Effect of choline kinase inhibitor hexadecyltrimethylammonium bromide on Plasmodium falciparum gene expression
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ABSTRACT:
ORGANISM(S): Plasmodium falciparum
SUBMITTER: Mohd Ridzuan Mohd Abd Razak
PROVIDER: E-GEOD-54775 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Investigations on the fundamental of malaria parasite biology, such as invasion, growth cycle, metabolism and cell signalling have uncovered a number of potential antimalarial drug targets, including choline kinase, a key enzyme involved in the synthesis of phosphatidylcholine, an important component in parasite membrane compartment. The effect on gene expression of Plasmodium falciparum K1 strain following 72 hours exposure to 2 μM (IC50 concentration) of the choline kinase inhibitor, hexadecyltrimethylammonium bromide (HDTAB) was evaluated by DNA microarray analysis. Genes important in P. falciparum intra-erythrocytic life cycle, such as invasion, cytoadherance and growth were among those affected by at least 2-fold changes in their expression levels compared with non HDTAB-treated control.
2014-02-08 | GSE54775 | GEO
Project description:The P. falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. The members of these gene families are uniformly positioned within heterochromatic domains of the genome and are thus subject to variegated expression. The best-studied example is that of the var gene family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). Transcriptional regulation of other subtelomeric gene families and their role in parasite biology is much less understood. Here, we investigated the mode of transcriptional control of var, rif, stevor, phist and pfmc-2tm families by comparative genome-wide transcriptional profiling of transgenic parasite lines. Our results establish a clear functional distinction between var and non-var transcriptional control mechanisms. Unlike var promoters, we find that promoters of non-var families are not silenced by default. Moreover, we show that mutually exclusive transcription is unique to the var gene family. 3D7 wild-type parasites were transfected with constructs carrying eight different promoters that drive expression of the drug-selectable marker hdhfr-gfp. Thereof seven promoters are members of the multigene families upsA var, upsB var, upsC var, rif, stevor, phistb and pfmc-2tm. The cam promoter was used as transfection-based control and also a wild-type 3D7 cell line was included as control. These nine cell lines were subjected to genome-wide transcriptional profiling. Parasites were synchronized to obtain an 8 hour growth window and were harvested at four consecutive timepoints (TP): TP1 (6-14 hours post-invasion (hpi)); TP2 (14-22 hpi); TP3 (22-30 hpi); TP4 (30-38 hpi) to monitor intra- and inter-family specific linkage of multigene family expression.
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Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.
2015-01-13 | E-GEOD-64887 | biostudies-arrayexpress
Project description:The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive immune escape, invasion of human liver and blood cells, and transmission through the female Anopholes mosquito. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. However, there is mounting evidence across a broad range of species that intergenic long non-coding RNA (lncRNA) and antisense RNA can regulate chromatin state and gene expression. To pursue such functional roles for lncRNAs in P. falciparum, we performed deep, strand-specific RNA sequencing of fifteen non-polyA-selected blood stage samples, and assembled and characterized the properties of 660 intergenic lncRNAs, 474 antisense RNAs, and 1381 circular RNAs (circRNAs). We further validated the non-canonical splice junctions of seven P. falciparum circRNAs, an emerging class of non-coding RNA with regulatory potential and unexplored functional significance in P. falciparum. Our comprehensive analysis of P. falciparum lncRNAs indicates a functional role for these transcripts; P. falciparum intergenic lncRNAs and antisense RNAs are developmentally regulated in a similar periodic fashion to annotated transcripts, and sense-antisense pair expression is significantly anti-correlated. Notable outliers include intergenic lncRNAs that strongly peak in expression during parasite invasion, such as the telomere-associated lncRNA-TARE family, antisense transcripts that drop in expression during parasite invasion, and a highly correlated, multi-exonic, antisense counterpart to P. falciparum Gametocyte Developmental Protein 1 (PfGDV1). Taken together, our results present over two thousand P. falciparum intergenic lncRNA, antisense, and circRNA candidates and highlight promising P. falciparum lncRNAs for future investigation. We harvested fifteen blood stage samples from two biological replicate time-courses. The first time-course comprised of eleven samples that finely map temporal changes during P. falciparum blood stage development. We harvested samples over 56 hours, at roughly 4-hour time intervals, from a tightly synchronized P. falciparum 3D7 parasite population. As the asexual blood stage is an approximately 48-hour cycle, this time-course allowed us to profile gene expression during RBC rupture and parasite invasion. The second time-course comprised of four samples harvested in synchronous P. falciparum 3D7 parasites approximately four hours before and after the ring to trophozoite and trophozoite to schizont morphological stage transitions, which occur during the blood stage at 24 hours post invasion (hpi) and 36 hpi, respectively.
2015-05-08 | E-GEOD-57439 | biostudies-arrayexpress
Project description:The malaria parasite Plasmodium falciparum relies on clonally variant gene expression in order to escape immune recognition and secure continuous proliferation during blood stage infection. Here, we studied the role of heterochromatin protein 1 (HP1), an evolutionary conserved regulator of heritable gene silencing, in the biology of P. falciparum blood stage parasites. We demonstrate that conditional PfHP1 depletion de-represses hundreds of heterochromatic virulence genes and disrupts the elusive mechanism underlying mutually exclusive expression and antigenic variation of PfEMP1. Intriguingly, we also discovered that the PfHP1-dependent regulation of an ApiAP2 transcription factor controls the switch from asexual parasite proliferation to sexual differentiation. This uncovers the first mechanistic insight into the unknown pathway triggering gametocyte conversion and establishes a new concept of HP1-dependent cell fate decision in unicellular eukaryotes. P. falciparum 3D7 parasites expressing endogenous PfHP1-GFP-DD were grown in presence of 4nM WR/625nM Shield-1 (3D7/HP1ON) or 4nM WR (3D7/HP1OFF). RNA extracted from these samples at eleven consecutive time points each was processed for microarray analysis.
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2010-05-05 | E-GEOD-13251 | biostudies-arrayexpress
Project description:The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. To examine the impact of P. falciparum infection on the mosquito midgut and carcass transcriptomes in the presence or absence of midgut bacteria, we used A. gambiae whole genome microarrays to compare the mRNA abundance of P. falciparum-infected and -naïve mosquitoes of antibiotic- and non-antibiotic treated cohorts. P. falciparum infection induced changes in the abundance of as many as 2,183 and 2,429 transcripts in whole mosquitoes belonging to a variety of functional groups in aseptic and septic mosquitoes. Ultimately, we were interested in identifying the genes involved in bacteria-independent anti-Plasmodium responses, and therefore we focused on transcripts displaying increased abundance in the parasite-infected aseptic midguts, placing a particular emphasis on those with predicted immune functions. Because of the central role of serine protease cascades in regulating insect immune defenses, we focused the remainder of our analysis on a clip-domain serine protease C2 (CLIPC2, AGAP004317) and a serine protease inhibitor 7 (SRPN7, AGAP007693) that were specifically upregulated in the parasite-infected, aseptic mosquito midgut. We showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Aseptic and septic midguts and carcasses from P. falciparum-infected A. gambiae vs aseptic and septic midguts and carcasses from uninfected, blood-fed A. gambiae. 3 biological replicates and 1 pseudo-replicate per each array.
2013-08-10 | E-GEOD-49690 | biostudies-arrayexpress
Project description:To study the bromodomain protein PfBDP1 in the malaria parasite P. falciparum, a transgenic parasite line was generated (PfBDP1DD) in which PfBDP1 was tagged with a haemagglutinin tag and a ligand regulatable FKBP destabilization domain that allowed conditional knockdown of PfBDP1 by removal of the stabilizing ligand Shld1 from cultures. Shld1 was removed 30 hours post invasion (hpi) and the cells allowed to reinvade fresh red blood cells. The effects of PfBDP1 knockdown on gene expression were assessed by transcriptional profiling on microarrays over 6 consecutive time points (8 hour intervals) in the intraerythrocytic developmental cycle (IDC). Two condition matched time course experiment, PfBDP1DD ON versus OFF Shld1. Transgenic P. falciparum 3D7 parasites (PfBDP1DD) expressing an endogeneous PfBDP1-HA-DD fusion protein were grown in the presence of 2.5 nM WR99210/500 nM Shld1 (PfBDP1DD_ON) or 2.5 nM WR99210 (PfBDP1DD_OFF). After invasion, RNA was extracted at 6 consectutive time points in 8 hour intervals during the IDC, starting at 4 hpi, and was processed for microarray analysis. Three matched biological replicates were performed for both conditions, independently grown and harvested. Each array represents one RNA sample.
2015-01-07 | E-GEOD-64688 | biostudies-arrayexpress