Article

Interactions between the ROP18 kinase and host cell proteins that aid in the parasitism of Toxoplasma gondii

Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei, China.
Acta tropica (Impact Factor: 2.52). 02/2012; 122(3):255-60. DOI: 10.1016/j.actatropica.2012.02.001
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Jian Du, Feb 01, 2015
1 Follower
 · 
105 Views
  • Source
    • "The AH109 yeasts were transformed with pGBKT7-ROM3 (A), pGBKT7 (B, negative control) and pCL1 (C, positive control), respectively. the reporter AH109 (Cheng et al., 2012). To generate a bait construct with the peptide AC domain without the signal sequence (Asn27–Cys211) of EtROM3 (GenBank DQ323509), the cDNA was amplified by PCR (sense 5 -CCGGAATTCAACATTTCACTGGACAAGTCG-3 , antisense 5 -CGGGATCCACACGTTACTGCGAACCCGCA–3 ) from E. tenella cDNA (Zheng et al., 2011), and inserted into the EcoRI–BamHI site of pGBKT7. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Invasion in several apicomplexan parasites, including Eimeria tenella, is accompanied by shedding of surface adhesins by intramembrane proteolysis mediated by rhomboid protease. We have previously identified E. tenella rhomboid 3 (EtROM3), but its precise role has not been elucidated. In this study, the interactions between EtROM3 and microneme (MIC) proteins were analyzed using the yeast two hybrid technique. The results showed that c-Myc-ROM3 fusion protein interacted with EtMIC4 protein in co-transformed AH109 yeasts, which was further confirmed by immunoprecipitation assay. Smaller EtMIC4 band from co-transformed cells suggested that EtROM3 was an active protease and involved in the cleavage of EtMIC4.
    Veterinary Parasitology 01/2014; 201(1-2). DOI:10.1016/j.vetpar.2014.01.010 · 2.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: It is not known the role of the virulent gene ROP18 polymorphisms in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis.
    Parasitology International 10/2013; 63(2). DOI:10.1016/j.parint.2013.10.012 · 2.11 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remains unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12 and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.
    Journal of Biological Chemistry 03/2014; 289(18). DOI:10.1074/jbc.M113.544718 · 4.57 Impact Factor