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Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite

ABSTRACT: The var multigene family encodes clonally variant surface antigens that are key to immune evasion and pathogenesis in the human malaria parasite, Plasmodium falciparum. Epigenetics and nuclear organization regulate the var gene family in a system of mutually exclusive expression; however, few factors have been shown to play a direct role in these processes. Thus, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for identification of novel factors associated with var genes in their natural chromatin context. A tagged, catalytically inactive Cas9 (“dCas9”) was targeted to the promoters or introns of a subset of var genes and subjected to immunoprecipitation followed by label-free LC-MS/MS. A non-targeted dCas9 served as a control.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

TISSUE(S): Erythrocyte

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Valentin SABATET

LAB HEAD: Damarys Loew

PROVIDER: PXD013131 | Pride | 2020-07-15

REPOSITORIES: pride

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			Action	DRS
	Design.xls	Xls
	H2273FD.msf	Msf
	H2273FD.raw	Raw
	H2274FD.msf	Msf
	H2274FD.raw	Raw

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Environmental inhibition of RNA Pol III reduces malaria parasite virulence (during asymptomatic infection)

Project description:During the dry season, in many African regions, persistent asymptomatic malaria cases have been linked to downregulated virulence gene expression encoded by the var gene family, resulting in low adhesion and increased circulation time of Plasmodium falciparum-infected erythrocytes 1. Several studies have investigated associations between parasite transcription and clinical severity, but the underlying process that results in lower virulence gene expression in asymptomatic malaria remains unknown. Here we report an RNA Polymerase III-dependent mechanism that in response to external stimuli, decreases var gene transcription and parasite multiplication rate. The singular expression of var gene members, while under tight epigenetic regulation by the parasite 2, is additionally controlled by a family of ncRNA, termed RUF6 3. We show that Pol III transcription of tRNAs and RUF6 is severely decreased in field isolates of asymptomatic individuals infected with P. falciparum during the dry season. We identified Maf1 as a negative effector of RUF6 ncRNA transcription and show that external stimuli depend on plasmodial Maf1 to trigger lower expression of RNA Pol III-transcribed genes. Our results uncover a TOR-independent unprecedented signaling pathway in a protozoan pathogen that is highly relevant for establishing asymptomatic subclinical parasite persistence in the dry season.

2024-06-25 | PXD040576 | Pride

Discovery of RUF6 ncRNA interacting proteins involved in P. falciparum immune evasion

Project description:RUF6 is a ncRNA gene family that is transcribed by RNA Polymerase III but actively regulates the Pol II-transcribed var virulence gene family. Understanding how RUF6 ncRNA connect to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including. Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression.

2022-11-15 | PXD036801 | Pride

Exploring the var gene interactome with CRISPR/dCas9

Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.

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Mutually Exclusive Transcription of Subtelomeric Gene Families in Plasmodium falciparum is Restricted to var Genes

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:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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