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Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: Organization of the protein domains affects the protein secretion pathway


Lalle, M; Rosati, M A; Bien, J; Hehl, A B; Pozio, E; Tosini, F (2011). Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: Organization of the protein domains affects the protein secretion pathway. Experimental Parasitology, 127(3):680-686.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Parasitology
04 Faculty of Medicine > Institute of Parasitology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
600 Technology
Language:English
Date:2011
Deposited On:16 Feb 2011 11:31
Last Modified:10 Nov 2016 14:13
Publisher:Elsevier
ISSN:0014-4894
Publisher DOI:https://doi.org/10.1016/j.exppara.2010.11.009
PubMed ID:21112325
Permanent URL: https://doi.org/10.5167/uzh-45256

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