Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.
International journal for parasitology
2014; 44 (11): 837-45
Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.
View details for DOI 10.1016/j.ijpara.2014.06.008
View details for PubMedID 25075445
Polyunsaturated fatty acids induce polarized submembranous F-actin aggregates and kill Entamoeba histolytica.
2013; 70 (5): 260-268
We have recently identified a novel galacto-glycerolipid (GGL) from the plant Oxalis corniculata that killed the human pathogen Entamoeba histolytica. In this study, we show that the anti-amoebic activity of GGL was due to the polyunsaturated fatty acid ?-linolenic acid (C18:3 ) side chain. Treatment of ?-linolenic acid to E. histolytica trophozoites disrupted the cytoskeletal network and led to polarization of F-actin at one end of the cells with prominent filopodial extensions. In addition, clustering of surface receptors and signaling molecules was also observed adjacent to the polarized actin similar to concanavalin-A-(Con-A) induced capping. But, in contrast to Con-A-induced capping, ?-linolenic acid induced caps were not shed and showed accumulation of long and numerous filopodia at the cap site. We found that ?-linolenic acid disrupts the actin cytoskeletal network, which led to the detachment of plasma membrane from the underlying cytoskeleton. A similar effect was observed with other dietary fatty acids such as linoleic acid (C18:2 ), arachidonic acid (C20:4 ), eicosapentaenoic acid (C20:5 ), and docosahexaenoic acid (C22:6 ). Our findings showed that dietary polyunsaturated fatty acids are powerful anti-amoebic agents that lead to disruption of the actin cytoskeleton. © 2013 Wiley Periodicals, Inc.
View details for DOI 10.1002/cm.21105
View details for PubMedID 23568815
Novel Isoforms of the Transport Regulator Klar
2013; 8 (2)
Klar is a regulator of microtubule-motor dependent transport processes in Drosophila, including nuclear migration, vesicle motility, and lipid-droplet transport. The single klar locus gives rise to multiple isoforms that presumably have unique functions. Up to now, three Klar isoforms (?, ?, ?) were known. Here we describe two novel isoforms, ? and ?, whose expression depends on a previously uncharacterized promoter. Klar ? and/or ? are widely expressed during development, including in the embryonic and larval nervous system as well as in ovaries. When we specifically ablate Klar ? and ? expression genetically, no gross organismal phenotypes are apparent. However, ectopic expression of these isoforms causes nuclear mispositioning in developing photoreceptors and in oocytes, demonstrating their biological activity. Our analysis identifies novel forms of the Klar protein and provides new tools for functionally dissecting the complex klar locus.
View details for DOI 10.1371/journal.pone.0055070
View details for Web of Science ID 000315602700013
View details for PubMedID 23457459
A Novel Galacto-Glycerolipid from Oxalis corniculata Kills Entamoeba histolytica and Giardia lamblia
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
2010; 54 (11): 4825-4832
Oxalis corniculata is a naturally occurring weed that has been used in traditional medicine for the cure of dysentery and diarrhea in India. One of the common causes of dysentery is due to infection by the protist pathogen Entamoeba histolytica. Bioactivity profiling of extracts from O. corniculata identified several compounds that showed antiamoebic activity in axenic cultures of E. histolytica. These were characterized by nuclear magnetic resonance, infrared, and mass spectrometry as (i) Oc-1, a mixture of saturated fatty acids C?? to C??; (ii) Oc-2, a mixture of long-chain alcohols C?? to C??; and (iii) Oc-3, a single compound that was a galacto-glycerolipid (GGL). Of the different compounds that were obtained, the strongest antiamoebic activity was found in GGL. The addition of GGL to E. histolytica xenic cultures containing other microbial flora from the large intestine did not affect its antiamoebic activity. Amoebicidal concentrations of GGL had no effect on intestinal microbial flora or on the mammalian cell line HEK-293. GGL was also found to be equally effective in killing another protist pathogen, Giardia lamblia, that causes diarrhea in humans. The importance of this study is based on the identification of novel natural products and the possibility of developing these compounds as active agents to treat at least two pathogenic parasitic intestinal infections endemic to tropical regions.
View details for DOI 10.1128/AAC.00546-10
View details for Web of Science ID 000284155000042
View details for PubMedID 20713666
Long term transcript accumulation during the development of dehydration adaptation in Cicer arietinum
2004; 135 (3): 1608-1620
Cool season crops face intermittent drought. Exposure to drought and other abiotic stresses is known to increase tolerance of the plants against subsequent exposure to such stresses. Storage of environmental signals is also proposed. Preexposure to a dehydration shock improved adaptive response during subsequent dehydration treatment in a cool season crop chickpea (Cicer arietinum). We have identified 101 dehydration-inducible transcripts of chickpea by repetitive rounds of cDNA subtraction; differential DNA-array hybridization followed by northern-blot analysis and analyzed their responses to exogenous application of abscisic acid (ABA). Steady-state expression levels of the dehydration-induced transcripts were monitored during the recovery period between 2 consecutive dehydration stresses. Seven of them maintained more than 3-fold of expression after 24 h and more than 2-fold of expression level even at 72 h after the removal of stress. Noticeably, all of them were inducible by exogenous ABA treatment. When the seedlings were subjected to recover similarly after an exposure to exogenous ABA, the steady-state abundances of 6 of them followed totally different kinetics returning to basal level expression within 24 h. This observation indicated a correlation between the longer period of abundance of those transcripts in the recovery period and improved adaptation of the plants to subsequent dehydration stress and suggested that both ABA-dependent and -independent mechanisms are involved in the maintenance of the messages from the previous stress experience.
View details for DOI 10.1104/pp.104.043141
View details for Web of Science ID 000222692700041
View details for PubMedID 15247380