DNA topoisomerase IIIβ promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia

Abstract

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3β) with similarity to the mammalian topoisomerase IIIβ. We found that TOP3β was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3β can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3β can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3β increased expression of cwp1 - 3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3β. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3β gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3β may be functionally conserved, and involved in inducing Giardia cyst formation.

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

“…We have developed a CRISPR/Cas9 system to successfully disrupt the mlf and top3β genes [ 44 , 57 ]. We further used this system for the targeted disruption of the mbf1 gene.…”

“…We confirmed the replacement of the mbf1 gene with the puromycin acetyltransferase (pac) gene by PCR and sequencing analysis of genomic DNA ( Figure 8 B,C and Figure S6 ), and found a successful disruption of the mbf1 gene by about 29% and a partial replacement of the mbf1 gene with the pac gene ( Figure 8 B,C). G418, an inhibitor of polypeptide synthesis, had cytotoxicity and was used to test the drug sensitivity of the top3β targeted disruption cell line [ 57 ]. We also used G418 to test the drug sensitivity of the MBF1td cell line and found that the viability of the MBF1td cell line decreased compared to the control cell line after G418 treatment, suggesting that MBF1td cell line exhibited increased sensitivity to G418 ( Figure 8 D).…”

“…Western blots were probed with anti-V5-HRP (Invitrogen, city, if any state (abbr. ), country), anti-HA monoclonal antibody (1/5000 in blocking buffer; MilliporeSigma, Burlington, MA, United States), anti-MBF1 (1/10,000 in blocking buffer) (see below in this paper), anti-CWP1 (1/10,000 in blocking buffer) [ 20 ], anti-Myb2 (1/5000 in blocking buffer) [ 23 ], anti-α-tubulin (1/10,000 in blocking buffer, Sigma), anti-ISCS (1/10,000 in blocking buffer) [ 57 ], anti-RAN (1/10,000 in blocking buffer) [ 24 ], or preimmune serum (1/5000 in blocking buffer), and detected with HRP-conjugated goat anti-mouse IgG (1/5000; Thermo Fisher Scientific, Waltham, MA, USA) or HRP-conjugated goat anti-rabbit IgG (1/5000; Pierce) and enhanced chemiluminescence (Merck Millipore, MilliporeSigma, Burlington, MA, USA).…”

“…The stable cell lines were cultured in growth medium or inoculated into encystation medium with puromycin and harvested and lysed after 24 h as previously described [ 57 ]. The cell lysates were incubated with anti-HA antibody conjugated to beads as previously described [ 57 ]. For reciprocal immunoprecipitation experiments, anti-MBF1 was used to do immunoprecipitation.…”

“…For reciprocal immunoprecipitation experiments, anti-MBF1 was used to do immunoprecipitation. The lysates were incubated with 2 μg of anti-MBF1 antibody or preimmune serum for 2 h and then incubated with protein G plus/protein A-agarose (Merck) for 1 h. Proteins from the beads were analyzed by Western blotting using anti-HA monoclonal antibody (1/5000 in blocking buffer; Sigma), or anti-MBF1, anti-Myb2 (1/5000 in blocking buffer) [ 23 ], anti-ISCS (1/10,000 in blocking buffer) [ 57 ] as previously described [ 57 ].…”