Method for deletion of variant surface antigen genes at subtelomeric region of Plasmodium falciparum.
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ABSTRACT:
ORGANISM(S): Plasmodium falciparum 3D7
PROVIDER: GSE296188 | GEO | 2025/05/07
REPOSITORIES: GEO
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Project description:Background: The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes to each VSA gene. Methods: All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray. Results: From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly upregulated gene in rosetting parasites was ITvar9 (19 to 429-fold upregulated over six time points). Only one rif gene (IT4rifA_042) was upregulated by more than 4-fold (5-fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were upregulated by more than 2-fold. 49 non-VSA genes were upregulated in rosetting parasites by more than 3-fold in at least two time-points, although none as markedly as ITvar9. Conclusions: We demonstrate that the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.
2011-07-01 | GSE28990 | GEO
Project description:Background: The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes to each VSA gene. Methods: All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray. Results: From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly upregulated gene in rosetting parasites was ITvar9 (19 to 429-fold upregulated over six time points). Only one rif gene (IT4rifA_042) was upregulated by more than 4-fold (5-fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were upregulated by more than 2-fold. 49 non-VSA genes were upregulated in rosetting parasites by more than 3-fold in at least two time-points, although none as markedly as ITvar9. Conclusions: We demonstrate that the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation. Plasmodium falciparum parasites, strain IT/R29, were selected for (R29R+) or against (R29R-) rosetting. Both cultures (R29R+ and R29R-) were tightly synchronised before a timecourse experiment was performed. 6 samples, named time points 1 to 6, were taken every 8 hours. 12μg of RNA from the R29 non-rosetting parasites at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both rosetting and non-rosetting parasites were then used for cDNA synthesis. For microarray hybridizations,each cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample. The solution was loaded on a microarray slide and hybridized for 14–16 h.
2011-07-01 | E-GEOD-28990 | biostudies-arrayexpress
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.
2011-09-01 | E-GEOD-31829 | biostudies-arrayexpress
Project description:Epigenetic regulation is recognized as a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite P. falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the bromodomain proteins PfBDP1 and PfBDP2 (BDP1/BDP2 core complex) was shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7 (P. falciparum bromodomain protein 7), because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, Stevor, and PfMC2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and identified PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.
2021-11-09 | GSE186984 | 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.
2011-09-02 | GSE31829 | GEO
Project description:Genomic variation is an inherent phenomena observed among members of same species belonging to different geographical locations. In case of P. falciparum, an apicomplexan protozoan parasite, its 22.8 MB nuclear genome is known to display vast genetic diversity in the subtelomeric compartments having but not exclusively variant gene families like var, rifins and stevors and examples in other elements of the genome have recently been documented. Microarrays, relies solely on the genomic sequence information to capture the relevant transcript abundance and needs to consider these variations into account for revealing true transcriptional variation.Here, we describe the designing strategy of a custom P. falciparum 15K array using Agilent platform to study the transcriptome of Indian field isolates for which genome sequence information is limited. Array contains probes representing genome sequence of two distinct geographical isolates (i.e 3D7 and HB3) and subtelomeric var gene sequence of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts by performing a 244K array experiment representing multiple probes per gene/transcript. Array performance was evaluated and validated using RNA materials from P. falciparum clinical isolates obtained directly from patients with differing clinical conditions due to malaria infection.Due to pre probe screening large percentage (91 %) of the represented transcripts could be detected from Indian P. falciparum isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. Plasmodium falciparum isolates were collected from patients (n=13) with differing clinical conditions. The patients exhibited symptoms categorized as uncomplicated (n=6) or complicated malaria (n=7). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to evaluate the performance of the array.
2016-01-17 | E-GEOD-54253 | biostudies-arrayexpress
Project description:Genomic variation is an inherent phenomena observed among members of same species belonging to different geographical locations. In case of P. falciparum, an apicomplexan protozoan parasite, its 22.8 MB nuclear genome is known to display vast genetic diversity in the subtelomeric compartments having but not exclusively variant gene families like var, rifins and stevors and examples in other elements of the genome have recently been documented. Microarrays, relies solely on the genomic sequence information to capture the relevant transcript abundance and needs to consider these variations into account for revealing true transcriptional variation.Here, we describe the designing strategy of a custom P. falciparum 15K array using Agilent platform to study the transcriptome of Indian field isolates for which genome sequence information is limited. Array contains probes representing genome sequence of two distinct geographical isolates (i.e 3D7 and HB3) and subtelomeric var gene sequence of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts by performing a 244K array experiment representing multiple probes per gene/transcript. Array performance was evaluated and validated using RNA materials from P. falciparum clinical isolates obtained directly from patients with differing clinical conditions due to malaria infection.Due to pre probe screening large percentage (91 %) of the represented transcripts could be detected from Indian P. falciparum isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains.
2016-01-17 | GSE54253 | GEO
Project description:Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, PfMORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (AP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PfAP2-MRP and PF3D7_0420300, PF3D7_0613800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1, PfEELM2, and PfISWI). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation.
2024-11-05 | GSE239393 | GEO
Project description:Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (AP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800 , and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1, PfEELM2, and PfISWI). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation.
2024-08-14 | PXD044256 | Pride
Project description:Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. Data revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA. The vast majority of the genome shows an unexpected pattern of enrichment of H3K4me3 and H3K9ac at intergenic regions and depletion at genes. Chip-chip (and cDNA) from Plasmodium falciparum strain NF54 asexual blood stages with H3, H3K4me3, H3K9me3 and H3K9ac
2010-05-19 | E-GEOD-15728 | biostudies-arrayexpress