Identification of Distinct Interacting Proteins of Giardia lamblia Centrins Using Yeast Two-Hybrid Assays.

Giardia lamblia, a protozoan parasite that causes diarrhea in humans, contains two centrins implicated in cellular morphology and cell division. To identify proteins interacting with each G. lamblia centrin (GlCent), we performed a yeast two-hybrid assay. The coding sequences of the two centrins, GlCent1 (GL50803_6744) and GlCent2 (GL50803_104685), were cloned into pGBKT7 as bait constructs. A G. lamblia cDNA library, driven by pGADT7, was transformed into yeast strains expressing each bait protein. Initial screening on selective media yielded 17 and 23 candidate clones for GlCent1 and GlCent2, respectively. After retransformation and interaction strength analysis using growth assays, 10 GlCent1-interacting clones and 5 GlCent2-interacting clones were selected. Their interactions were validated using co-immunoprecipitation and immunofluorescence assays in transgenic Giardia cells co-expressing hemagglutinin-tagged centrins and myc-tagged candidate proteins. The results showed that ubiquitin protein ligase 1 (GL50803_9779) and preimplantation protein 3 (GL50803_3417) were GlCent1-binding proteins, whereas two hypothetical proteins (GL50803_4239 and _27739) interacted with GlCent2. Proteins interacting with the two centrins transiently co-localized during cell division. These findings suggest that GlCent1 and GlCent2 might participate in cell division by associating with distinct partner proteins in Giardia.