Characterization of a Secretory Type Theileria parva Glutaredoxin Homologue Identified by Novel Screening Procedure

Thomas Ebel,Jamesf S Middleton,Angelika Frisch,Joachim Lipp

doi:10.1074/jbc.272.5.3042

Abstract

The schizont stage of the protozoan parasite Theileria parva induces features characteristic of tumor cells in infected bovine T-cell lines. Most strikingly T. parva-infected cell lines acquire unlimited growth potential in vitro. Their proliferative state is entirely dependent on the presence of a viable parasite within the host cell cytoplasm. It has been postulated that parasite proteins either secreted into the host cell or expressed on the parasite surface membrane are involved in the parasite-host cell interaction. We used an in vitro transcription-translation-membrane translocation system to identify T. parva-derived cDNA clones encoding secretory or membrane proteins. Within 600 clones we found one encoding a 17-kDa protein which is processed by microsomal membranes to a 14-kDa protein (11E), presumably by signal peptidase. The processed form is expressed in the T-cell line TpM803 harboring viable parasites. By immunolocalization we show that the 11E protein mostly resides within the parasite, often in close vicinity to membranous structures, but in addition it appears at the surface membrane. Amino acid sequence comparison suggests that 11E belongs to the glutaredoxin family, but is unique so far in containing a signal sequence for endoplasmic reticulum membrane translocation.

Highlights

Theileria parva is an obligate intracellular protozoan parasite that infects cattle and causes a usually fatal disease called East Coast Fever
Identification of cDNA Clone 11E—We were interested in determining proteins which are directly involved in the interaction of T. parva and its host the bovine T-lymphocyte
Since the schizont stage of T. parva is obligate intracellular and cannot be cultured in a free form, we developed a general method to identify proteins which enter the secretory pathway of eucaryotic cells

Summary

Introduction

This reversibility of the system implies that parasite factors, most likely proteins, induce and maintain host cell transformation. In eucaryotes most secretory and surface proteins use a common intracellular transport pathway. This pathway is entered by translocation of the nascent polypeptide across the ER membrane [4, 5]. For proteins of different protozoan parasites it has been shown that they can be translocated across microsomal membranes and processed by a signal peptidase (8 –10). We have employed an in vitro transcription-translationtranslocation assay to identify secretory and membrane proteins of the T. parva schizont. 600 individual T. parva cDNA clones were expressed in the cell-free system and screened for translation products, which were translocated across microsomal membranes. Higher resolution analysis by immuno-electron microscopy suggests that 11E is predominantly associated with intraparasitic membrane structures and the surface membrane

Methods

Results

Conclusion