| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 107 === Giardia lamblia is a widely distributed intestinal protozoan parasite. People ususally get infected by drinking contaminated water or having contaminated fruits. There are two stages of life cycle in G. lamblia, trophozoites and cysts. When G. lamblia enters hosts, it lives in the trophzoite form, and encysts to form an infectious cyst in intestine.
Autophagy is a self-degradative process and an important mechanism in regulation of cell cycle in organisms. During autophagy, lysosomes will combine with autophagosomes to be the autophagolysosomes. AcPh (acid phosphatase) is an enzyme stored in lysosomes, and it is also stored in autophagosomes. Proteasome breaks peptide bonds by chemical reaction to degrade unnecessary or damaged proteins. RPN11 is a component of lid in proteasome. BIP protein is a chaperone protein and marker of ER (endoplasmic reticulum) stress. ATG8 protein is named LC3II protein in mammals, and is related to autophagy. FYVE protein promotes formation of autophagosomes in human. Our lab found that MLF protein located in the vesicles that ATG8 and FYVE protein located in, and we suggested that MLF protein is related to autophagy. Because reactive oxygen species (ROS) will cause autophagy in mammals, we tried to detect ROS level by adding drugs.
We analyzed AcPh expression trophozoites, and we observed increased BIP, RPN11, ATG8 and MLF protein expression and reduced CWP1 protein expression. We further added autophagy inhibiting drugs or oxidative-stress promoting drugs and growth-inhibiting drugs in mammals above. Chloroquine inhibits the fusion of autophagosomes with lysosomes by affecting pH value in lysosomes, and causes increased autophagosome formation. Nocodazole interferes microtubules in cells and causes lysosomal damage, resulting in an increase in autophagosome formation. MG132 is a proteasome inhibitor that interrupts the function of 26S proteasome. MG132 makes LC3b transform to LC3b-II and promotes autophagy and induces cell apoptosis. DTT is a strong reducing agent, it causes ER stress by interfering the folding proteins and promotes autophagy. G418 is an analog of Geneticin. G418 interrupts the elongation step in protein translation and blocks polypeptide synthesis and. G418 causes cell death because of lack of necessary proteins. Puromcyin causes premature chain termination during translation to inhibit protein translation. Curcumin is an anti-inflammatory drugs. It disrupts signal transfer and inhibits tumor cell proliferation. Scr7 is an inhibitor of non-homologous end joining (NHEJ) and enhances the efficiency of CRISPR-Cas9 genome editing. Rapamycin, an inhibitor of mTOR, makes increased LC3b-II protein expression and causes autophagy. The unpaired electron of H2O2 causes oxidative stress to damage cells. E.coli can induce autophagy in mammal cells, and can be used to test xenphagy in Giardia. We measured ROS level and used 2′,7′-Dichlorofluorescin diacetate. We found that ROS level elevated after starvation, Chloroquine, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin, H2O2 and E.coli treatment. A research suggested that Cysteine-HCl is a ROS scavenger and reduces ROS. The TYI-33 medium we cultured G. lamblia also has Cysteine-HCl. We removed Cysteine-HCl of TYI-33 medium, and we found that ROS levels increased in the TYI-33 medium without Cysteine-HCl.
Next we found increased BIP protein expression by starvation, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin, H2O2 and E.coli treatment. These drugs cause oxidative stress in G. lamblia. We suggested that oxidative stress can increase BIP protein expression in G. lamblia. We found increased RPN11 protein expression by starvation, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin and E.coli treatment. We suggested that these drugs will influence the function of 26S proteasome. We also found increased MLF protein expresson by curcumin, Scr7, rapamycin and H2O2 treatment. We suggested that these drugs promote autophagy in G. lamblia. We found increased ATG8 and FYVE protein expression by curcumin, Scr7, rapamycin and H2O2 treatment. We suggested that these drugs cause autophagy in G. lamblia. We also tested AcPh expression trophozoites by MG132, DTT, Scr7, H2O2 and E.coli treatment. The expression of AcPh is increased after MG132, DTT, Scr7, H2O2 and E.coli treatment. The TYI-33 medium we cultured G. lamblia has Cysteine-HCl. We removed Cysteine-HCl of TYI-33 medium, and we found that ROS increases and increased BIP, RPN11, ATG8 proteins expression after removal of Cysteine-HCl.
Our data provide evidence that AcPh、RPN11、BIP、MLF、ATG8 and FYVE protein expression increased after autophagy inhibitor treatment. We also found that ROS level increased after autophagy inhibitor treatment.
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