Heterologous expression of Cryptosporidium parvum vaccine candidate GP60 in Tetrahymena thermophila.

EUGENIA ELGUERO; TOMAZIC ML; MONTES, GUADALUPE; C. B. NUDEL; CLARA NUDEL; LEONHARD SCHNITTGER; A. D. NUSBLAT

Camerino

Conferencia; Conference on Ciliate Molecular Biology 2015; 2015

Bovine cryptosporidiosis is mainly caused by the zoonotic apicomplexan Cryptosporidium parvum. The disease is responsible for a considerable morbidity and mortality of pre-weaned calves around the world. Infected animals excrete large amounts of the infective stage, the oocyst, into the environment. In humans, the infection is known to be a major cause of infant death in developing and considered emergent in industrialized countries. So far there are neither fully effective chemotherapeutic treatments nor vaccines available. It is currently considered that the development of a vaccine to control bovine cryptosporidiosis is preferable to that of other control measures as it also limits oocyst dissemination into the environment. Prominent vaccine candidates of many pathogenic apicomplexan protozoans are surface proteins containing glycosylphosphatidylinositol (GPI) anchors. In Plasmodium falciparum, the principal GPI-anchored surface proteins of sporozoites and schizonts, the circumsporozoite protein (CSP) and the major surface proteins (MSP-1 and -2), respectively, have been shown to confer variable degrees of or even complete protection against challenge in rodent models. Additionally, it has been shown that recombinant i antigen, a GPI-anchored protein of Ichthyophthirius multifiliis, protects freshwater fish against Ichtyophihiriasis (white spot disease) caused by this parasite. Likewise, the GPI-anchored antigen GP60 (alias: GP40/15) is an immunodominant  antigen and represents a prominent vaccine candidate of C. parvum;  it  was therefore expressed in the ciliate T. thermophila for future inclusion into a subunit vaccine. T. thermophila as an eukaryotic expressions system exhibits multiple advantages one of which is the expression of GPI-anchored proteins including the GPI moiety as a post translational modifications. Episomal expression of the GP60 antigen of Cryptosporidium parvum could be verified in whole cell extract as well as the membrane enriched GPI fraction of T. thermophila as assessed by western blot analysis. Additionally, processing of the GP60 antigen by cleavage into the mature 40 kDa protein by an unknow protease of the ciliate was observed. Also a preliminary biochemical analysis of GPI proteins encoded by T. thermophila was performed. Membrane fractions enriched in GPI-proteins were isolated using the Triton X-114 phase-partitioning procedure and subsequently analyzed on Coomasie-stained SDS-PAGE gels. In the final fraction a band of 35 kda representing a potential GPI-protein was excised and its amino acid sequence analyzed by mass spectrometry (HPLC-MS). The protein constitutes a yet uncharacterized GPI-anchored protein of T. thermophila as determined by identification of a GPI-anchor motif using bioinformatic tools. The presented results support the potential of Tetrahymena for the production of subunit vaccines.