Exploring the virulence gene interactome withCRISPR/dCas9 in the human malaria parasite

Abstract

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR‐based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl‐CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene‐associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin‐associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183

“…To begin to explore whether inhibition of ACS could affect these downstream pathways, we studied the localization of ACS using an endogenous GFP-tagged ACS parasite line (Fig S9a-b). ACS-GFP localized to the cytosol, although it was unclear whether it is also expressed in the nucleus in P. falciparum (Fig 3c), as previously observed in apicomplexan parasites (27,30,31). To verify this localization, we stained wild-type parasites with ACS immune serum.…”

“…Here, we have conclusively demonstrated for the first time that CoA-PanAm is the active metabolite that inhibits ACS. With the previously suggested role of ACS in regulating the acetylome, transcriptome and metabolome (including fatty acid elongation) (27,28,30,31,39) and the corresponding cytoplasmic/perinuclear (this study) and nuclear localization of ACS for these functions (31), it could be hypothesized that these pathways are affected by CoA-PanAms.…”

The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum

“…However, although we have identified a panel of PfHMGB1-binding target genes at their upstream regions in P. falciparum , the transcriptional activities of these genes are not directly controlled by this factor. A recent study also shows that PfHMGB1 is not present in the protein complex binding to var gene loci ( 45 ). Therefore, differential expression of those genes in the Pfhmgb1 -KO line is a consequence of nuclear reorganization.…”

Peculiarities of Plasmodium falciparum Gene Regulation and Chromatin Structure

“…A repressed state in contrast—extensively studied on the example of var genes—is maintained by heterochromatin protein HP1, histone deacetylase HDA2 and the histone lysine methyltransferase SET2 and marked by H3K9me3 rendering these genes heterochromatic [ 94 , 95 , 96 , 97 ]. Furthermore, a chromatin-remodeling enzyme [PF3D7_0624600] and sirtuin proteins influence chromatin condensation [ 98 , 99 ], and the incorporation of noncoding RNAs complement the var gene-switching mechanism by silencing the gene locus via its sense—activating via its antisense—lncRNA [ 90 , 100 ]. Sexual commitment is known to be regulated by the epigenetic cascade starting with Ap2-G expression being repressed by HP1, which is evicted upon gametocyte development 1 (GDV1) association to heterochromatin, and GDV1 itself is controlled by the gdv1 antisense RNA [ 35 , 97 , 101 , 102 ].…”

“…In a pioneering study, Ji and Arnot identified and classified the first SWI2/SNF2 enzyme (Snf2L) with approximately 60% sequence homology to the ATPase domain of the yeast ISWI remodeler [ 127 ]. Since then, only a few studies addressed apicomplexan remodelers [ 98 , 128 ]. A genome-wide mutagenesis screen proposed one of the plasmodial remodelers to be essential and another two as putatively essential in asexual blood stages [ 9 ].…”