A myeloid leukemia factor homolog is involved in tolerance to stresses and stress-induced protein metabolism in Giardia lamblia.

BACKGROUND: The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia. RESULTS: In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF. CONCLUSIONS: Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.

A myeloid leukemia factor homolog involved in encystation-induced protein metabolism in Giardia lamblia.

BACKGROUND: Giardia lamblia differentiates into resistant cysts as an established model for dormancy. Myeloid leukemia factor (MLF) proteins are important regulators of cell differentiation. Giardia possesses a MLF homolog which was up-regulated during encystation and localized to unknown cytosolic vesicles named MLF vesicles (MLFVs). METHODS: We used double staining for visualization of potential factors with role in protein metabolism pathway and a strategy that employed a deletion mutant, CDK2m3, to test the protein degradation pathway. We also explored whether autophagy or proteasomal degradation are regulators of Giardia encystation by treatment with MG132, rapamycin, or chloroquine. RESULTS: Double staining of MLF and ISCU or CWP1 revealed no overlap between their vesicles. The aberrant CDK2m3 colocalized with MLFVs and formed complexes with MLF. MG132 increased the number of CDK2m3-localized vesicles and its protein level. We further found that MLF colocalized and interacted with a FYVE protein and an ATG8-like (ATG8L) protein, which were up-regulated during encystation and their expression induced Giardia encystation. The addition of MG132, rapamycin, or chloroquine, increased their levels and the number of their vesicles, and inhibited the cyst formation. MLF and FYVE were detected in exosomes released from culture. CONCLUSIONS: The MLFVs are not mitosomes or encystation-specific vesicles, but are related with degradative pathway for CDK2m3. MLF, FYVE, and ATG8L play a positive role in encystation and function in protein clearance pathway, which is important for encystation and coordinated with Exosomes. GENERAL SIGNIFICANCE: MLF, FYVE, and ATG8L may be involved an encystation-induced protein metabolism during Giardia differentiation.

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

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.

Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia.

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.