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Identification of invasion proteins of Cryptosporidium parvum


Host cell interactions and invasion by Cryptosporidium is a complex process mediated by zoites ligand–host cell receptors. Knowledge of proteins involved in this process will enable entry level inhibitors to be tried as therapeutic agents. In the present study, invasion proteins of Cryptosporidium parvum were studied in vitro. Cryptosporidium sporozoites membrane proteins were isolated and Cy5 dye labelled. They were then allowed to interact with the intact host cells. The interacting proteins were identified using 2-dimensional gel electrophoresis followed by mass spectrometry analysis. Sixty-one proteins were identified including twenty-seven previously reported invasion proteins. The newly identified proteins such as serine/threonine protein kinase, PI4 kinase, Hsp105 and coiled coil may have their roles in the parasitic invasion process. Thus, a new approach was used in the study to identify the probable proteins involved in invasion and/or host–parasite interactions. The advantage of this method is that it takes only a months’ time instead of decades to identify these proteins involved in invasion process.

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  1. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V (2004) Complete genome sequence of the apicomplexa, C. parvum. Science 304:441–445

  2. Arrowood MJ, Sterling CR, Healey MC (1991) Imunofluorescent microscopical visualization of trails left by gliding Cryptosporidium parvum sporozoites. J Parasitol 77:315–317

  3. Baker RP, Wijetilaka R, Urban S (2006) Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria. PLoS Pathog 2:113

  4. Barker IK, Carbonnel PL (1974) Cryptosporidium agni sp. n. from lambs and Cryptosporidium bovissp. n. from a calf with observations on the oocyst. Z Parasitenk 44:289–298

  5. Barnes DA, Bonnin A, Huang JX, Gousset L, Wu J, Gut J, Doyle P, Dubremetz JF, Ward H, Petersen C (1998) A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Mol Biochem Parasitol 30:93–110

  6. Barta JR, Thompson RC (2006) What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends Parasitol 22:463–468

  7. Bhopale GM (2003) Pathogenesis of toxoplasmosis. Comp Immunol Microbiol Infect Dis 26:213–222

  8. Billker O, Lourido S, Sibley LD (2009) Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5:612–622

  9. Blackman MJ (2008) Malarial proteases and host cell egress: an ‘emerging’ cascade. Cell Microbiol 10:1925–1934

  10. Blanshard C, Jackson AM, Shanson DC, Francis N, Gazzard BG (1992) Cryptosporidiosis in HIV seropositive patients. Q J Med 85:813–823

  11. Brossier F, Jewett TJ, Sibley LD, Urban S (2005) A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc Natl Acad Sci USA 102:4146–4151

  12. Butaeva F, Paskerova G, Entzeroth R (2006) Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeusalbidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium. Tsitologiia 48:695–704

  13. Cevallos AM, Zhang X, Waldor MK, Jaison S, Zhou X, Tzipori S, Neutra MR, Ward HD (2000) Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infect Immun 68:4108–4116

  14. Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124:755–766

  15. Current WL, Reese NC, Ernst JV, Bailey WS, Heyman MB, Weinstein WM (1983) Human cryptosporidiosis in immunocompetent and immunodeficient persons: studies of an outbreak and experimental transmission. N Engl J Med 308:1252–1257

  16. Deng M, Templeton TJ, London NR, Bauer C, Schroeder AA, Abrahamsen MS (2002) Cryptosporidium parvum genes containing thrombospondin type 1 domains. Infect Immun 70:6987–6995

  17. Didigu CA, Doms RW (2012) Novel approaches to inhibit HIV entry. Viruses 4:309–324

  18. Dubremetz JF (2007) Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell Microbiol 9:841–848

  19. Elliott DA, Clark DP (2000) Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface. Infect Immun 68:2315–2322

  20. Fayer R, Speer CA, Dubey JP (1990) General biology of Cryptosporidium. In: Dubey JP, Speer CA, Fayer R (eds) Cryptosporidiosis of man and animals. CRC Press, Boca Raton, pp 1–29

  21. Forney JR, Yang S, Healey MC (1996) Protease activity associated with excystation of Cryptosporidium parvum oocysts. J Parasitol 82:889–892

  22. Forney JR, Vaughan DK, Yang S, Healey MC (1998) Actin-dependent motility in Cryptosporidium parvums porozoites. J Parasitol 84:908–913

  23. Gondeau C, Corradin G, Heitz F, Le Peuch C, Balbo A, Schuck P, Kajava AV (2009) The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer. Mol Biochem Parasitol 165:153–161

  24. Hunter PR, Nichols G (2002) Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin Microbiol Rev 15:145–154

  25. Jenkins MC, Fayer R, Tilley M, Upton SJ (1993) Cloning and expression of a cDNA encoding epitopes shared by 15 and 60 kilodalton proteins of Cryptosporidium parvum sporozoites. Infect Immun 61:2377–2382

  26. Kappe SHI, Buscaglia CA, Bergman LW, Coppens I, Nussenzweig V (2004) Apicomplexan gliding motility and host cell invasion: overhauling the motor model. Trends Parasitol 20:13–16

  27. Khramtsov NV, Tilley M, Blunt DS, Montelone BA, Upton SJ (1995) Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70. J Eukaryot Microbiol 42:416–422

  28. Kulangara C, Kajava AV, Corradin G, Felger I (2009) Sequence conservation in Plasmodium falciparum alpha-helical coiled coil domains proposed for vaccine development. PLoS ONE 4:e5419

  29. Kun JF, Hibbs AR, Saul A, McColl DJ, Coppel RL, Anders RF (1997) A putative Plasmodium falciparum exported serine/threonine protein kinase. Mol Biochem Parasitol 85:41–51

  30. Martin DN, Uprichard SL (2013) Identification of transferrin receptor 1 as a hepatitis C virus entry factor. Proc Natl Acad Sci 25:10777–10782

  31. Mazurie AJ, Alves JM, Ozaki LS, Zhou S, Schwartz DC, Buck GA (2013) Comparative genomics of Cryptosporidium. Int J Genomics. doi:10.1155/2013/832756

  32. McNamara CW, Lee MC, Lim CS, Lim SH, Roland J, Nagle A, Simon O, Yeung BK, Chatterjee AK, McCormack SL, Manary MJ, Zeeman AM, Dechering KJ, Kumar TR, Henrich PP, Gagaring K, Ibanez M, Kato N, Kuhen KL, Fischli C, Rottmann M, Plouffe DM, Bursulaya B, Meister S, Rameh L, Trappe J, Haasen D, Timmerman M, Sauerwein RW, Suwanarusk R, Russell B, Renia L, Nosten F, Tully DC, Kocken CH, Glynne RJ, Bodenreider C, Fidock DA, Diagana TT, Winzeler EA (2013) Targeting Plasmodium PI (4) K to eliminate malaria. Nature 504:248–253

  33. Morgan-Ryan UM, Fall A, Ward LA, Hijjawi N, Sulaiman I, Fayer R, Thompson RC, Olson M, Lal A, Xiao L (2002) Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens. J Eukaryot Microbiol 49:433–440

  34. Navin TR, Hardy AM (1987) Cryptosporidiosis in patients with AIDS. J Infect Dis 155:150

  35. Nelson JB, O’Har SP, Small AJ, Tietz PM, Choudhury AK, Pagano RE, Chen XM, LaRusso NF (2006) Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains. Cell Microbiol 8:1932–1945

  36. Nesterenko MV, Tilley M, Upton SJ (1995) Ametallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites. Microbios 83:77–88

  37. Nesterenko MV, Woods KM, Upton SJ (1997) Effects of manganese salts on the AIDS-related pathogen, Cryptosporidium parvum in vitro and in vivo. Biol Trace Elem Res 56:243–253

  38. Nesterenko MV, Woods K, Upton SJ (1999) Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell. Biochim Biophys Acta 7:165–173

  39. O’Connor RM, Burns PB, Ha-Ngoc T, Scarpato K, Khan W, Kang G, Ward H (2009) Polymorphic mucin antigens CpMuc4 and CpMuc5 are integral to Cryptosporidium parvum infection in vitro. Eukaryot Cell 8:461–469

  40. O’Hara SP, Chen XM (2011) The cell biology of Cryptosporidium infection. Microbes Infect 13:721–730

  41. Okhuysen PC, Chappell CL (2002) Cryptosporidium virulence determinants—are we there yet? Int J Parasitol 32:517–525

  42. Okhuysen PC, Chappell CL, Kettner C, Sterling CR (1996) Cryptosporidium parvum metallo-aminopeptidase inhibitors prevent in vitro excystation. Antimicrob Agents Chemother 40:2781–2784

  43. Olugbile S, Villard V, Bertholet S, Jafarshad A, Kulangara C, Roussilhon C, Frank G, Agak GW, Felger I, Nebie I, Konate K, Kajava AV, Schuck P, Druilhe P, Spertini F, Corradin G (2011) Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope. Vaccine 16:7090–7099

  44. Peng MM, Xiao L, Freeman AR, Arrowood MJ, Escalante AA, Weltman AC, Ong CSL, Mackenzie WR, Lal AA, Beard CB (1997) Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles. Emerg Infect Dis 3:567–573

  45. Pereira SJ, Ramirez NE, Xiao L, Ward LA (2002) Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs. J Infect Dis 186:715–718

  46. Perkins ME, Riojas YA, Wu TW, Le Blancq SM (1999) CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host-parasite boundary in intracellular stages. Proc Natl Acad Sci USA 11:5734–5739

  47. Petersen C, Gut J, Doyle PS, Crabb JH, Nelson RG, Leech JH (1992) Characterization of a >900,000-M(r) Cryptosporidium parvum sporozoite glycoprotein recognized by protective hyperimmune bovine colostral immunoglobulin. Infect Immun 60:5132–5138

  48. Putignani L, Possenti A, Cherchi S, Pozio E, Crisanti A, Spano F (2008) The thrombospondin-related protein CpMIC1 (CpTSP8) belongs to the repertoire of micronemal proteins of Cryptosporidium parvum. Mol Biochem Parasitol 157:98–101

  49. Riggs MW, Stone AL, Yount PA, Langer RC, Arrowood MJ, Bentley DL (1997) Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites. J Immunol 15:1787–1795

  50. Shaw MK, Roos DS, Tilney LG (2002) Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii. Microbes Infect 4:119–132

  51. Siddiki AMAMZ (2012) Proteome analysis of Cryptosporidium parvum and C. hominis using two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Iran J Biotechnol 10:198–207

  52. Siddiki AMAMZ (2013) Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry. J Vet Sci 14:107–114

  53. Spano F, Putignani L, Naitza S, Puri C, Wright S, Crisanti A (1998) Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family. Mol Biochem Parasitol 1:147–162

  54. Strong W, Gut J, Nelson RG (2000) Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infect Immun 68:4117–4134

  55. Sylte MJ, Suarez DL (2009) Influenza neuraminidase as a vaccine antigen. Curr Top Microbiol Immunol 333:227–241

  56. 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 (2006) A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science 15:1776–1780

  57. Thompson RC, Olson ME, Zhu G, Enomoto S, Abrahamsen MS, Hijjawi NS (2005) Cryptosporidium and cryptosporidiosis. Adv Parasitol 59:77–158

  58. Tosini F, Agnoli A, Mele R, Gomez Morales MA, Pozio E (2004) A new modular protein of Cryptosporidium parvum, with ricin B and LCCL domains, expressed in the sporozoite invasive stage. Mol Biochem Parasitol 134:137–147

  59. Trasarti E, Pizzi E, Pozio E, Tosini F (2007) The immunological selection of recombinant peptides from Cryptosporidium parvum reveals 14 proteins expressed at the sporozoite stage, 7 of which are conserved in other apicomplexa. Mol Biochem Parasitol 152:159–169

  60. Upton SJ, Tilley M, Brillhart DB (1994) Comparative development of Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines. FEMS Microbiol Lett 15:233–236

  61. Valentini E, Cherchi S, Possenti A, Dubremetz JF, Pozio E, Spano F (2012) Molecular characterisation of a Cryptosporidium parvum rhoptry protein candidate related to the rhoptry neck proteins TgRON1 of Toxoplasma gondii and PfASP of Plasmodium falciparum. Mol Boichem Parasitol 183:94–99

  62. Valigurova A, Jirku M, Koudela B, Gelnar M, Modry D, Slapeta J (2008) Cryptosporidia: epicellular parasites embraced by the host cell membrane. Int J Parasitol 38:913–922

  63. Wanyiri J, Ward H (2006) Molecular basis of Cryptosporidium-host cell interactions: recent advances and future prospects. Future Microbiol 1:201–208

  64. Wanyiri JW, Techasintana P, O’Connor RM, Blackman MJ, Kim K, Ward HD (2009) Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvuminfection in-vitro. Eukaryot Cell 8:470–477

  65. Wetzel DM, Schmidt J, Kuhlenschmidt MS, Dubey JP, Sibley LD (2005) Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infect Immun 73:5379–5387

  66. Woods KM, Tilley M, Iseli A, Upton SJ, Montelone BA, Khramtsov NV (1999) Sequence of the gene encoding hsp90e from Cryptosporidium parvum. DNA Seq 10:339–342

  67. Zhu G, Keithly JS (1997) Molecular analysis of a P-Type ATPase from Cryptosporidium parvum. Mol Biochem Parasitol 90:307–316

  68. Zhu G, LaGier MJ, Stejskal F, Millership JJ, Cai X, Keithly JS (2002) Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 18:79–89

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This study was supported by a grant sanctioned by Indian Council of Medical Research (ICMR), Department of Health research, Ministry of Health and Family Welfare, Government of India. Preeti Singh acknowledges the ICMR for providing Senior Research fellowship. We thank Professor Jai Shree Paul, Dr. Anil Verma and Dr. Reena Kumari, Jawaharlal Nehru University, New Delhi for helping in MS analysis.

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Correspondence to Alagiri Srinivasan.

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Alagiri Srinivasan and Bijay Ranjan Mirdha have equally contributed to the work.

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Singh, P., Mirdha, B.R., Srinivasan, A. et al. Identification of invasion proteins of Cryptosporidium parvum . World J Microbiol Biotechnol 31, 1 (2015). https://doi.org/10.1007/s11274-015-1936-9

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  • Cryptosporidium parvum
  • HCT-8 cell line
  • Cy5 dye
  • Mass spectrometry