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TurboID identification of evolutionarily divergent components of the nuclear pore complex in the malaria model Plasmodium berghei

ABSTRACT: Twenty years since publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium NPC (nuclear pore complex) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry we identify four known Nups (138, 205, 221, and the bait 313) and verify interaction with the putative FG Nup637; we assign five proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by GFP tagging. Based on gene deletion attempts, all new Nups Nup176, 269, 335, 390, and 434 are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit two incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC4. The protocols developed here showcase the power of proximity-labeling for elucidating protein complex composition and annotation in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position.

INSTRUMENT(S): LTQ Orbitrap, Orbitrap Fusion Lumos

ORGANISM(S): Plasmodium

DISEASE(S): Malaria

SUBMITTER: Sushma Ambekar

LAB HEAD: Gunnar Mair

PROVIDER: PXD034491 | Pride | 2022-08-21

REPOSITORIES: Pride

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Project description:Analysis of transcriptional response to Plasmodium berghei ANKA infection in cerebral malaria susceptible C57BL/6 mouse brains as well as cerebral malaria resistant BXH2 mice which carry a severe hypomorphic Irf8-R294C allele. Interferon Regulatory Factor 8 (IRF8) is required for development, maturation and expression of anti-microbial defenses of myeloid cells. BXH2 mice harbor a loss-of-function allele at Irf8 (Irf8-R294C) that causes susceptibility to infection with intracellular pathogens, including Mycobacterium tuberculosis. We report that XH2 are completely resistant to the development of cerebral malaria following Plasmodium berghei ANKA infection. Comparative transcriptional profiling of brain RNA as well as chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) was used to identify IRF8-regulated genes whose expression is associated with pathological acute neuroinflammation. Genes up-regulated by infection were strongly enriched for IRF8 binding sites, suggesting that IRF8 acts as a transcriptional activator in inflammatory programs. These lists were enriched for myeloid-specific pathways, including interferon responses, antigen presentation and Th1 polarizing cytokines. We show that inactivation of several of these downstream target genes confers protection against experimental cerebral malaria. We also report strong overlap between genes bound and regulated by IRF8 during cerebral malaria and genes regulated in the lungs of M. tuberculosis infected mice. This IRF8-dependent network contains several genes recently identified as risk factors in acute and chronic human inflammatory conditions. In summary, this work defines a common core of IRF8-bound genes forming a critical inflammatory host-response network. Comparison of whole brain transcript profiles for wildype C57BL/6 mice versus severely hypomorphic Irf8-R294C BXH2 mice following experimental infection with Plasmodium berghei ANKA (d7). Baseline (d0) profiles are also compared. Pleaes note that each sample record represents 2-4 replicates and sample data table contains mean, standard error of the mean (SEM), and quality for the replicates. The non_normalized data matrix contains raw data for each replicate (total 12 samples).

Project description:Control of malaria is threatened by emerging parasite resistance to artemisinin drug (ART) therapies. The molecular details of how Plasmodium malaria parasites response to ART and how this relates to resistance is not clear. To determine how parasites respond to ART by altering gene expression, we performed a transcriptomic study of dihydroartemisinin (DHA) response in P. falciparum K1 strain and in P. berghei ANKA strain. Microarray data from DHA-treated P. falciparum trophozoite stage parasites were compared with data from other ART treatments. Genes with consistent changes in expression were identified, which includes notably down-regulation of cytosolic ribosomal protein genes. RNA-seq data revealed a similar pattern of transcriptomic change, although the pattern was much clearer in that more than one-third of P. falciparum trophozoite genes are differentially expressed with greater statistical support for down-regulation of ribosomal protein genes. The poor overlap of differentially-expressed genes between microarray and RNA-seq and less-well defined patterns for the former suggests that the accuracy of microarray is limited by technological bias. The trophozoite response to DHA is overall âring-likeâ and less âtrophozoite-likeâ, which is consistent with previous findings that Plasmodium can enter a quiescent ring-like state to resist ART. RNA-seq data from DHA-treated P. falciparum rings reveal a more muted response, although there is considerable overlap of differentially expressed genes with DHA-treated trophozoites. In contrast, P. falciparum schizonts are unresponsive to DHA, suggesting that the protective response acts mainly to arrest parasite development through the G2/M checkpoint. The transcriptional response of P. berghei to DHA treatment in vivo in infected mice is strikingly similar to the P. falciparum in vitro ring and trophozoite responses, in which ribosomal protein genes are notably down-regulated. These results suggest Plasmodium species respond to DHA in the same way. This knowledge could be applied to outwit the parasite to deliver more effective artemisinin therapies, and maybe hinder the development of drug resistance. Two condition drug-treatment experiment, Dihydroartemisinin vs. Vehicle control treatment with matched reference untreated controls. Biological replicates: 5 independently grown and harvested experimental culture replicates. One replicate of treatment/reference time-point per array.

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TurboID identification of evolutionarily divergent components of the nuclear pore complex in the malaria model Plasmodium berghei

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