Transcriptomic analysis of Plasmodium PBANKA, PBSLTRiP-KO, PB268-KO parasite infected and uninfected host cell
ABSTRACT: Liver stage of malaria parasite exports SLTRiP and PB268 to the cytosol of parasite infected host cell. To know the host genes perturbed by WT-PBANKA, SLTRiP-KO and PB268-KO parasite growth, we did transcriptomic sequencing of infected host cells. We did mRNA sequencing of four samples for comparative analysis of WT and PB-knockout parasites infected host cells at 22 hours of post sporozoites infection. mRNA profiles of Plasmodium PBANKA, PBSLTRiP-KO, PB268-KO parasite infected and uninfected HepG2 cells after 22hrs of sporozoites infections were generated by deep sequencing using Illumina GAIIx.
Project description:Liver stage of malaria parasite exports SLTRiP and PB268 to the cytosol of parasite infected host cell. To know the host genes perturbed by WT-PBANKA, SLTRiP-KO and PB268-KO parasite growth, we did transcriptomic sequencing of infected host cells. We did mRNA sequencing of four samples for comparative analysis of WT and PB-knockout parasites infected host cells at 22 hours of post sporozoites infection. Overall design: mRNA profiles of Plasmodium PBANKA, PBSLTRiP-KO, PB268-KO parasite infected and uninfected HepG2 cells after 22hrs of sporozoites infections were generated by deep sequencing using Illumina GAIIx.
Project description:Malaria’s cycle of infection requires parasite transmission between a mosquito vector and a vertebrate host. Plasmodium regulates transmission by translationally repressing specific mRNAs until their products are needed. We demonstrate that the Plasmodium yoelii Pumilio-FBF family member Puf2 allows the sporozoite to retain its infectivity in the mosquito salivary glands while awaiting transmission. Puf2 mediates this critical feature solely through its RNA-Binding Domain (RBD) likely by protecting and silencing specific mRNAs. Puf2 storage granules are distinct from stress granules and P-bodies and dissolve rapidly after infection of hepatocytes, likely releasing the protected and silenced transcripts for ‘just-in-time’ translation by early exoerythrocytic forms (EEFs). Further corroborating this model, pypuf2- sporozoites have no apparent defect in host infection early after invading the salivary glands, but become progressively noninfectious and subsequently prematurely transform into EEFs during prolonged salivary gland residence. In contrast, the premature overexpression of Puf2 in oocysts causes striking deregulation of sporozoite maturation, resulting in fewer oocyst sporozoites that are non-infectious and unable to colonize the salivary glands. Maintenance of Puf2 expression in liver stage parasites produces no phenotype, suggesting that a window of permissive expression exists. Finally, by conducting the first comparative RNAseq analysis of Plasmodium sporozoites, we have uncovered that Puf2 may play a role in both the protection of specific transcripts as well as RNA turnover via the CCR4/Not complex. These findings uncover requirements for maintaining a window of opportunity for the malaria parasite to accommodate the unpredictable moment of transmission from mosquito to vertebrate host. Wild-type (Py17XNL) and pypuf2 -salivary gland sporozoites
Project description:We show that an ongoing malaria blood stage infection impairs the establishment of Plasmodium sporozoites in hepatocytes and that secondary infections can only be established after a previous infection has been cleared from circulation. Using control mice, mice infected with sporozoites only, mice infected by iRBCs only or mice reinfected, we show that this impairment is not due to an effect of the acquired host immune response or to a decrease in host cell survival. Instead, an ongoing blood stage infection leads to a significant increase in the expression of hepcidin, a peptide hormone that is secreted by the liver and controls body iron homeostasis. A rapid increase of hepcidin levels during blood stage infection causes sequestration of iron in storage forms within cells of reticuloendothelial system decreasing its availability in hepatocytes, where it is required for Plasmodium sporozoite establishment.
Project description:Many eukaryotic developmental and cell fate decisions are effected post-transcriptionally that mechanistically involve RNA binding proteins as regulators of translation of key mRNAs. In the unicellular eukaryote malaria parasite, Plasmodium, one of the most dramatic changes in cell morphology and function occurs during transmission between mosquito and human host. In the mosquito salivary glands, Plasmodium sporozoites are slender, motile and remain infectious for several weeks; only after transmission and liver cell invasion, does the parasite rapidly transform into a round, non-motile exo-erythrocytic form (EEF) that gives rise to thousands of infectious merozoites to be released into the blood stream. Here we demonstrate a Plasmodium homolog of the RNA binding protein, Pumilio, as a key regulator of the sporozoite to EEF transformation. In the absence of Pumilio-2 (Puf2) Plasmodium berghei sporozoites initiate early stage EEF development inside mosquito salivary glands with characteristic morphological changes; puf2- salivary gland sporozoites lose gliding motility, cell traversal ability and are less infective. Global expression profiling confirmed that transgenic parasites exhibit genome-wide transcriptional adaptations typical for Plasmodium intra-hepatic development. The data demonstrate that Puf2 is a key player in regulating developmental control, and imply that transformation of salivary gland-resident sporozoites into early liver stage parasites is regulated by a post-translational mechanism.
Project description:Systemic injection of salivary glands P. berghei ANKA GFP-sporozoites into IFNAR-/- mice or salivary glands extracts from non-infected mosquitoes into wild-type C57BL/6 mice. Data obtained were compared with part of hybridizations from experiment E-TABM-839
Project description:In the rodent malaria parasite Plasmodium berghei, we found that an AP2 family transcription factor designated AP2-L plays a critical role in the development of the liver stage. Using DNA microarray analysis we showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Gene expression of P. berghei sporozoites was compared between wild type and AP2-G KO parasites. Five biologically independent experiments were performed for each genotype.
Project description:After entering their mammalian host via the bite of an Anopheles mosquito, Plasmodium sporozoites migrate to the liver where they traverse several hepatocytes before invading the one inside which they will develop and multiply into thousands of merozoites. Although this constitutes an essential step in malaria infection, the requirements of Plasmodium parasites in liver cells and how they use the host cell for their own survival and development are poorly understood. To gain new insights into the molecular host-parasite interactions that take place during malaria liver infection, we have used high-throughput microarray technology to determine the transcriptional profile of P. yoelii-infected hepatocytes that were collected from P. yoelii-infected mice 24 and 40 h after infection. This in vivo microarray expression was compared with the microarray analysis of in vitro infected hepatoma cells infected with closely related rodent malaria parasite P. berghei. Differential expression patterns for host genes identify genes and pathways involved in the host response to rodent Plasmodium parasites. Keywords: gene expression Total RNA from sorted liver stage (LS) infected hepatocytes were isolated from PyGFP-infected BALB/c mice as described in Tarun et al (2008). RNA from LS-infected hepatocytes were isolated at two time points post-infection (pi): 24 hr pi (LS24) and 40 hr pi (LS40). As control, RNA from hepatocytes isolated from mock-infected mice (infected with salivary gland extract) at the same time points was also isolated following the same procedure. Total RNA was then subjected to two rounds of linear amplification using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion) according to manufaturer’s directions. The quality of total and amplified RNAs were examined with the Agilent 2100 BioanalyzerTM (Agilent Technologies) and the quantity of the RNA samples was assessed using a Nanodrop ND-1000 spectrophotometer prior to microarray hybridization. For each timepoint two independent biological replicates were obtained.
Project description:To facilitate pre-eythrocytic malaria vaccine and drug target identification, a comprehensive transcriptome analysis of the parasites liver stages (LS) was undertaken. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared to other parasite life cycle stages. The analysis reveals ~2000 genes active during LS development. Keywords: Stage comparison, time course Overall design: 1. The study profiled gene expressions in samples from 7 stages of malaria parasite P. yoelii life cycle: a. ooSpz: Sporozoites from infected A. stephensi mosquitoes midguts (harvested 10 days after mosquito feeding) b. sgSpz: Sporozoites infected A. stephensi mosquitoes salivary glands (harvested 15 days after mosquito feeding) c. LS24: Isolated liver stage infected hepatocytes 24 hrs after in vivo infection d. LS40: Isolated liver stage infected hepatocytes 40 hrs after in vivo infection e. LS50: Isolated liver stage infected hepatocytes 50 hrs after in vivo infection f. Schz: Purified erythrocytic schizonts g. BS: mixed erythrocytic stages when parasitemia was at 5-10% 2. Samples from Liver stage parasites and Sporozoites were mainly compared to samples from mixed erythrocytic and/or schizont stage parasites with various technical replicates, including dye swaps in some comparisons.
Project description:mRNA Transcripts expression profile of Plasmodium berghei infected host HepG2 cells with and without SUMO1 over expression. To check the changes in the transcript profiles upon SUMO1 over expression which is responsible for Plasmodium berghei parasite's growth arrest in host HepG2 cells. Agilent one-color experiment, Organism: Homo sapiens, Custom Agilent 8x60k Human Whole Genome Microarray Gene expression (AMADID: 039494), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442).
Project description:We report the dual RNA-sequencing of host and pathogen transcriptomes during Plasmodium berghei liver-stage development in vitro. Unlike traditional transcriptomic approaches that analyze RNA reads separately from host and pathogen, a dual-approach maps the mixed reads to each annotated genome within samples of pathogen-infected host cells. This is a powerful method, as host and pathogen transcriptomes can be analyzed simultaneously. We have taken advantage of this dual-RNA sequencing approach in order to gain insight into Plasmodium liver stage development within host hepatocytes. Huh7.5.1 hepatocytes were infected in vitro with P. berghei sporozoites freshly dissected from infected Anopheles stephansi mosquitos, and cells were collected throughout liver-stage development. This included samples collected at time zero (uninfected hepatocytes and sporozoites before infection), time 24 hours post infection (when the sporozoites have transformed into trophozoites), and time 48-50 hours post infection (when the trophozoites have transformed into liver-stage schizonts). Overall design: Dual RNA-sequencing of P. berghei-GFP and human RNA in infected and uninfected Huh7.5.1 cells