Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica.
The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 microM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired.
Fisher
Ohad
O
Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, POB 9649, 31096 Haifa, Israel.
Siman-Tov
Rama
R
Ankri
Serge
S
D044503:5-Methylcytosine / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002467:Cell Nucleus / Q000201:enzymology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004748:Entamoeba histolytica / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D018503:Genome, Protozoan; D006801:Humans; D008100:Liver Abscess / Q000469:parasitology; D008647:Mesocricetus; D008969:Molecular Sequence Data; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014774:Virulence / Q000187:drug effects
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012333:RNA, Messenger; D044503:5-Methylcytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine