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T. gondii is an obligate intracellular parasite, belonging to the Phylum Apicomplexa, infecting all warm-blooded animals including humans. During host cell invasion, specialized cytoskeletal and secretory organelles play a pivotal role. ROP18, as a member of the ROP2 family, has been identified as a key virulence factor mediating pathogenesis in T. gondii. Here, we identify an ER-resident protein, Derlin2, a factor implicated in the removal of misfolded proteins from the ER for cytosolic degradation, as a component of the machinery required for ER-associated protein degradation (ERAD). We identified Derlin2 interacting with ROP18 by yeast two-hybrid screening system. The interaction between ROP18 and Derlin2 was further confirmed through in vitro GST pull-down and in vivo immunoprecipitation assays. By immunofluorescence assay, we found that ROP18 co-localized with Derlin2 in the endoplasmic reticulum. Using overexpression and knockdown approaches, we demonstrated that Derlin2 was required for T. gondii ROP18 degradation. Consistently, cycloheximide chase experiments showed that the degradation of ROP18 relied on the Derlin2, but not Derlin1. These results indicate that interaction between Derlin2 and ROP18 is functionally relevant and leads ultimately to degradation of ROP18. The finding provides the basis for future studies on Derlin2-dependent ERAD of T. gondii ROP18 and subsequent antigen generation.

Toxoplasma gondii, a ubiquitous and obligate intracellular pathogen, belonging to the phylum Apicomplexa, is capable of infecting a broad range of warm-blooded hosts including birds and mammals that is nearly worldwide. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines is of urgent need. The plant-like calcium-dependent protein kinases (CDPKs) expressed by T. gondii, play important roles in cell invasion, gliding motility, egress and some other developmental processes, in which T. gondii CDPK3 (TgCDPK3) has been implicated as an important virulence factor. In this study, the immune protective function of recombinant TgCDPK3 (rTgCDPK3) against experimental toxoplasmosis in BALB/c were evaluated. We divided the mice into different dose groups of vaccines and all immunizations with purified rTgCDPK3 protein were injected by intramuscular at weeks 0, 2, and 4 in BALB/c mice. The rTgCDPK3 vaccine provided protection was correlated with the development of humoral and cellular immune responses demonstrated through the antigen-specific spleen cell proliferation, release of Th1 cytokines IFN-γ, and the production of the high titers of IgG antibody with a predominance of IgG2a over IgG1. Vaccination with rTgCDPK3 conferred partial protection against acute toxoplasmosis, as demonstrated by prolonged survival rate after lethal challenge. Additionally, the amount of brain tissues cysts in vaccinated mice led to 46.5% reduction compared with non-vaccinated ones. These data demonstrated that rTgCDPK3 inoculation prevents or attenuates the harmful influence of T. gondii infection, and it is a potential vaccine candidate against toxoplasmosis.

Serine/threonine kinases secreted from rhoptry organelles are important virulence factors for Toxoplasma gondii. Among rhoptry proteins, the ROP18 kinase has been identified as a key virulence determinant mediating pathogenesis in T. gondii; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, the interactions between the ROP18 kinase of Toxoplasma gondii and the host cell proteins were analyzed using a yeast two-hybrid technique. The cMyc-ROP18(25-251) fusion proteins expressed by pGBKT7 plasmids in AH109 yeast were bound to host cell proteins from a human fetal brain cDNA library transformed to AH109 yeast using a mating method. Using these selection procedures, we identified seven host proteins that had not previously been reported to interact with ROP18 such as DDB1, TOR1AIP1, integrin, SLC3A2, TPST2, DERL2 and OCIAD1. These host proteins are associated with DNA repair, transcriptional regulation, translation modification, protein degradation and cell adhesion. Our data strongly support the hypothesis that the secreted kinase ROP18 is involved in several complex cellular pathways for the invasion and commandeering of host functions.