A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia

Abstract

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia

“…We further investigated the role of Spo11 in encystation using our CRISPR/Cas9 system that successfully disrupted the mlf , top3β , and mbf1 genes [ 23 , 25 , 54 ]. The CRISPR/Cas9 system was delivered by cotransfection of plasmid DNA with gRNA and Cas9 into Giardia and by the establishment of the stably transfected cell line with puromycin selection ( Figure 7 A).…”

“…The stable cell lines cultured in growth medium were inoculated into encystation medium with puromycin and harvested and lysed after 24 h as previously described [ 23 ]. The cell lysates were incubated with anti-HA antibody conjugated to beads as previously described [ 23 ]. For reciprocal immunoprecipitation experiments, anti-WRKY or anti-MYB2 was used to do immunoprecipitation.…”

“…The lysates were incubated with 2 μg of anti-WRKY, anti-MYB2 antibody or preimmune serum for 2 h and then incubated with protein G plus/protein A-agarose (Merck Millipore, MilliporeSigma, Burlington, MA, USA) for 1h. Proteins from the beads were analyzed by Western blotting using anti-HA monoclonal antibody (1/5000 in blocking buffer; MilliporeSigma, Burlington, MA, USA), or anti-WRKY (1/5000 in blocking buffer) [ 19 ], anti-MYB2 (1/5000 in blocking buffer) [ 21 ], anti-ISCS (1/10,000 in blocking buffer) [ 25 ], as previously described [ 23 ].…”

“…The pPSpo11 stable cell line and 5′Δ5N-Pac control cell lines cultured in growth medium were inoculated into encystation medium and harvested after 24 h and the assay was performed as previously described [ 23 , 25 ]. The chromatin extracts were incubated with anti-HA antibody conjugated to beads (MilliporeSigma, Burlington, MA, USA), as previously described [ 18 ].…”

“…Previously, our lab identified and investigated the role of molecules involved in inducing cwp gene expression. These include eight transcription factors, MYB2 (named from myeloblastosis), GARP1 (named from the maize GOLDEN2, Arabidopsis response regulator, and Chlamydomonas Psr1), ARID1 (named from AT-rich interaction domain), WRKY, E2F1 (named from adenovirus E2 gene promoter binding factor), Pax1 (named from paired box), Pax2, and multiprotein bridging factor 1 (MBF1), and two topoisomerases (TOP2 and TOP3β) [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”