Project description:The parasite Plasmodium falciparum is responsible for severe malaria, which remains a major cause of death, particularly in sub-Saharan Africa. The reference strain NF54 (or its subclone 3D7) is commonly used for controlled human malaria infection (CHMI), but recently strains with a different geographic and genomic background have become available for CHMI, including 7G8, which was subcloned from the Brazilian isolate IMTM22 in 1984 (Burkot TR et al. 1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg 78 (3):339-41. doi: 10.1016/0035-9203(84)90114-7). To provide the first RNA-seq reference dataset for 7G8 ring stage parasites and validate our qPCR primer set targeting all var gene variants above 6 kb encoded in the 7G8 genome according to current guidelines and best practices, we thawed an aliquot from the Sanaria 7G8 parasite working cell bank (Lot: SAN03-021214 dated 20. February 2014) thawed and cultured for 13 parasite generations at a hematocrit of 5% in human O+ erythrocytes. Parasites were kept synchronous by regular sorbitol treatment. Ring-stage infected erythrocytes were lysed with Trizol, and total RNA was purified using column-based purification (Qiagen RNeasy Mini Mit), including DNase treatment on the column and control for absence of genomic DNA contamination by qPCR. Total RNA samples were cleared of human globin mRNA using magnetic bead isolation technology (GLOBINclear kit). After quality control of RNA samples and optimized preparation of cDNA libraries for AT-rich genomes for Illumina sequencing, RNA-seq was performed at BGI Genomics (Shenzhen, China) on the HiSeq 4000 to generate 100 bp paired-end sequencing reads.
Project description:This study examined the difference in gene expression pattern between early ring stage parasites grown in medium with or without 15 mM sodium L-lactate supplementation for 5 hours using RNA-seq approach.
Project description:P. berghei ANKA parasites were collected from the blood at 74h after infection of wild-type and TCRδ-/- mice with 2x10^4 sporozoites (samples were pooled from 3 mice/ group) and mRNA was sequenced by RNA-seq.
Project description:Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the Plasmodium genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis.
Project description:The ability to sense, respond and adapt to changes in nutrient availability is a survival requisite. This might be particularly important for malaria parasites, which encounter major alterations in nutrients levels throughout infection. How these parasites deal with nutrient fluctuations and maintain infection without killing their hosts before transmission remains unknown. Here, we show that blood-stage malaria parasites respond to dietary-restriction through a rearrangement of their transcriptome accompanied by a significant reduction in their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN, a putative AMP-activated kinase homologue, as a master regulator that senses host nutrients both in vitro and in vivo, and mediates the transcriptional response to the host nutritional status. Diet-responsive genes include the plant-like ApiAP2 transcription regulators, which appear to act as downstream effectors of KIN. Overall, these findings reveal key components of a parasite nutrient-sensing mechanism that is critical to modulate replication and virulence in malaria parasites.
Project description:To profile cell-cycle progression and the asynchronous process of differentiation, we performed single-cell RNA-sequencing (scRNA-Seq) of T. gondii using Seq-Well (Gierahn et al., 2017). Wild-type or ∆BFD1 parasites were grown under unstressed or stressed conditions (24, 48 or 72 h), mechanically released from host cells, and analyzed.
Project description:The Purpose of this series of experiments is to identify copy number variations, duplications, and deletions in human embryonic stem (hES) cell lines deposited to the National Stem Cell bank and reveal the difference between different hES cell lines. CGH can achieve these aims at higher resolution. Keywords: comparative genomic hybridization