טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTovy Ayala
SubjectThe Function of Ehmeth an E.histolytica Dnmt2 homolog, and
the Meaning of its Interaction with Enolase
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

The unicellular Entamoeba histolytica has a single DNA methyltransferase, Ehmeth. This protein belongs to the Dnmt2 protein family. The function of Dnmt2 proteins in high eukaryotes is under debate, as it was demonstrated that Dnmt2 is a tRNA methyltransferase rather than a DNA methyltransferase. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. We addressed this subject for Ehmeth by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase was shown to have both a cytoplasmic and a nuclear localization in the parasite. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG which suggest that glucose metabolism controls the non-glycolytic function of enolase. Glucose as a sensor of the environment is highly significant to E. histolytica which is exposed to adverse nutrient conditions in the human host. Consequently, we examined the effect of glucose starvation on E. histolytica pathogenesis and on the enolase inhibitory effect over Ehmeth.  Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-Ehmeth complex, and to a significant reduction of the tRNAAsp methylation in the parasite. Moreover, we observed that the virulence of the parasite could be enhanced by glucose starvation. To gain insights into how the virulence is regulated by glucose starvation, we analyzed differential protein expression levels in control versus glucose starved trophozoites. Surprisingly, A and cysteine proteinase 5, two important virulence factors were down regulated by starvation. The effect of glucose depletion on virulence can be reproduced in strains silenced for amoebapore and for cysteine proteinase. This effect is lost in the light lectin subunit Ehlgl1 silenced strain. These data emphasize the unexpected role of glucose starvation on the modulation of E. histolytica virulence and the involvement of Ehlgl1 in this phenomenon. This effect of GS on E. histolytica supports the idea that epigenetic modifications involving DNA methylation are sensitive to environmental stresses. The effect of stress conditions on E. histolytica DNA and tRNA methylation has never been investigated so far. Therefore, we examined the DNA methylation status of the parasite following nitric oxide, hydrogen peroxide, and heat shock stress, by using a new method based on differential-high resolution melting analysis. The results indicate that nitrosative stress, but not oxidative and heat shock, impairs the DNA methylation status.