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: 2.27). 02/2012; 122(3):255-60. DOI: 10.1016/j.actatropica.2012.02.001
Source: PubMed


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.

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    • "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. "
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