EhMLBP is an essential constituent of the Entamoeba histolytica epigenetic machinery and a potential drug target.
EhMLBP has been identified as a protein that specifically binds to methylated long interspersed element (LINE) retrotransposons and rDNA in Entamoeba histolytica. EhMLBP is unique to Entamoeba parasites, which makes this protein a possible drug target for treating amebiasis. In the work described here, we evaluated this potential. Downregulation of EhMLBP using antisense technology resulted in trophozoites with impaired growth and cytopathic activity. This indicated that EhMLBP is an essential protein. With a view to identifying new antiamebic agents, we tested the effect of distamycin A, a drug with known antimalarial activity, on the growth of the parasite and on the ability of EhMLBP to bind to DNA. Distamycin A (IC(50) = 13 microM) efficiently inhibited the growth of E. histolytica. Indeed, distamycin A at a concentration of 5-20 microM inhibited the binding of EhMLBP to methylated LINE DNA in vitro. As an additional approach to identify molecules that inhibit EhMLBP activity, a selective biopanning assay was performed using the DNA-binding domain of EhMLBP and the Ph.D.-12 phage display peptide library. Remarkably, four out of the 11 phages selected after three rounds of biopanning expressed the peptide 'SYFDQNERWGAP' (Pept3) at their surface. The binding of EhMLBP to Pept3 was confirmed by ELISA. Phage expressing Pept3 inhibited the binding of EhMLBP to RT LINE DNA. The growth of E. histolytica transfectants expressing Pept3 was significantly impaired compared with that of trophozoites expressing a scrambled version of Pept3. These results highlight EhMLBP as an essential constituent of the parasite E. histolytica and a novel target for antiamebic chemotherapy.
Lavi
Tal
T
Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Siman-Tov
Rama
R
Ankri
Serge
S
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D019175:DNA Methylation; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004214:Distamycins / Q000494:pharmacology*; D015536:Down-Regulation; D004748:Entamoeba histolytica / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004749:Entamoebiasis / Q000188:drug therapy* / Q000469:parasitology; D044127:Epigenesis, Genetic / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D019151:Peptide Library; D011485:Protein Binding / Q000187:drug effects; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D016372:RNA, Antisense / Q000235:genetics; D053058:Trophozoites / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism
D000962:Antimalarials; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D004214:Distamycins; D019151:Peptide Library; D015800:Protozoan Proteins; D016372:RNA, Antisense; C030000:stallimycin