טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKushnir Oded
SubjectThe Effect of Environmental Changes on the Epigenetic
Machinery of Entamoeba histolytica
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

                  E.histolytica is an anaerobic parasitic protozoan that infects humans. As a result of the host cellular environment and immune system, E.histolytica is exposed to different environmental modifications, such as heat shock (HS), nitrosative and oxidative stresses.  Our group found that the formation of m5C in E.histolytica is catalyzed by Ehmeth; a protein which belongs to the DNMT2 family. In E.histolytica there is a protein that interacts with some specific methylated sequences present in the parasite, like the RT LINE, it has been identified and further characterized. This protein (EhMLBP) is an important regulator of the parasite's virulence. The effect of environmental stresses on the DNA methylation/ methylated DNA recognition apparatus of the parasite were not addressed to date.

                  The DNA methylation status of the parasite following treatment with nitric oxide (NO), hydrogen peroxide (H2O2), HS and culture with E.coli O55 was investigated.

                  The LRRP gene (XM_644299) has been previously showed to be methylated and was used to track the changes that occur in the DNA of E.histolytica exposed to different stresses. The methylation level of LRRP and Actin in trophozoites treated with NO, H2O2 or HS was determined by D-HRMA. NO but not H2O2 or HS led to the demethylation of LRRP. In contrast, NO and HS were both able to induce the demethylation of Actin. After two months of recovery the methylation status of these genes returned to its original level. A preliminary result indicates that NO inhibits directly the Ehmeth activity. This maybe the mechanism that led to demethylation of LRRP in trophozoites treated with NO.

            Growth of E.histolytica in presence of E.coli O55 for more than a year increased the overall DNA methylation of the parasite but didn't affect the methylation status of the LRRP gene.

            In addition, a bioinformatic study of EhMLBP using a novel homology method (HHpred) was performed. This bioinformatic analysis revealed the presence of a heat shock domain in EhMLBP suggesting a role of this protein during heat shock. I found that EhMLBP has an even distribution within the nucleus in trophozoites overexpressing EhMLBP. The same distribution was also observed in trophozoites following heat shock. Finally, I observed that constitutive over-expression of EhMLBP protects trophozoites against heat shock.

            In summary this work indicates that DNA methylation is modulated by environmental stresses in E.histolytica. In addition, EhMLBP has a decisive role in the defense of the parasite against HS.