טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLavi Tal
SubjectCharacterization of a Novel Methylated DNA Binding Protein
in the Protozoan Parasite Entamoeba histolytica
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

Amebic dysentery, or gastrointestinal amebiasis, is caused by the protozoan Entamoeba histolytica. It is the third leading cause of mortality due to parasitic disease in humans (after malaria and schistosomiasis). Almost 10 % of the world population are carriers of the parasite. However, only less than 10% of the carriers develop symptoms. The precise conditions that trigger the process of differentiation into a virulent amoeba are still unknown. In our lab we are examining these conditions, which appear to be reversible. We have gained evidence showing that this trigger may be the result of epigenetic modifications in the genome that bring heritable changes in gene expression without a change in the DNA sequence. We hypothesize that these modification in E.histolytica, mainly DNA methylation combined with chromatin condensation, could be part of the flexible, inherited mechanism that control virulence in the parasite. In E.histolytica, DNA methylation was found predominantly in repetitive elements. On this basis, we have isolated a nuclear protein, named EhMLBP, using methylated reverse transcriptase gene of the Long Interspersed Nuclear Element as bait. EhMLBP has no homology to other known classical methylated DNA Binding Proteins and is the first report of a DNA methylated binding activity in protozoa. Later, we have revealed its essential role for the parasite growth and virulence via knocking down its expression. With a potential to identify new anti-amebic agents, we found a 12-mer peptide, using the Phage-Display screen that inhibits EhMLBP binding activity and significantly impaired the parasite growth. We managed to identify more EhMLBP DNA targets, and revealed the presence of a consensus motif that includes a stretch of adenines ("A tracts"). It was shown that EhMLBP recognition is dependant on its binding motif rather the DNA methylation status of its gene target; however the later one significantly increases its DNA-protein binding.

EhMLBP expression was strongly induced following 1 hour of 42°C heat shock stress. This upregulation appeared to be mediated by the binding of heat shock transcription factor to the EhMLBP promoter. We were surprised to see that under heat shock conditions the parasite viability of downregulated EhMLBP amoebas was impaired compared to the control amoebas.  In an effort to reveal the molecular regulation of its binding activity, two E.histolytica interacting proteins were found using the Yeast Two Hybrid screen. Only one of these proteins appeared to bind with EhMLBP DNA target and is assumed to antagonize with EhMLBP binding activity.