Characterization of function of MLF, ATG8 and FYVE containing protein in Giardia lamblia

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Giardia lamblia is an early-branching and widely distributed intestinal protozoan parasites. People get infected by drinking the contaminated water. G. lamblia have two stages in the life cycle: a binucleate trophozoite and a quadrinucleate cyst. G. lamblia trophozoite parasitizies in small intestine and encysts by PH value changes. Autophagy is a self-degradative process of organisms that is an important balance of cell cycle in development and metabolism. In the previous reference, ATG8 was associated with the autophagosomes formation in Saccharomyces cerevisiae, and can be used as markers of autophagy. FYVE protein promotes autophagosomes formation in human and interacts with human autophagy-related protein-LC3 .We found localization of myeloid leukemia factor (MLF) in the vesicles. We’ve discovered autophagosome maker protein LC3/ATG8 related protein and FYVE protein that may transport vesicles in the Giardia by search genome database. This research will determine G. lamblia autophagy related mechanism. We observed ATG8 and FYVE proteins both located in MLF vesicles. MLF over expression can induce CWP1 protein level. In earlier experiments, we found the G. lamblia cell cycle relate CDK2 protein is cytosolic, but CDK2dm with a deletion of 49-1742a.a localized in MLF contained vesicles. To test the characteristics of these vesicles, we performed the starvation analysis, and treated PBS to cause starvation response in Giardia wild type WB strain. We found increased levels of MLF protein expression. We tested various autophagy inhibitors. Chloroquine inhibits the fusion of autophagosomes with lysosomes, and causes increased autophagosome formation. Nocodazole causes lysosomal damage, and decreases fusion ability of autophagosomes with lysosomes, resulting in an increase in autophagosome formation. MG132 is a proteasome inhibitor, that increases damage proteins and autophagy formation. Wortmannin is phagophore inhibitor and can inhibit autophagy formation. Dithiothreitol (DTT) can interfere the folding of proteins in ER, and induce expression of autophagosme maker protein-LC3. Puromycin (PU) and G418 will cause Giardia death. E. coli can induce autophagy (Xenophagy) in mammal cell, and can be used to test Xenophagy in Giardia. First, we treated chloroquine, nocodazole, MG132, wortmannin, DTT, G418, puromycin (PU), and E. coli in the Giardia wild type WB cells. We found increased numbers of MLF vesicles and increased MLF protein expression by chloroquine, nocodazole, MG132, wortmannin, DTT, G418, puromycin and E. coli treatment. The expression of BIP protein was also increased in the chloroquine treatment experimental group. Then we tested the starvation effect in pATG8 experession strain. Increased numbers of ATG8 vesicles and increased ATG8 protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the pATG8 expression strain, and found increased numbers of ATG8 vesicles and increased ATG8 protein expression. Using immunoprecipitation methods, we found that ATG8 proteins interacted with BIP. We transfected human LC3b gene in Giardia and created Giardia’s human LC3b expression strain-pLC3b. We found pLC3b interacted with G. lamblia MLF protein. The results showed that human LC3b was similar to G. lamblia ATG8 protein. Next we tested the starvation effect in pFYVE experession strain. Increased numbers of FYVE vesicles and increased FYVE protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the pFYVE expression strain, and found increased numbers of FYVE vesicles and increased FYVE protein expression. We created Giardia’s mutation strain-pFYVEm1, and found that pFYVEml expression strain, expressed lower level of CWP1 protein than pFYVE expression strain. Using immunoprecipitation methods, we found that FYVE proteins interacted with BIP, and FYVE protein also interacted with ATG8 protein. MLF protein may be an autophagy related protein in Giardia. We also wanted to know the function of human MLF2 protein in Giardia. We transfected human MLF2 gene in Giardia and created Giardia’s human MLF2 expression strain-phMLF2. We tested the starvation effect in phMLF2 experession strain. Increased numbers of hMLF2 and increased hMLF2 protein expression was found. We treated chloroquine, nocodazole, MG132, DTT, G418, and E. coli in the phMLF2 expression strain, and found increased numbers of hMLF2 vesicles and increased hMLF2 protein expression. Using immunoprecipitation methods, we found hMLF2 proteins interacted with G. lamblia MLF protein. We treat chloroquine in the pCDK2dm expression strain, and found increased numbers of CDK2dm vesicles and increased CDK2dm protein expression. Next, we transfected pMLFHA plasmid in the CDK2dm expression strain. And found increased level of MLF and CDK2dm proteins and number of those vesicles, but reduced CWP1 protein expression. We provide evidence that the MLF、ATG8 and FYVE protein expression increased and numbers of vesicles by autophagy inhibitors trement. Using a mutant protein model CDK2dm, we also know that vesicles may transport mutant protein. Key words: Giardia lamblia, Autophagy, ATG8, FYVE domain, MLF, Chloroquine