Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Mobile genetic elements in protozoan parasites.

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtleomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

Pathogenesis of infection by Entamoeba histolytica.

Entamoeba histolytica, a protozoan parasite, is the etiologic agent of amoebiasis in humans. It exists in two forms—the trophozoite which is the active, dividing form, and the cyst which is dormant and can survive for prolonged periods outside the host. In most infected individuals the trophozoites exist as commensals. In a small percentage of infections, the trophozoites become invasive and penetrate the intestinal mucosa, causing ulcers. The trophozoites may reach other parts of the body—mainly liver, where they cause tissue necrosis, leading to life-threatening abscesses. It is thought that pathogenesis of infection by Entamoeba histolytica is governed at several levels, chief among them are (i) adherence of trophozoite to the target cell, (ii) lysis of target cell, and (iii) phagocytosis of target cell. Several molecules which may be involved in these processes have been identified. A lectin inhibitable by galactose and N-acetyl-D-galactosamine is present on the trophozoite surface. This is implicated in adherence of trophozoite to the target cell. Various amoebic pore-forming proteins are known, of which 5kDa protein (amoebapore) has been extensively studied. These can insert into the lipid bilayers of target cells, forming ion-channels. The phagocytic potential of trophozoites is directly linked to virulence as measured in animal models. Factors like association of bacteria with trophozoites also influence virulence. Thus, pathogenesis is determined by multiple factors and a unifying picture taking into account the relative contributions of each factor is sought. Recent technical advances, which includes the development of a transfection system to introduce genes into trophozoites, should help to understand the mechanism of pathogenesis in amoebiasis.