Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: organization of the protein domains affects the protein secretion pathway.
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.
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. marco.lalle@iss.it
Marco
M
Lalle
Maria Adelaide
MA
Rosati
Justina
J
Bien
Adrian B
AB
Hehl
Edoardo
E
Pozio
Fabio
F
Tosini
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016785:Cryptosporidium parvum / Q000235:genetics / Q000378:metabolism*; D005872:Giardia / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
C514023:Cpa135 protein, Cryptosporidium parvum; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins