Identification and molecular characterization of GRA8, a novel, proline-rich, dense granule protein of Toxoplasma gondii

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Abstract

We have generated two monoclonal antibodies (MAbs 17.9 and A3.2) against Toxoplasma gondii, both of which localize to the dense granules of tachyzoites by immunoelectron microscopy. MAb 17.9 is directed against GRA6, a previously described 32 kDa dense granule protein. MAb A3.2 is directed against a novel 38 kDa dense granule protein, which we refer to as GRA8. GRA8 is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. The cDNA sequence encoding GRA8 was determined by screening a T. gondii cDNA expression library with MAb A3.2. The deduced amino acid sequence of GRA8 consists of a polypeptide of 267 amino acids, with no significant homology to any other known protein. The sequence contains an amino terminal signal peptide, three degenerate proline-rich repeats in the central region and a potential transmembrane domain near the carboxy terminus. The most striking feature of GRA8 is its remarkably high proline content (24%).

Introduction

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. The pathology of toxoplasmosis is due to repeated cycles of host cell invasion and lysis by the actively dividing form of the parasite, the tachyzoite. The apical end of the tachyzoite contains an elaborate set of cytoskeletal structures and secretory organelles which are thought to function in host cell invasion and establishment of infection. Among the three classes of secretory organelles in the tachyzoite, the micronemes and rhoptries discharge their contents from the apical end of the tachyzoite, whereas the dense granules discharge from the apical, lateral and posterior surfaces of the parasite [1], [2]. Microneme proteins are released first, upon contact with the host cell and are thought to function in host cell recognition and attachment [1], [3], [4], [5], [6]. The contents of the rhoptries are released next and may function in the formation of the parasitophorous vacuole (PV) [1], [6], [7], [8], [9]. Finally, the dense granule proteins are exocytosed both during and after invasion into the PV. The exocytosed dense granule proteins either remain soluble in the lumen of the vacuole or become associated with the PV membrane or the tubular-reticular network of membranes within the PV (see section 4 below). Dense granule proteins are thought to modify the environment within the PV, thereby functioning in intracellular survival and replication [10].

To identify previously uncharacterized proteins on the surface of the parasite or within its secretory organelles, we have generated monoclonal antibodies (MAbs) against a membrane fraction of T. gondii tachyzoites. We describe, in this report, the use of one of these MAbs to identify and characterize a novel dense granule protein, GRA8.

Section snippets

Parasite culture

Human foreskin fibroblasts (HFFs) (CRL 1634, ATCC, Rockville, MD) and T. gondii tachyzoites (RH strain) were cultured as previously described [11]. Prior to use, tachyzoites were filtered through a 10 μm polycarbonate filter (Poretics, Livermore CA).

Antigen preparation and MAb production

A crude membrane extract was prepared, as previously described [12], with the following modifications. Parasites were washed twice (4 min, 1000×g) with 135 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.75 mM KH2PO4, pH 7.4 (PBS) and resuspended in PBS at a

Antigens recognized by MAbs A3.2 and 17.9

In the course of screening for MAbs against novel surface or secretory proteins of T. gondii tachyzoites, we identified two hybridoma supernatants (A3.2 and 17.9) that showed a similar punctate distribution within the tachyzoite by immunofluorescence (see below), but reacted with different sized antigens (38 and 32 kDa, respectively) on western blots. The hybridomas were expanded, cloned and rescreened. The corresponding MAbs were purified from ascitic fluid. Purified MAbs A3.2 and 17.9

Discussion

We report here on the identification and molecular characterization of GRA8, a novel dense granule protein of T. gondii. GRA8 was identified using a MAb which recognizes a 38 kDa tachyzoite protein on western blots and which localizes to the dense granules by immunoelectron microscopy. The sequence of GRA8 is highly proline-rich (24% overall) and contains both an N-terminal signal sequence and a potential C-terminal transmembrane domain.

There are nine dense granule proteins previously

Acknowledgements

We thank Bill Church and Jamie Yturriondobeitia of Green Mountain Antibodies (Burlington, VT) for assistance with MAb production and purification; Con Beckers, Murry Stein, Mary Tierney, Doug Taatjes and Marilyn Wadsworth for helpful discussions; Michael Wichroski for critical comments on the manuscript; and Tim Hunter of the Vermont Cancer Center DNA Sequencing Facility for excellent sequencing services provided. This work was supported in part by PHS grants AI42355 (GW) and CA22435 (Vermont

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    Note: Nucleotide sequence data reported in this paper have been submitted to GenBank with the accession number AF150729.

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