Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis

BMC Infectious Diseases

Shenyi He²  Xing-Quan Zhu¹  Qunli Zhao²  Hua Cong²  Huaiyu Zhou²  Lin Wang²  Yali Han²  Yang Bai²  Min Sun²  Min Meng²  Gang Lv²  Aihua Zhou³  Guanghui Zhao² 

[1] State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province, P. R. China;Department of Parasitology, Shandong University School of Medicine, Jinan, Shandong Province 250012, P R China;Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University School of Medicine, Jinan, Shandong Province 250021, P R China

关键词: Toxoplasmosis;    Vaccine;    Bioinformatics;    Cathepsin proteases;    Toxoplasma gondii;   

Others  :  1148505
DOI  :  10.1186/1471-2334-13-207

 received in 2013-03-01, accepted in 2013-05-01,  发布年份 2013

【 摘 要 】

Background

Toxoplasma gondii, an obligate intracellular apicomplexan parasite, infects a wide range of warm-blooded animals including humans. T. gondii expresses five members of the C1 family of cysteine proteases, including cathepsin B-like (TgCPB) and cathepsin L-like (TgCPL) proteins. TgCPB is involved in ROP protein maturation and parasite invasion, whereas TgCPL contributes to proteolytic maturation of proTgM2AP and proTgMIC3. TgCPL is also associated with the residual body in the parasitophorous vacuole after cell division has occurred. Both of these proteases are potential therapeutic targets in T. gondii. The aim of this study was to investigate TgCPB and TgCPL for their potential as DNA vaccines against T. gondii.

Methods

Using bioinformatics approaches, we analyzed TgCPB and TgCPL proteins and identified several linear-B cell epitopes and potential Th-cell epitopes in them. Based on these results, we assembled two single-gene constructs (TgCPB and TgCPL) and a multi-gene construct (pTgCPB/TgCPL) with which to immunize BALB/c mice and test their effectiveness as DNA vaccines.

Results

TgCPB and TgCPL vaccines elicited strong humoral and cellular immune responses in mice, both of which were Th-1 cell mediated. In addition, all of the vaccines protected the mice against infection with virulent T. gondii RH tachyzoites, with the multi-gene vaccine (pTgCPB/TgCPL) providing the highest level of protection.

Conclusions

T. gondii CPB and CPL proteases are strong candidates for development as novel DNA vaccines.

【 授权许可】

   
2013 Zhao et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Dubey JP, Su C: Population biology of Toxoplasma gondii: what’s out and where did they come from. Memórias do Instituto Oswaldo Cruz 2009, 104(2):190-195.
• [2]Dubey JP: The history of Toxoplasma gondii–the first 100 years. J Eukaryot Microbiol 2008, 55(6):467-475.
• [3]Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet 2004, 363(9425):1965-1976.
• [4]Robert-Gangneux F, Darde ML: Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 2012, 25(2):264-296.
• [5]Luft BJ, Hafner R, Korzun AH, Leport C, Antoniskis D: Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome. N Engl J Med 1993, 329(14):995-1000.
• [6]Afonso C, Paixão VB, Costa RM: Chronic Toxoplasma infection modifies the structure and the risk of host behavior. PLoS One 2012, 7(3):e32489 p.
• [7]Dass SA, Vasudevan A, Dutta D, Soh LJ, Sapolsky RM: Protozoan parasite Toxoplasma gondii manipulates mate choice in rats by enhancing attractiveness of males. PLoS One 2011, 6(11):e27229 p.
• [8]Haroon F, Händel U, Angenstein F, Goldschmidt J, Kreutzmann P: Toxoplasma gondii actively inhibits neuronal function in chronically infected mice. PLoS One 2012, 7(4):e35516 p.
• [9]Bhopale GM: Development of a vaccine for toxoplasmosis: current status. Microbes and Infection 2003, 5(5):457-462.
• [10]Hiszczynska-Sawicka E, Holec-Gasior L, Kur J: DNA vaccines and recombinant antigens in prevention of Toxoplasma gondii infections—current status of the studies. Wiadomości Parazytologiczne 2009, 55(2):125-139.
• [11]Liu Q, Singla LD, Zhou H: Vaccines against Toxoplasma gondii: status, challen ges and future directions. Hum Vaccin Immunother 2012, 8(9):1305-1308.
• [12]Barrett AJ, Kirschke H: Cathepsin B, Cathepsin H, and Cathepsin L. Methods Enzymol 1981, 80:535-561.
• [13]Amuthan G, Biswas G, Zhang SY, Klein-Szanto A, Vijayasarathy C: Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion. EMBO J 2001, 20(8):1910-1920.
• [14]Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA: Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Investig 2000, 106(9):1127-1137.
• [15]Dou Z, Carruthers VB: Cathepsin proteases in Toxoplasma gondii. Advances in Experimental Medicine and Biology 2011, 712:49-61.
• [16]Que X, Ngo H, Lawton J, Gray M, Liu Q: The cathepsin B of Toxoplasma gondii, toxopain-1, is critical for parasite invasion and rhoptry protein processing. J Biol Chem 2002, 277(28):25791-25797.
• [17]Parussini F, Coppens I, Shah PP, Diamond SL, Carruthers VB: Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol Microbiol 2010, 76(6):1340-1357.
• [18]Miranda K, Pace DA, Cintron R, Rodrigues JC, Fang J: Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol Microbiol 2010, 76(6):1358-1375.
• [19]Huang R, Que X, Hirata K, Brinen LS, Lee JH: The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin. Mol Biochem Parasitol 2009, 164(1):86-94.
• [20]Harper JM, Huynh MH, Coppens I, Parussini F, Moreno S: A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Molecular Biology of the Cell 2006, 17(10):4551-4563.
• [21]El Hajj H, Papoin J, Cérède O, Garcia-Réguet N, Soête M: Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes. Eukaryotic Cell 2008, 7(6):1019-1028.
• [22]Rabenau KE, Sohrabi A, Tripathy A, Reitter C, Ajioka JW: TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2. Mol Microbiol 2001, 41(3):537-547.
• [23]Brydges SD, Sherman GD, Nockemann S, Loyens A, Däubener W: Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol Biochem Parasitol 2000, 111(1):51-66.
• [24]Que X, Wunderlich A, Joiner KA, Reed SL: Toxopain-1 is critical for infection in a novel chicken embryo model of congenital toxoplasmosis. Infect Immun 2004, 72(5):2915-2921.
• [25]Teo CF, Zhou XW, Bogyo M, Carruthers VB: Cysteine protease inhibitors block Toxoplasma gondii microneme secretion and cell invasion. Antimicrob Agents Chemother 2007, 51(2):679-688.
• [26]Larson ET, Parussini F, Huynh MH, Giebel JD, Kelley AM: Toxoplasma gondii cathepsin l is the primary target of the invasion inhibitory compound LHVS. J Biol Chem 2009, 284(39):26839-26850.
• [27]Gao J, Faraggi E, Zhou Y, Ruan J, Kurgan L: BEST: improved prediction of B-cell epitopes from antigen sequences. PLoS One 2012, 7(6):e40104 p.
• [28]Van Regenmortel MH: What is a B-cell epitope? Methods in Molecular Biology 2009, 524:3-20.
• [29]Tong JC, Tammi MT: Prediction of protein allergenicity using local description of amino acid sequence. Front Biosci 2008, 13:6072-6078.
• [30]Carter JM, Loomis-Price L: B cell epitope mapping using synthetic peptides. Current Protoc Immunol 2004. Chapter 9:Unit 9.4
• [31]Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982, 157(1):105-132.
• [32]Welling GW, Weijer WJ, van der Zee R, Welling-Wester S: Prediction of sequential antigenic regions in proteins. FEBS Lett 1985, 188(2):215-218.
• [33]Subramani A, Floudas CA: Structure prediction of loops with fixed and flexible stems. The Journal of Physical Chemistry. B 2012, 116(23):6670-6682.
• [34]Gershoni JM, Stern B, Denisova G: Combinatorial libraries, epitope structure and the prediction of protein conformations. Immunol Today 1997, 18(3):108-110.
• [35]El-Kady IM: T-cell immunity in human chronic toxoplasmosis. J Egypt Soc Parasitol 2011, 41(1):17-28.
• [36]Bhasin M, Lata S, Raghava GP: Searching and mapping of T-cell epitopes, MHC binders, and TAP binders. Methods in Molecular Biology 2007, 409:95-112.
• [37]Vider-Shalit T, Louzoun Y: MHC-I prediction using a combination of T cell epitopes and MHC-I binding peptides. J Immunol Methods 2011, 374(1–2):43-46.
• [38]Zhou H, Min J, Zhao Q, Gu Q, Cong H: Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant. Vaccine 2012, 30(10):1800-1806.
• [39]Cui X, Lei T, Yang D, Hao P, Li B, Liu Q: Toxoplasma gondii immune mapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplasmosis. Vaccine 2012, 30(13):2282-2287.
• [40]Min J, Qu D, Li C, Song X, Zhao Q: Enhancement of protective immune responses induced by Toxoplasma gondii dense granule antigen 7 (GRA7) against toxoplasmosis in mice using a prime-boost vaccination strategy. Vaccine 2012, 30(38):5631-5636.
• [41]Constant S, Pfeiffer C, Woodard A, Pasqualini T, Bottomly K: Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells. The Journal of Experimental Medicine 1995, 182(5):1591-1596.
• [42]Chaturvedi P, Yu Q, Southwood S, Sette A, Singh B: Peptide analogs with different affinites for MHC alter the cytokine profile of T helper cells. Int Immunol 1996, 8(5):745-755.
• [43]Romano P, Giugno R, Pulvirenti A: Tools and collaborative environments for bioinformatics research. Briefings in Bioinformatics 2011, 12(6):549-561.
• [44]Martin DM, Berriman M, Barton GJ: GOtcha: a new method for prediction of protein function assessed by the annotation of seven genomes. BMC Bioinforma 2004, 5:178. BioMed Central Full Text
• [45]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(4):458-464.
• [46]Mannie MD: Do holes in the T-cell repertoire have a center-surround regulatory structure? A rationale for the bifurcation of the Th1 and Th2 pathways of differentiation. Medical Hypotheses 1997, 8(3):261-265.
• [47]Schaeffer EB, Sette A, Johnson DL, Bekoff MC, Smith JA, Grey HM, Buus S: Relative contribution of “determinant selection” and “holes in the T-cell repertoire” to T-cell responses. Proc Natl Acad Sci USA 1989, 86(12):4649-4653.