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

Cellular and proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica

AbstractEntamoeba histolytica is the protozoan parasite agent of amoebiasis, an infectious disease of the human intestine and liver. Specific active pathogenic factors are secreted toward the external milieu upon interaction of the parasite with human tissue. Trafficking dynamics and secretion of these factors is not known and characterization of the dynamics interplay of subcellular compartments such as the ER or Golgi apparatus is still pending. In this work, we took advantage of cell fractionation and a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. Over 1500 proteins were identified and the two top categories contained components of trafficking machinery and GTPases. Trafficking related proteins account for over 100 markers from the ER, Golgi, MVB, and retromers. The lack of important components supporting Golgi polarization was also highlighted. The data further describe principal components of the endosomal traffic highlighting Alix in isolated vesicles and during parasite division.Biological significanceThis work represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established

Substrate-bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine 1-phosphate uridyltransferase and a catalytic mechanism for acetyl transfer

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU

Comparative proteomic analyses of avirulent, virulent and clinical strains of mycobacterium tuberculosis identify strain-specific patterns

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen

Live Cells Imaging and Comparative Phosphoproteomics Uncover Proteins from the Mechanobiome in Entamoeba histolytica

International audienceEntamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis in humans. This amoeba invades human tissues by taking advantage of its actin-rich cytoskeleton to move, enter the tissue matrix, kill and phagocyte the human cells. During tissue invasion, E. histolytica moves from the intestinal lumen across the mucus layer and enters the epithelial parenchyma. Faced with the chemical and physical constraints of these diverse environments, E. histolytica has developed sophisticated systems to integrate internal and external signals and to coordinate cell shape changes and motility. Cell signalling circuits are driven by interactions between the parasite and extracellular matrix, combined with rapid responses from the mechanobiome in which protein phosphorylation plays an important role. To understand the role of phosphorylation events and related signalling mechanisms, we targeted phosphatidylinositol 3-kinases followed by live cell imaging and phosphoproteomics. The results highlight 1150 proteins, out of the 7966 proteins within the amoebic proteome, as members of the phosphoproteome, including signalling and structural molecules involved in cytoskeletal activities. Inhibition of phosphatidylinositol 3-kinases alters phosphorylation in important members of these categories; a finding that correlates with changes in amoeba motility and morphology, as well as a decrease in actin-rich adhesive structures

Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica▿

rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions −86 to +123 (rDNA I) and positions −101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions −53 to −68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions

Data set for the proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica

Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1%) were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs) and functional descriptions (KEGG and GO terms) allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1]

A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite <i>Entamoeba histolytica</i>

<div><p>Investigations of human parasitic diseases depend on the availability of appropriate <i>in vivo</i> animal models and <i>ex vivo</i> experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as <i>Entamoeba histolytica</i>, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids <i>E. histolytica</i> crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver <i>in vitro</i> model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. <i>E. histolytica</i> invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of <i>E. histolytica</i> with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by <i>E. histolytica</i>.</p></div

Human cytokine and growth factor profile in the absence or the presence of <i>E. histolytica</i>.

<p>(A–D) ELISA quantification in compartments S1, S2 and S3 of the 3D-liver model (Model) or the setup without LSEC (no LSEC), in the absence or the presence of <i>E. histolytica</i> (− or + Eh). Samples used for ELISA assays were prepared as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004381#ppat-1004381-g004" target="_blank">Figure 4</a>. (A) Amounts of IL-6 at 3 h, (B) IL-1β, (C) galectin-1 and (D) galectin-3 at 6 h of incubation with fresh serum-free medium containing or not virulent <i>E. histolytica</i>. Graphs with standard deviation and statistical evaluation by One way ANOVA, p<0.01*, <0.001**, <0.0001*** for 3–5 independent experiments. None of the cytokines was detected in the COL-I matrix control without hepatic cells. n/d = not detected. (E, F) Summary of the results from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004381#ppat-1004381-g004" target="_blank">Figure 4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004381#ppat-1004381-g005" target="_blank">Figure 5</a>, for the setup without LSEC (E) and the 3D-liver model (F), showing the complexity of the pro-inflammatory response induced by <i>E. histolytica</i> in the 3D-liver model.</p

Human released or cell surface-associated proteins identified in the medium on top of the 3D-liver model during <i>E. histolytica</i> invasion (specific for the 3D-liver model with amoebae).

<p>Proteome data after BLAST search with the <i>H. sapiens</i> UniProt database (samples from 9 biological replicates) and two-stage filtering (i.e. elimination of bovine matches and subtraction of proteins identified in the model without amoebae) identified 139 proteins having at least one human-specific peptide (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004381#ppat.1004381.s005" target="_blank">Table S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004381#ppat.1004381.s006" target="_blank">S2</a>). Further analysis was performed to identify secreted and cell membrane-associated proteins performing a literature search and information contained in the UniProtKB/Swiss-Prot database. For the 24 proteins found, their main function (given by the UniProtKB/Swiss-Prot database) is indicated in the table. Database accession numbers are provided. The proteins were ranked according to the number of samples in which they were found (decreasing from top to bottom). Bold-labelled entries correspond to those proteins for which hepatic cells are known to be the exclusive or a main site of synthesis. The numbers given are average from all samples. PSMS is peptide spectrum matches, MW molecular weight.</p><p>Human released or cell surface-associated proteins identified in the medium on top of the 3D-liver model during <i>E. histolytica</i> invasion (specific for the 3D-liver model with amoebae).</p