COMPARISON OF THE GIARDIA LAMBLIA TROPHOZOITE AND CYST TRANSCRIPTOME
ABSTRACT: To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription. Six cysts and three trophozoites life cycle stages were analyzed.
Project description:Giardia causes more episodes of illness worldwide than any other parasite. It is a flagellated cyst-forming enteric pathogen that inhabits the lumen of the small intestine and although it is clear that more and less virulent strains do occur, pathogenesis and virulence of Giardia remains poorly understood. Identification of Giardia virulence factors is highly desirable since their expression would be the best indicator for determining disease outcome. The advent of quantitative proteomics has been timely in investigating which proteins are likely to be secreted virulence factors. Here we successfully apply such analyses to the whole parasite and to the supernatants derived from the parasite, in order to ascertain a repertoire of secreted proteins. In our initial analysis we compared replicates of Giardia cells and culture supernatants from the two major lineages which cause human disease – assemblage A and assemblage B. The genome of Giardia is believed to contain open reading frames which could encode as many as 6000 proteins. In our study we confirm expression of ~1600 proteins from each assemblage, the vast majority of which being common to both lineages. To look for actual enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet. This simple method defines a small group of enriched proteins, putatively secreted at a steady state by cultured growing Giardia trophozoites of both assemblages. This secretome contains a high proportion annotated to have N’ terminus signal peptides, with some showing evidence of having recently evolved under strong positive selective pressure. The most abundant secreted proteins include known virulence factors such as the Cathepsin B cysteine proteases and members of a Giardia superfamily of Cysteine Rich Proteins which comprises VSPs, HCMPs and a new class of virulence factors, the Giardia Tenascins. Since we also find that physiological function of human enteric epithelial cells can be disrupted by soluble factors even in the absence of the trophozoites we are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia derived soluble mediators.
Project description:We have performed a DNA microarray analysis of gene expression in trophozoites which were adapted to grow in a media depleted from glucose for one month and on glucose-starved trophozoites which were recovered with the addition of glucose.
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).
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
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:To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription. Overall design: Six cysts and three trophozoites life cycle stages were analyzed.
Project description:We analyzed ~27nt small RNAs from Entamoeba invadens trophozoites, 24h cysts, 72h cysts, and excysting cells (8h) E. invadens trophozoites were induced to encyst by incubation in low glucose media, and parasites harvested at 0, 24 and 72h. A subset of the 72h cysts were induced to excyst, and parasites harvested at 8h. Total RNA was extracted for each sampleand small RNA libraries constructed and sequenced as below
Project description:Acanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for sugar polymers to show that purified cyst walls of A. castellanii retain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight cysteines each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for sugar polymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and sugar polymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and sugar polymers to which they bind are mostly inaccessible in the ectocyst layer and ostioles. In summary, these results show the most abundant A. castellanii CWPs are three sets of lectins, which localize to either the ectocyst layer (Jonah) or endocyst layer and ostioles (Luke and Leo).
Project description:microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. Here, we perform a comprehensive analysis of miRNAs in the zoonotic parasite E. canadensis G7, one of the causative agents of the neglected disease cystic echinococcosis. Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. As a result, we found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed significant regulated miRNAs between life cycle stages of E. canadensis G7. We confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. This study will provide valuable information for better understanding the complex biology of this parasite and could help to find new potential targets for therapy and/or diagnosis. Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For each sample type, two libraries were constructed from two independent samples in order to have biological replicates.