Date Published: March 11, 2019
Publisher: Public Library of Science
Author(s): Zi-Qi Lin, Soo-Wah Gan, Szu-Yu Tung, Chun-Che Ho, Li-Hsin Su, Chin-Hung Sun, Rodney D. Adam.
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.
Giardia lamblia is a common cause of waterborne diarrhea due to contamination of water with human or animal feces [1,2]. Although most cases have self-limiting diarrhea, a subset of patients may develop chronic giardiasis or irritable bowel syndrome after giardiasis . Chronic giardiasis in children may lead to malnutrition and cognitive impairment [4–6]. 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 [9–11]. Synthesis of at least two CWPs is coordinately induced during encystation, possibly by signaling molecules and transcription factors, including CDK2, MYB2, WRKY, PAX1, and E2F1 [12–16]. Histone modifications or epigenetic mechanisms may be involved in CWP expression and cyst formation [17,18].
The protozoan G. lamblia that can form cysts to survive in the environment and to infect hosts, is a unique model diverging from the commonly studied eukaryotes. Studies in Giardia provide information for biology of the unicellular eukaryote, particularly of regulation of cell differentiation. Establishment and application of gene knockout systems are important works for studies of this organism. 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). The disruption of mlf gene can be achieved by partial replacement of the mlf gene with the pac gene by HR (Fig 7A). To further improve the disruption efficiency, we added Scr7, an NHEJ inhibitor, to increase HR, and found an increase of the disruption efficiency on mlf gene from about 32% to about 53%, compared with the system without Scr7 (Figs 2B, 3B, and S6 Fig, strategy 2). Scr7 also worked in mammalian cells to increase knock-in efficiency via CRISPR/Cas9-coupled HR [39,40,41]. Canonical NHEJ works by DNA ligase IV and XRCC mediated joining of dsDNA break . Alternative NHEJ works by DNA ligase I/III . We only found one DNA ligase gene (open reading frame 7649) with low homology to ligase IV and III. Since Scr7 can inhibit DNA ligase IV, and also DNA ligase III [40,58], it is possible that Scr7 can target Giardia DNA ligase. This awaits future work to explore. A XRCC-like gene was found from Giardia genome database (open reading frame 6918) but also with very low homology. Scr7 may inhibit Giardia DNA repair since Scr7 can inhibit Giardia growth (S6 Fig). In addition, Scr7 can increase gene disruption efficiency in our system, suggesting that Giardia may have an unusual NHEJ pathway which still can be inhibited by Scr7. The importance of NHEJ for eukaryotic genome stability also suggests the presence of NHEJ in Giardia, which awaits further studies to clarify .