A noncanonical GATA transcription factor of Entamoeba histolytica modulates genes involved in phagocytosis.

In this paper, we explored the presence of GATA in Entamoeba histolytica and their function as regulators of phagocytosis-related genes. Bioinformatics analyses evidenced a single 579 bp sequence encoding for a protein (EhGATA), smaller than GATA factors of other organisms. EhGATA appeared phylogenetically close to Dictyostelium discoideum and Schistosoma mansoni GATA proteins. Its sequence predicts the presence of a zinc-finger DNA binding domain and an AT-Hook motif; it also has two nuclear localization signals. By transmission electron and confocal microscopy, anti-EhGATA antibodies revealed the protein in the cytoplasm and nucleus, and 65% of nuclear signal was in the heterochromatin. EhGATA recombinant protein and trophozoites nuclear extracts bound to GATA-DNA consensus sequence. By in silico scrutiny, 1,610 gene promoters containing GATA-binding sequences appeared, including Ehadh and Ehvps32 promoters, whose genes participate in phagocytosis. Chromatin immunoprecipitation assays showed that EhGATA interact with Ehadh and Ehvps32 promoters. In EhGATA-overexpressing trophozoites (NeoGATA), the Ehadh and Ehvps32 mRNAs amount was modified, strongly supporting that EhGATA could regulate their transcription. NeoGATA trophozoites exhibited rounded shapes, high proliferation rates, and diminished erythrophagocytosis. Our results provide new insights into the role of EhGATA as a noncanonical transcription factor, regulating genes associated with phagocytosis.

Corrigendum: The sole DNA ligase in Entamoeba histolytica is a high-fidelity DNA ligase involved in DNA damage repair.

[This corrects the article DOI: 10.3389/fcimb.2018.00214.].

The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.

The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolytica encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H2O2 treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H2O2 treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.

Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

The R2R3 Myb protein family in Entamoeba histolytica.

The MYB DNA-binding domain is conserved in vertebrates, plants, and fungi. This domain mediates the DNA-binding activity of proteins (that have transcription factor activity) in a sequence-specific manner and is also used for the protection of telomeric regions. The MYB DNA-binding domain contains three imperfect conserved repeats of 52 amino acids (R1, R2, and R3). Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids. In order to understand the role of Myb transcription factors in Entamoeba histolytica, we searched for MYB DNA-binding domain containing proteins using the amino acid sequence of human c-Myb as the query. We found 34 putative MYB DNA-binding domain containing proteins, which clustered into three monophyletic groups. Family I members conserve only the R2 and R3 repeats in their MYB DNA-binding domain and were dubbed in this report as EhMybR2R3. Family II includes single-repeat proteins related to human telomeric binding proteins. Family III is predicted to comprise proteins with one single repeat where the region corresponding to the conserved tryptophan of the third alpha helix is replaced by a (S)/(T)HAQK(Y)/(F)F motif; this family was named EhMybSHAQKYF. In this work, we focused on proteins that belong to the EhMybR2R3 family. RT-PCR analysis showed that EhMybR2R3 genes were differentially expressed in trophozoites grown in basal culture conditions. Purified rEhMyb10 protein, belonging to the EhMybR2R3 family, was able to bind a consensus Myb recognition element in vitro. In addition, using nuclear extracts from trophozoites of E. histolytica, we were able to detect Myb DNA-binding activity to this sequence. Our in silico surveys demonstrated that this consensus sequence is present in E. histolytica gene promoters. Interestingly, these promoters include different families of genes that are related to signal transduction, vesicular transport, heat shock response, and virulence. Thus, Myb putative transcription factors in E. histolytica could be involved in the transcriptional regulation of genes participating in several different pathways.