Extensive transcriptome analysis identifies essential genes sustaining intestinal invasion by Entamoeba histolytica
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Entamoeba histolytica
SUBMITTER: Nancy Guillen
PROVIDER: E-MTAB-4882 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Entamoeba histolytica is the parasite causing amoebiasis, an infectious disease targeting the intestine and liver of humans. The molecules involved in amoeba adaptation to different iron conditions during the invasive process are unknown. To investigate the effects of iron availability on gene expression in E. histolytica, we determined by microarray experiments the gene expression profile of parasites grown in the presence of different iron concentrations. Conditions included low amounts of iron, iron starvation and iron starvation supplemented with hemoglobin (Hb). Genes encoding important proteins involved in iron metabolism, reported in other organism such as bacteria, were identified for the first time in E. histolytica. The influence of iron on the amount of these transcripts was confirmed by quantitative real-time PCR and their protein products were analyzed by Western blots
2014-06-30 | E-MTAB-1158 | biostudies-arrayexpress
Project description:The ability of Entamoeba histolytica to phagocytose host cells correlates to observed virulence in vivo. To better understand the mechanism of phagocytosis we used paramagnetic beads coated with host ligand and sorted trophozoites based on phagocytic ability. Gene expression was then measured in both the sorted phagocytic and non-phagocytic populations using a custom Affymetrix chip for E. histolytica. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. PMID 23045476 M280 Streptavidin Dynabeads (Invitrogen) were labeled with 20ug/mL biotinylated Human C1q (Quidel). Entamoeba histolytica (strain HM1) were washed twice with PBS then resuspended with the labeled beads at a 10:1 ratio of beads to trophozoites. Samples were incubated at 37°C for 45 minutes, washed twice with agitation to remove adherent beads, then resuspended in MACS buffer. Samples were loaded into magnetic columns (Miltenyi Biotec) and trophozoites were seperated according to manufacturer's protocols. phagocytic vs. non-phagocytic Entamoeba histolytica populations
2012-10-16 | E-GEOD-41604 | biostudies-arrayexpress
Project description:2 million E. histolytica HM1:IMSS was treated with 0.5% DMSO for 3 h and 2 million E. histolytica HM1:IMSS was treated with 1 M-5M auranofin for 3 h
2012-02-03 | E-MEXP-3494 | biostudies-arrayexpress
Project description:Evaluation of transcript levels of two strains E.Histolytica (virulent and avirulent) in two conditions (in culture or in contact with a human colon explant)
2012-08-01 | E-MTAB-1201 | biostudies-arrayexpress
Project description:Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMK) with proposed roles that include both amebic response to the environment and immune evasion. In the later case, the process requires several elements --these include a large gene family encoding antigenically distinct surface proteins and the expression of one variant antigen at a time by a single pathogen. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilized for non-redundant functions by the parasite. For laser capture microdissection analysis, harvested trophozoites were allowed to adhere to metal framed PEN membrane slides (Molecular Devices, Sunnyvale, CA) for 15 mins at 37°C in TYI-S-33 media. Subsequently, single cells were captured from the PEN slides using a Pix Cell II laser capture microdissection system equipped with an Olympus microscope (Arcturus Engineering, Mountain View, CA).
2010-11-01 | E-GEOD-19064 | biostudies-arrayexpress
Project description:We previously showed that epigenetic transcriptional silencing of the amoebapore gene (Ehap-a) in E. histolytica G3 trophozoites was accompanied by the down-regulation of two other members of the saposin-like gene family (Ehap-b, and EhSaplip1). Comparison of the transcriptomes of G3 trophozoites with those of the parent HM-1:IMSS strain revealed 72 additional transcripts that were either down- or up-regulated at least 7 fold. One of the polyA+, up-regulated (>200) transcripts (459.m00030), had the potential to encode a 66 amino acid protein. Antibodies raised against a synthetic N-terminal peptide of the gene did not recognize any such protein in trophozoite lysates. Trophozoite cultures grown with the proteasome inhibitor MG-132 to prevent protein degradation, also did not have any detectable protein. Transfections with a plasmid in which the Ehactin gene promoter elements were used to flank the 459.m gene, significantly increased their level transcript but again, no protein was detected. Transfectants in which the 459.m ORF was placed downstream to the GST gene, expressed a fused protein which reacted with the antibody against the m.459 peptide. Constructs with the CAT reporter gene placed under the 459.m gene promoter element or fused to the C-terminal sequence of the 459.m ORF were prepared to determine if the higher levels of 459.m transcript found in the G3 trophozoites were due to changes in the stability of the transcript or increased transcription. No difference was observed in the very weak expressions seen in both G3 and HM-1:IMSS transfectants. Similarly, transfectants with a plasmid construct in which the 459.m gene was placed under its regulatory elements, did not induce an increase in the transcript levels. Our conclusions are that the transcript of the 459.m gene is an mRNA-like ncRNA and that modulations of transcript levels, especially of hypothetical genes, may not correlate with changes in the proteome. Samples include two replicates of the HM-1:IMSS strain and the amoebapore a silenced G3 strain (G3(A))
2011-08-30 | E-GEOD-28883 | biostudies-arrayexpress
Project description:Entamoeba histolytica is a human pathogen, responsible for amoebic dysentery and invasive extraintestinal disease. The parasite faces several types of stress within the host, and to establish a successful infection it must generate a robust adaptive response against host defense mechanisms. In order to obtain comprehensive information of gene expression changes in E. histolytica under growth stress, we have (1) optimized the proteomics protocol to increase the protein coverage in E. histolytica, and (2) integrated proteomic data with transcriptomic analysis under the same conditions. We have applied this approach to better understand the cellular response during serum-starvation. Label-free quantitative proteomics was performed, and compared with mRNA levels based on RNA-seq data to decipher regulation at translational and transcriptional levels. Across all samples, 2344 proteins were identified, which is an improvement over the maximum recorded number in E. histolytica proteomic studies so far. A total of 127 proteins were found to be differentially expressed and associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport, which revealed proteomic signatures to distinguish serum-starved from normal trophozoites. Gal/GalNAc-inhibitable lectin, Lgls, Hgl3 and Igl were repeatedly identified as significantly altered in serum-stress condition. Further, integration of transcriptomic and proteomic data revealed instances of post-transcriptional regulation. Six highly expressed transcripts had low corresponding protein expression, indicating translational repression. Conversely, eleven transcripts showed much greater downregulation compared with their corresponding proteins, indicating translational induction, or increased stability of these proteins during serum stress in E. histolytica. This multi-omics approach enables more refined gene expression analysis that would not be possible at the mRNA or protein levels alone. Our study provides important data to further understand the adaptive response of E. histolytica to growth stress.
2022-09-23 | PXD031921 | Pride
Project description:Amoebiasis is a disease caused by the protozoan parasite, Entamoeba histolytica, with or without clinical symptoms . It has been suggested that pathogenic amoebae use three major virulence factors: Gal/GalNAc lectin, cysteine proteinases and amoebapores. However, these molecules cannot exclusively be responsible for amoebic virulence, because most of them are found in pathogenic E. histolytica as well as in non-pathogenic E. dispar, a not close correlation has been found in amoebic strains with different virulence phenotype. Due to the high genetic variability of E. histolytica isolates and strains, differential gene expression might simply reflect inter-isolate genetic variation rather than specific differences linked to virulence. In the current study, we obtained a non-virulent UG10 mutant derived from E. histolytica HM-1:IMSS. Comparing the transcriptome of both strains, no differences in gene expression of the classical virulence factors were observed. RAB family GTPase and AIG1 family members showed higher transcriptional levels in the virulent strain than in the non-virulent mutant. Our study suggests a number of novel gene products in E. histolytica with a role yet to determine in cell physiology and involvement in the pathogenesis process.
2015-05-15 | E-MTAB-3525 | biostudies-arrayexpress
Project description:incubation for 15 min and 30 min of human liver sinusoidal cell cultures with virulent or virulence-attenuated Entamoeba histolytica or incubation without parasites added
2011-01-01 | E-MEXP-2572 | biostudies-arrayexpress
Project description:Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix (ECM). Transcriptional silencing of the E. histolytica matrix surface metalloprotease EhMSP-1 produces hyper-adherent, less motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1 silenced phenotype, here we compared the phosphoproteome of EhMSP-1 silenced and vector control trophozoites using quantitative mass spectrometry based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1 silenced and control cells, including EhCoactosin, a member of the ADF/Cofilin family of actin binding proteins, which was hyperphosphorylated at serine 147 (S147). Regulated over-expression of wild type, phosphomimetic (S147D), and non phosphorylatable (S147A) EhCoactosin variants was used to test if S147 phosphorylation functions in control of E. histolytica actin dynamics. Each of the over-expressed proteins co37 localized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing EhCoactosin S147D formed more and poorly coordinated small membrane protrusions compared to control or S147A expressing cells, while trophozoites overexpressing EhCoactosin S147A were significantly more motile within a model of mammalian ECM. Therefore, although EhCoactosin’s actin-binding ability appeared unaffected by S147 phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1 silenced trophozoites, and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness.
2020-07-15 | PXD018276 | Pride