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UGO is a multipass membrane protein that is key to sense and transduce cues for schizont egress and gametocyte activation in malaria parasites

ABSTRACT: Many critical events in the Plasmodium lifecycle depend on tight regulation of cGMP homeostasis by guanylyl cyclases (GC) and phosphodiesterases including egress of schizonts from the host erythrocyte and activation of gametocytes upon a mosquito blood-feed. Both processes are triggered by distinct cues but similarly depend on GCα for cGMP production. How these cues are integrated by the same pathway remains a mystery. Here we show that in gametocytes, temperature-dependent epistatic interactions between phosphodiesterases prevent activation prior to a blood-feed. We demonstrate that GCα interacts with two multipass membrane cofactors, UGO and SLF, in schizonts and gametocytes. While SLF is important to regulate GCα basal activity, UGO is essential for schizont egress and gametocyte activation but not for GCα basal activity. Instead, UGO is key to upregulate GCα in response to signals that lower the tension of parasite membranes, including the mosquito molecule xanthurenic acid or a rise in extracellular pH. Altogether our results suggest that intramembrane sensing of distinct signals by UGO represents a focal point to enhance GCα activity and trigger timely schizont egress and activation of gametogenesis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Plasmodium Berghei Anka

TISSUE(S): Gametocyte

SUBMITTER: alexandre Hainard

LAB HEAD: Mathieu Brochet

PROVIDER: PXD035569 | Pride | 2023-04-28

REPOSITORIES: Pride

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			Action	DRS
	20201023_LU50HMC101_pl20109_RR_15.raw	Raw
	20201023_LU50HMC101_pl20109_RR_15__F012851_.mzid.gz	Mzid
	20201023_LU50HMC101_pl20109_RR_15__F012851_.mzid_20201023_LU50HMC101_pl20109_RR_15__F012851_.MGF	Mzid
	20201023_LU50HMC91_pl20109_RR_10.raw	Raw
	20201023_LU50HMC91_pl20109_RR_10__F012846_.mzid.gz	Mzid

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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.

Project description:Purpose: In malaria parasites, the regulation of mRNA translation, storage and degradation is a critical aspect of gene expression, especially during the life stage transition. Since the DEAD-box helicases are involved in RNA metabolism, we wanted to determine whether pfdozi disruption led to altered mRNA abundance in P. falciparum. Methods: In this study, we functionally characterized the DEAD-box RNA helicase PfDOZI in the human malaria parasite Plasmodium falciparum and discovered its functions on mRNA metabolism during development. We generate a pfdozi gene knockout (KO) parasite line. 1). The transcriptomic analysis via RNAseq to the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(10h, 20h, 30h, and 40h post-invasion) and the sexual stage (gametocytes) between KO and wildtype (WT) 3D7 parasite line were performed in triplicate. Total RNA was isolated from the purified parasites using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Differentially expressed genes are determined by DESeq2 with Padj < 0.01 and absolute log2 fold change higher than 1. 2). To investigate the function of pfdozi in stress response, both KO and WT parasites at the schizont stage were cultured under stress conditions. RNAs were harvested for the transcriptomic analysis via RNAseq. 3). To identify the mRNA targets of PfDOZI in schizonts and gametocytes, we performed RNA-immunoprecipitation (RIP) coupled with next-generation sequencing from the PfDOZI::GFP parasites. A gaussian mixture model with a set of stringent criteria, including a read-depth coverage greater than 10, a fold enrichment ratio greater than 2, and posterior probabilities higher than 0.95 in both replicates. Results: 1). RNA-seq analysis identified up-regulation of 18, 4, 7 and 130 genes and down-regulation of 104, 44, 28 and 15 genes in the Δpfdozi parasite compared to WT 3D7 (fold change ≥ 2 and adjusted P (Padj) < 0.01) at ring (10h), early trophozoite (20h), late trophozoite (30h) and schizont (40h) stage, respectively. Our results showed that pfdozi disruption led to the upregulation of invasion-related genes in schizonts; 2). RNA-seq analysis revealed that the effect of pfdozi disruption on gametocytes was substantially more profound than in asexual stages, with 842 downregulated and 752 upregulated transcripts (fold change ≥2, Padj < 0.01) in the Δpfdozi gametocyte. About 80% of these markedly reduced transcripts are gametocyte/ookinete-specific transcripts. Genes related to microtubule/cytoskeleton, cellular respiration, and crystalloid were significantly enriched among the downregulated genes, which agrees with the defective morphogenesis of the Δpfdozi gametocytes. 3). RNAseq analysis showed that under the stress conditions, the WT parasites showed three-fold more upregulated transcripts (591) than downregulated (149), suggesting that significantly more transcripts were protected from degradation under stress conditions. In contrast, the Δpfdozi parasites had a much higher proportion (57%, 463) of downregulated than that of upregulated transcripts (43%, 346) under such stress conditions. 4). The RIP-seq analysis collectively identified the potential target mRNAs of the PfDOZI complex(es) in P. falciparum schizonts and gametocytes, identified 823 and 1377 transcripts as potential target mRNAs of the PfDOZI complexes in schizonts and gametocytes, respectively. Our data also showed significant overlaps with genes whose expression was disturbed upon pfdozi disruption. Conclusions: Collectively, we have demonstrated that PfDOZI, likes its metazoan orthologs, is a versatile regulator of mRNA metabolism, and these functions are dynamically regulated during development with stage-specific characteristics.

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UGO is a multipass membrane protein that is key to sense and transduce cues for schizont egress and gametocyte activation in malaria parasites

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