Abstract
Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia.
Highlights
Giardia lamblia is a common cause of waterborne diarrhea due to contamination of water with human or animal feces [1,2]
We found that knockdown of mlf gene by the CRISPR/Cas9 system resulted in a significant decrease of cwp gene expression and cyst formation
We have developed a CRISPR/Cas9 system to disrupt mlf gene using two plasmids to express Cas9 and gRNA (Figs 2A and 3, strategy 1)
Summary
Giardia lamblia is a common cause of waterborne diarrhea due to contamination of water with human or animal feces [1,2]. Most cases have self-limiting diarrhea, a subset of patients may develop chronic giardiasis or irritable bowel syndrome after giardiasis [3]. Giardia can serve as a unique protozoan model for eukaryotic cell differentiation because its life cycle has been completed with the test tube [1,7]. Like many other organisms that adapt to inhospitable environments by persisting in a dormant state, G. lamblia undergoes differentiation from a pathogenic trophozoite form into a resistant infectious cyst form [1,8]. The cysts are protectively walled by cyst wall proteins (CWPs) and resistant to hypotonic lysis by fresh water and gastric acid [9,10,11]. Histone modifications or epigenetic mechanisms may be involved in CWP expression and cyst formation [17,18]
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