DNA methylation and targeting of LINE retrotransposons in Entamoeba histolytica and Entamoeba invadens.

In this study, we have isolated by affinity chromatography, using anti-m5C antibody as a ligand, a DNA encoding reverse transcriptase of LINE retrotransposon (RT LINE) in both Entamoeba invadens and Entamoeba histolytica. RT LINE transcripts were detected in E. histolytica but were absent from E. invadens. The methylation status of genomic copies of E. invadens RT LINE was confirmed by bisulfite analysis. In contrast, all the genomic copies of the E. histolytica RT LINE analyzed in this study were not methylated. Many of these genomic copies diverge from the RT LINE isolated by m5C affinity chromatography by a number of mutations that includes conversion of C to T and G to A. These mutations are reminiscent of the conversion of C to T (and G to A on the complementary DNA strand) that occurred during primate evolution in Alu elements following accelerated deamination of methylated cytosines. E. invadens and E. histolytica RT LINEs isolated by affinity chromatography were cloned in a pEhAct Neo vector, amplified in E. coli GM2163 (dam-dcm) and transformed into E. histolytica. Bisulfite analysis of transfected amoeba showed the presence of m5C in E. invadens RT LINE replicated in E. histolytica, but not in E. histolytica RT LINE or in the neomycine phosphotransferase gene, which is also carried by the pEhAct Neo vector. These results suggest the existence of a specific mechanism based on DNA methylation that controls retrotransposons in these parasites.

Epigenetic and classical activation of Entamoeba histolytica heat shock protein 100 (EHsp100) expression.

The protozoan parasite Entamoeba histolytica expresses a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, the 5' region of E. histolytica heat shock protein 100 (5'EHsp100) was isolated by affinity chromatography with 5-methylcytosine antibodies as ligand. The methylation status of 5'EHsp100 was confirmed by sodium bisulfite sequencing. We showed that the expression of EHsp100 was induced by heat shock, 5-azacytidine (5-AzaC), an inhibitor of DNA methyltransferase and Trichostatin A (TSA), an inhibitor of histone deacetylase. The effect of TSA on EHsp100 expression was rapidly reversed by removing the drug from the culture. In contrast, EHsp100 expression was still detectable one month after removing 5-AzaC from the media. Whereas 5-AzaC and TSA caused demethylation in the promoter region of EHsp100, no demethylation was observed following heat shock. Remarkably, DNA that includes three putative heat shock elements identified in the promoter region of EHsp100 bound to a protein of 37kDa present in the nuclear fraction of heat-shocked trophozoites but absent in the nuclear fraction of 5-AzaC and TSA treated trophozoites. Our data suggest that EHsp100 expression can be regulated by both a classical and an epigenetic mechanism.

A chimeric peptide composed of a dermaseptin derivative and an RNA III-inhibiting peptide prevents graft-associated infections by antibiotic-resistant staphylococci.

Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K(4)-S4(1-13)(a) is a 13-residue dermaseptin derivative (DD(13)) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD(13)-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis (MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD(13)-RIP and DD(13) were equally effective, and that the chimeric peptide but not DD(13) was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD(13) act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.