Identification of host proteins interacting with the integrin-like A domain of Toxoplasma gondii micronemal protein MIC2 by yeast-two-hybrid screening.

BACKGROUND

Toxoplasma gondii is an obligate intracellular protozoan, causing the important zoonosis toxoplasmosis. This parasite utilizes a unique form of locomotion called gliding motility to find and invade host cells. The micronemal adhesin MIC2 plays critical roles in these processes by binding to substrates and host cell receptors using its extracellular adhesive domains. Although MIC2 is known to mediate important interactions between parasites and host cells during invasion, the specific host proteins interacting with MIC2 have not been clearly identified. In this study, we used a yeast-two-hybrid system to search for host proteins that interact with MIC2.

METHODS

Different adhesive domains of MIC2 were cloned into the pGBKT7 vector and expressed in fusion with the GAL4 DNA-binding domain as baits. Expression of bait proteins in yeast cells was analyzed by immuno-blotting and their autoactivation was tested via comparison with the pGBKT7 empty vector, which expressed the GAL4 DNA binding-domain only. To identify host proteins interacting with MIC2, a mouse cDNA library cloned into a GAL4 activation-domain expressing vector was screened by yeast-two-hybrid using the integrin-like A domain of MIC2 (residues 74-270) as bait. After initial screening and exclusion of false positive hits, positive preys were sequenced and analyzed using BLAST analysis and Gene Ontology Classifications.

RESULTS

Two host proteins that had not previously been reported to interact with T. gondii MIC2 were identified: they are LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) and RNaseH2B (ribonuclease H2 subunit B). Gene Ontology analysis indicated that these two proteins are associated with many cellular processes, such as lysosome maturation, signaling transduction, and RNA catabolism.

CONCLUSION

This study is the first one to report interactions between Toxoplasma gondii MIC2 and two host proteins, LAMTOR1 and RNaseH2B. The data will help us to gain a better understanding of the function of MIC2 and suggest that MIC2 may play roles in modulating host signal transduction and other biological processes in addition to binding host cells.