJ: Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii. J Biol Chem 2010, 285:15644-15652.
• [15]Saouros S, Dou Z, Henry M, Marchant J, Carruthers VB, Matthews S: Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain. J Biol Chem 2012, 287:36029-36040.
• [16]Tonkin ML, Roques M, Lamarque MH, Pugnière M, Douguet D, Crawford J, Lebrun M, Boulanger MJ: Host cell invasion by apicomplexan parasites: insigts from the co-structure of AMA1 with RON2 peptide. Science 2011, 333:463-467.
• [17]Lekutis C, Ferguson DJ, Boothroyd JC: Toxoplasma gondii: identification of a developmentally regulated family of genes related to SAG2. Exp Parasitol 2000, 96:89-96.
• [18]Cunha-Júnior JP, Silva DA, Silva NM, Souza MA, Souza GR, Prudencio CR, Pirovani CP, Cezar M, Cascardo J, Barbosa BF, Goulart LR, Mineo JR: A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection. Immunobiology 2010, 215:26-37.
• [19]Fux B, Rodrigues CV, Portela RW, Silva NM, Su C, Sibley D, Vitor RW, Gazzinelli RT: Role of cytokines and major histocompatibility complex restriction in mouse resistance to infection with a natural recombinant strain (type I-III) of Toxoplasma gondii. Infect Immun 2003, 71:6392-6401.
• [20]Liu S, Tobias R, McClure S, Styba G, Shi Q, Jackowski G: Removal of endotoxin from recombinant protein preparations. Clin Biochem 1997, 30:455-463.
• [21]Behnke MS, Khan A, Wootton JC, Dubey JP, Tang K, Sibley LD: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc Natl Acad Sci USA 2011, 108:9631-9636.
• [22]Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 1992, 89:10915-10919.
• [23]He XL, Grigg ME, Boothroyd JC, Garcia KC: Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily. Nat Struct Biol 2002, 9:606-611.
• [24]Crawford J, Grujic O, Bruic E, Czjzek M, Grigg ME, Boulanger MJ: Structural characterization of the bradyzoite surface antigen (BSR4) from Toxoplasma gondii, a unique addition to the surface antigen glycoprotein 1-related superfamily. J Biol Chem 2009, 284:9192-9198.
• [25]Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM: AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996, 8:477-486.
• [26]Melo F, Devos D, Depiereux E, Feytmans E: ANOLEA: a www server to assess protein structures. Proc Int Conf Intell Syst Mol Biol 1997, 5:187-190.
• [27]Melo F, Feytmans E: Assessing protein structures with a non-local atomic interaction energy. J Mol Biol 1998, 277:1141-1152.
• [28]Ramachandran GN, Ramakrishnan C, Sasisekharan V: Stereochemistry of polypeptide chain configurations. J Mol Biol 1963, 7:95-99.
• [29]Marim FM, Silveira TN, Lima DS Jr, Zamboni DS: A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells. PLoS One 2010, 5:e15263.
• [30]Mineo TW, Oliveira CJ, Gutierrez FR, Silva JS: Recognition by Toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum. Immunol Cell Biol 2010, 88:825-833.
• [31]Sherman MP, Aeberhard EE, Wong VZ, Griscavage JM, Ignarro LJ: Pyrrolidine dithiocarbamate inhibits induction of nitric oxide synthase activity in rat alveolar macrophages. Biochem Biophys Res Commun 1993, 191:1301-1308.
• [32]Béla SR, Oliveira Silva DA, Cunha-Júnior JP, Pirovani CP, Chaves-Borges FA, Reis de Carvalho F, Carrijo de Oliveira T, Mineo JR: Use of SAG2A recombinant Toxoplasma gondii surface antigen as a diagnostic marker for human acute toxoplasmosis: analysis of titers and avidity of IgG and IgG1 antibodies. Diagn Microbiol Infect Dis 2008, 62:245-254.
• [33]Santana SS, Silva DA, Vaz LD, Pirovani CP, Barros GB, Lemos EM, Dietze R, Mineo JR, Cunha-Junior JP: Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis. Immunol Lett 2012, 143:193-201.
• [34]Silva DA, Silva NM, Mineo TW, Pajuaba Neto AA, Ferro EA, Mineo JR: Heterologous antibodies to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain. Vet Parasitol 2002, 107:181-195.
• [35]Moreno B, Collantes-Fernández E, Villa A, Navarro A, Regidor-Cerrillo J, Ortega-Mora LM: Occurrence of Neospora caninum and Toxoplasma gondii infections in ovine and caprine abortions. Vet Parasitol 2012, 187:312-318.
• [36]Leng J, Butcher BA, Denkers EY: Dysregulation of macrophage signal transduction by Toxoplasma gondii: past progress and recent advances. Parasite Immunol 2009, 31:717-728.
• [37]Buzoni-Gatel D, Werts C: Toxoplasma gondii and subversion of the immune system. Trends Parasitol 2006, 22:448-452.
• [38]Leng J, Butcher BA, Egan CE, Abdallah DS, Denkers EY: Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered macrophage activation. J Immunol 2009, 182:489-497.
• [39]Boothroyd JC: Toxoplasma gondii: 25 years and 25 major advances for the field. Int J Parasitol 2009, 39:935-946.
• [40]Kong JT, Grigg ME, Uyetake L, Parmley S, Boothroyd JC: Serotyping of Toxoplasma gondii infections in humans using synthetic peptides. J Infect Dis 2003, 187:1484-1495.
• [41]Taylor S, Barragan A, Su C, Fux B, Fentress SJ, Tang K, Beatty WL, Hajj HE, Jerome M, Behnke MS, White M, Wootton JC, Sibley LD: A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 2006, 314:1776-1780.
• [42]Parmley SF, Gross U, Sucharczuk A, Windeck T, Sgarlato GD, Remington JS: Two alleles of the gene encoding surface antigen P22 in 25 strains of Toxoplasma gondii. J Parasitol 1994, 80:293-301.
• [43]Jung C, Lee CY, Grigg ME: The SRS superfamily of Toxoplasma surface proteins. Int J Parasitol 2004, 34:285-296.
• [44]Velge-Roussel F, Dimier-Poisson I, Buzoni-Gatel D, Bout D: Anti-SAG1 peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines. Parasitology 2001, 123:225-233.
• [45]Mineo JR, McLeod R, Mack D, Smith J, Khan IA, Ely KH, Kasper LH: Antibodies to Toxoplasma gondii major surface protein (SAG-1, P30) inhibit infection of host cells and are produced in murine intestine after peroral infection. J Immunol 1993, 150:3951-3964.
• [46]Gazzinelli RT, Eltoum I, Wynn TA, Sher A: Acute cerebral toxoplasmosis is induced by in vivo neutralization of TNF-alpha and correlates with the down-regulated expression of inducible nitric oxide synthase and other markers of macrophage activation. J Immunol 1993, 151:3672-3681.
• [47]Das S, Mukhopadhyay D: Intrinsically unstructured proteins and neurodegenerative diseases: conformational promiscuity at its best. IUBMB Life 2011, 63:478-488.
• [48]Ivanyi-Nagy R, Darlix JL: Intrinsic disorder in the core proteins of flaviviruses. Protein Pept Lett 2010, 17:1019-1025.
• [49]Dosztanyi Z, Csizmok V, Tompa P, Simon I: The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J Mol Biol 2005, 347:827-839.
• [50]Chen K, Kurgan LA, Ruan J: Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs. BMC Struct Biol 2007, 7:25.
• [51]Tompa P, Szasz C, Buday L: Structural disorder throws new light on moonlighting. Trends Biochem Sci 2005, 30:484-489.
• [52]Tompa P: The interplay between structure and function in intrinsically unstructured proteins. FEBS Lett 2005, 579:3346-3354.
• [53]Feng ZP, Zhang X, Han P, Arora N, Anders RF, Norton RS: Abundance of intrinsically unstructured proteins in P. falciparum and other apicomplexan parasite proteomes. Mol Biochem Parasitol 2006, 150:256-267.
• [54]Carvalho FR, Silva DA, Cunha-Júnior JP, Souza MA, Oliveira TC, Béla SR, Faria GG, Lopes CS, Mineo JR: Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera. Clin Vaccine Immunol 2008, 15:1265-1271.
• [55]Björkman C, Uggla A: Serological diagnosis of Neospora caninum infection. Int J Parasitol 1999, 29:1497-1507.
• [56]Staubli D, Nunez S, Sager H, Schares G, Gottstein B: Neospora caninum immunoblotting improves serodiagnosis of bovine neosporosis. Parasitol Res 2006, 99:648-658.
• [57]Silva DA, Lobato J, Mineo TW, Mineo JR: Evaluation of serological tests for the diagnosis of Neospora caninuminfection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity withToxoplasma gondii. Vet Parasitol 2007, 143:234-244.
• [58]Yan H, Yan H, Tao Y, Chen H, Li G, Gong W, Jiao H, Tian F, Ji M: Application and expression of Toxoplasma gondii surface antigen 2 (SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia coli strain. Trans R Soc Trop Med Hyg 2012, 106:356-362.
• [59]Mishima M, Xuan X, Shioda A, Omata Y, Fujisaki K, Nagasawa H, Mikami T: Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1. J Vet Med Sci 2001, 63:433-438.
• [60]Machado AV, Caetano BC, Barbosa RP, Salgado AP, Rabelo RH, Garcia CC, Bruna-Romero O, Escriou N, Gazzinelli RT: Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity. Vaccine 2010, 28:3247-3256.
• [61]Mendes EA, Caetano BC, Penido ML, Bruna-Romero O, Gazzinelli RT: MyD88-dependent protective immunity elicited by adenovirus 5 expressing the surface antigen 1 from Toxoplasma gondii is mediated by CD8(+) T lymphocytes. Vaccine 2011, 29:4476-4484.
• [62]Miller CM, Boulter NR, Ikin RJ, Smith NC: The immunobiology of the innate response to Toxoplasma gondii. Int J Parasitol 2009, 39:23-39.
• [63]Pollard AM, Knoll LJ, Mordue DG: The role of specific Toxoplasma gondii molecules in manipulation of innate immunity. Trends Parasitol 2009, 25:491-494.
• [64]Butcher BA, Fox BA, Rommereim LM, Kim SG, Maurer KJ, Yarovinsky F, Herbert DR, Bzik DJ, Denkers EY: Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS Pathog 2011, 7:e1002236.
• [65]Sacks D, Sher A: Evasion of innate immunity by parasitic protozoa. Nat Immunol 2002, 3:1041-1047.
• [66]Niedelman W, Gold DA, Rosowski EE, Sprokholt JK, Lim D, Farid Arenas A, Melo MB, Spooner E, Yaffe MB, Saeij JP: The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response. PLoS Pathog 2012, 8:e1002784.
• [67]Aliberti J, Reis e Sousa C, Schito M, Hieny S, Wells T, Huffnagle GB, Sher A: CCR5 provides a signal for microbial induced production of IL-12P40 by CD8 alpha+ dendritic cells. Nat Immunol 2000, 1:83-87.
• [68]Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A: TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 2005, 308:1626-1629.
• [69]Koblansky AA, Jankovic D, Oh H, Hieny S, Sungnak W, Mathur R, Hayden MS, Akira S, Sher A, Ghosh S: Recognition of Profilin by Toll-like Receptor 12 Is Critical for Host Resistance to Toxoplasma gondii. Immunity 2013, 38:119-130.
• [70]Andrade WA, Souza Mdo C, Ramos-Martinez E, Nagpal K, Dutra MS, Melo MB, Bartholomeu DC, Ghosh S, Golenbock DT, Gazzinelli RT: Combined Action of Nucleic Acid-Sensing Toll-like Receptors and TLR11/TLR12 Heterodimers Imparts Resistance to Toxoplasma gondii in Mice. Cell Host Microbe 2013, 13:42-53.
• [71]Plattner F, Soldati-Favre D: Hijacking of host cellular functions by the Apicomplexa. Annu Rev Microbiol 2008, 62:471-487.
• [72]Lambert H, Barragan A: Modelling parasite dissemination: host cell subversion and immune evasion by Toxoplasma gondii. Cell Microbiol 2010, 12:292-300.
• [73]Saeij JP, Coller S, Boyle JP, Jerome ME, White MW, Boothroyd JC: Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 2007, 445:324-327.
• [74]Ong YC, Reese ML, Boothroyd JC: Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. J Biol Chem 2010, 285:28731-28740.
• [75]Saeij JP, Boyle JP, Boothroyd JC: Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol 2005, 21:476-481.
• [76]Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama H, Kubo E, Ito H, Takaura M, Matsuda T, Soldati-Favre D, Takeda K: A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J Exp Med 2009, 206:2747-2760.
• [77]Jensen KD, Wang Y, Wojno ED, Shastri AJ, Hu K, Cornel L, Boedec E, Ong YC, Chien YH, Hunter CA, Boothroyd JC, Saeij JP: Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe 2011, 9:472-483.
• [78]Rosowski EE, Lu D, Julien L, Rodda L, Gaiser RA, Jensen KD, Saeij JP: Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J Exp Med 2011, 208:195-212.