Processing and secretion of ROP13: A unique Toxoplasma effector protein

Abstract

Like most intracellular pathogens, Toxoplasma synthesizes and secretes an arsenal of proteins to successfully invade its host cell and hijack host functions for intracellular survival. The rhoptries are key secretory organelles that inject proteins into the host cell where they are positioned to coopt host processes, although little is known regarding how these proteins exert their functions. We show here that the rhoptry protein ROP13 is synthesized as a pre-pro-protein that is processed in the parasite. Processing occurs at a conserved SϕXE cleavage site as mutagenesis of glutamic acid to alanine at the P1 position disrupts ROP13 maturation. We also demonstrate that processing of the prodomain is not necessary for rhoptry targeting and secretion. While gene disruption reveals that ROP13 is not essential for growth in fibroblasts in vitro or for virulence in vivo, we find that ROP13 is a soluble effector protein that can access the cytoplasm of host cells. Exogenously expressed ROP13 in human cells remains cytosolic but also appears toxic, suggesting that overexpression of this effector protein is disrupting some function within the host cell.

Identification of three novel Toxoplasma gondii rhoptry proteins

“…Rhoptry proteins contain a classic eukaryotic signal peptide for entrance into the secretory pathway and are trafficked from the endoplasmic reticulum through the Golgi by a conserved pathway before being packaged into the apically located secretory organelles (Sadak et al, 1988; Bradley and Boothroyd, 1999; Bradley et al, 2004; Carey et al, 2004; Hajj et al, 2006b, 2007; Turetzky et al, 2010). N-terminal pro-domains have been implicated in rhoptry protein sorting and indeed several rhoptry proteins exhibit N-terminal processing.…”

“…N-terminal pro-domains have been implicated in rhoptry protein sorting and indeed several rhoptry proteins exhibit N-terminal processing. However, the failure to remove the pro-domain does not seem to disrupt targeting (Bradley et al, 2002; Miller et al, 2003; Turetzky et al, 2010). Other rhoptry proteins do not appear to be processed and the mechanism by which they are targeted to the rhoptries is unknown.…”

Novel N -Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

“…Slides were permeabilized with PBS-0.2% Triton X-100 at room temperature and blocked with PBS-0.2% Triton X-100 -3% BSA at room temperature. For this set of experiments, dense granule antibody GRA1 (mouse) was kindly provided by Marie-France Cesbron-Delauw and Corinne Mercier (Institut Jean Roget) (4, 6), rhoptry antibody ROP13 was kindly provided by Peter Bradley (University of California Los Angeles) (46), and cathepsin antibodies (to visualize the acidocalcisome), cathepsin-like protein (CPL), and the microneme antibody M2 were kindly provided by Vern Carruthers (University of Michigan) (16), and anti-SAG1 antibody was from Invitrogen. Samples were stained with primary antibodies (GRA1, ROP13, CPL, M2, and SAG1) for 1 h and secondary antibody (mouse and rabbit) overnight.…”

Protective Efficacy of a Toxoplasma gondii Rhoptry Protein 13 Plasmid DNA Vaccine in Mice

“…Among the above antigens, ROPs are effector proteins that modulate the host response to the parasite and play a key role in accessing the cytoplasm of host cells (21). Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for preventing several parasitic diseases, including toxoplasmosis.…”

“…ROP13, a novel rhoptry protein, shows no homology to any known proteins, lacks any identifiable domains, and is a soluble protein that is proteolytically processed en route to the rhoptries (1). It is possible to detect ROP13 in the host cell in evacuoles due to the protein's effector functions involved in host response to T. gondii (21). Hence, T. gondii ROP13 may represent a good vaccine candidate.…”