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 family of cyst proteins with epidermal growth factor repeats in Giardia lamblia.

BACKGROUND: Giardia lamblia parasitizes the human small intestine to cause diarrhea and malabsorption. It undergoes differentiation from a pathogenic trophozoite form into a resistant walled cyst form. Few cyst proteins have been identified to date, including three cyst wall proteins (CWPs) and one High Cysteine Non-variant Cyst protein (HCNCp). They are highly expressed during encystation and are mainly targeted to the cyst wall. METHODOLOGY AND PRINCIPAL FINDINGS: To identify new cyst wall proteins, we searched the G. lamblia genome data base with the sequence of the Cryptosporidium parvum oocyst wall protein as a query and found an Epidermal Growth Factor (EGF)-like Cyst Protein (EGFCP1). Sequence analysis revealed that the EGF-like repeats of the EGFCP1 are similar to those of the tenascin family of extracellular matrix glycoproteins. EGFCP1 and HCNCp have a higher percentage of cysteine than CWPs, but EGFCP1 has no C-terminal transmembrane region found in HCNCp. Like CWPs and HCNCp, the EGFCP1 protein (but not transcript) was expressed at higher levels during encystation and it was localized to encystation-specific vesicles in encysting trophozoites. Like HCNCp, EGFCP1 was localized to the encystation-specific vesicles, cyst wall and cell body of cysts, suggesting that they may share a common trafficking pathway. Interestingly, overexpression of EGFCP1 induced cyst formation and deletion of the signal peptide from EGFCP1 reduced its protein levels and cyst formation, suggesting that EGFCP1 may help mediate cyst wall synthesis. We also found that five other putative EGFCPs have similar expression profiles and similar locations and that the cyst formation was induced upon their overexpression. CONCLUSIONS AND SIGNIFICANCE: Our results suggest that EGFCPs may function like cyst wall proteins, involved in differentiation of G. lamblia trophozoites into cysts. The results lead to greater understanding of parasite cyst walls and provide valuable information that helps develop ways to interrupt the G. lamblia life cycle.

Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia.

Myb family transcription factors are important in regulating cell proliferation, differentiation, and cell cycle progression. Giardia lamblia differentiates into infectious cysts to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. We have identified an encystation-induced Myb2 protein, which binds to the promoter regions of the cwp genes and myb2 itself in vitro. To elucidate the role of Myb2 in G. lamblia, we tested the hypothesis that Myb2 can activate encystation-induced genes. We found that overexpression of Myb2 resulted in an increase of expression of CWP1 at both protein and mRNA levels. Interestingly, the Myb2-overexpressing trophozoites had increased capability to differentiate into cysts. In cotransfection assays, Myb2 was able to transactivate the cwp promoters and its own promoter in vivo, suggesting that its gene can be positively autoregulated. Moreover, deletion of the N- or C-terminal domain resulted in a decrease of transactivation and autoregulation function of Myb2. We also found that the promoter of a newly identified encystation-induced gene, the giardial myeloid leukemia factor-like gene, has the Myb2 binding sites and that its mRNA levels were increased by Myb2 overexpression. Chromatin immunoprecipitation assays confirmed that Myb2 was bound to the promoters with its binding sites. Transfection of the myb2 antisense construct reduced the levels of the cwp1 transcripts and cyst formation. Our results suggest that Myb2 is a potent transactivator of the cwp genes and other endogenous genes and plays an important role in G. lamblia differentiation into cysts.