Unknown,Transcriptomics,Genomics,Proteomics

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Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

ABSTRACT: 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).

ORGANISM(S): Entamoeba histolytica

SUBMITTER: Carol Gilchrist

PROVIDER: E-GEOD-19064 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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))

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.

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Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

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