Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Abstract

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.The ancient eukaryotic human parasite, Entamoeba histolytica, is the causative agent of invasive amebiasis in humans infecting 10% of the world population and resulting in over 100,000 deaths annually, chiefly from amebic liver abscess (1). Infection is acquired through the ingestion of the quadranucleate cyst form of the organism from fecally contaminated food or water supplies. Excystation occurs in the small intestine, releasing immature trophozoites, which move into and colonize the nutrient-poor large intestine.As ancient eukaryotes, Entamoeba parasites contain only rudimentary endoplasmic reticulum and Golgi apparatus (2, 3). Despite this, vesicle trafficking pathways appear to be essential to the pathogenesis of these parasites as (i) uptake and digestions of nutrients by mature trophozoites in the large intestine, (ii) invasion of the intestinal epithelium, (iii) delivery of cyst wall components during encystation, and (iv) dissemination and establishment of extra-intestinal infections all rely on endocytosis and secretion. In addition, these parasites lack many traditional eukaryotic biochemical pathways, including the de novo biosynthesis of purine and pyrimidine nucleobases. As a result, these parasites are completely dependent upon their host for the supply of these essential preformed nutrients. In that regard, it has been reported previously that Entamoeba are capable of salvaging both purines and pyrimidines from the extracellular milieu during in vit...

Specific inhibition of bacterial RNase T2 by helix 41 of 16S ribosomal RNA

“…For example, when tomato cells are starved for phosphate, cells secrete RNase LE to digest extracellular RNAs and incorporate the digests as a phosphate source 3,4 . A similar phenomenon is observed in the human intestinal pathogen Entamoeba histolytica 5 .…”

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes

“…Therefore CLIP of HA-tagged U1A would increase the chances of detecting E complex components as well as B complex and C complex factors. To circumvent mRNA degradation by the numerous Entamoeba nucleases [43] during nuclear fractionation, and to ensure detection of RNA processing proteins bound to the RNA for MS/MS analysis, CLIP assays were carried out from nuclear extracts obtained from UV cross-linked amoebae.…”

A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis

“…Whereas RNase A proteins are restricted to vertebrates, and T1 RNases are restricted to fungi and Gram-positive bacteria (Dyer & Rosenberg, 2006; Sevcik et al ., 2002; Yoshida, 2001), the T2 RNases are widespread across the biological kingdoms, occurring in viruses, bacteria, protozoa, fungi, plants and animals (Deshpande & Shankar, 2002; Irie & Ohgi, 2001). T2 RNases have been most extensively studied in eukaryotes, where they have been implicated in nutritional scavenging of nucleotides and/or phosphate in protozoa, fungi and plants, senescence, self-incompatibility and defence against pathogens in plants, and regulation of membrane permeability in yeast (Deshpande & Shankar, 2002; Irie & Ohgi, 2001; MacIntosh et al ., 2001; McGugan et al ., 2007). Additionally, some fungal and animal virus T2 RNases have been shown to possess cytotoxic activity, which has potential for anti-carcinogenic and anti-angiogenic therapy (Hulst et al ., 1994; Schneider et al ., 1993; Schwartz et al ., 2007).…”