Expression of Cryptosporidium Parvum Cpa135/CpCCP1 Chimeras in Giardia Duodenalis: Organization of the Protein Domains Affects the Protein Secretion Pathway

Exp Parasitol. 2011 Mar;127(3):680-6. doi: 10.1016/j.exppara.2010.11.009. Epub 2010 Nov 26.

Abstract

Cpa135 is a multidomain antigenic protein secreted at the sporozoite stage of the Apicomplexa protozoan Cryptosporidium parvum. Previous studies have shown that the protozoan flagellate parasite Giardia duodenalis is a suitable system for the heterologous expression of secreted proteins of Apicomplexa. Here, we designed three different Cpa135 variants fused to a C-terminal HA tag in order to test their expression in G. duodenalis under the control of the inducible promoter of the cyst wall protein 1 gene (cwp1). The three Cpa135 chimeras encompassed different portions of the protein; CpaG encodes the entire polypeptide of 1574 amino acids (aa); CpaGΔC includes the first 826 aa at the N-terminus; and CpaGΔN consists in of the final 833 aa at the C-terminus. Immunoblot experiments showed that CpaG and CpaGΔN maintained the epitopes recognized by anti-C. parvum-specific human serum. The intracellular localization and transport of the three Cpa135 variants were studied by immunofluorescence in combination with G. duodenalis-specific antibodies. CpaGΔC was mainly accumulated in the endoplasmic reticulum and the intact form was also excreted in the medium. Differently, the Cpa135 chimeras possessing an intact C-terminus (CpaG and CpaGΔN) were transported towards the forming cyst wall possibly and were not detected in the medium. Furthermore, the full-length CpaG was incorporated into the cyst wall. The data presented suggest that the C-terminus of Cpa135, which includes a cysteine reach domain, could influence the secretion of the chimeric proteins.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Cryptosporidium parvum / genetics
  • Cryptosporidium parvum / metabolism*
  • Giardia / genetics
  • Giardia / metabolism*
  • Humans
  • Membrane Glycoproteins / biosynthesis*
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology
  • Mice
  • Microscopy, Fluorescence
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Transfection

Substances

  • Cpa135 protein, Cryptosporidium parvum
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins