Project description:To investigate the DNA-binding property of homeodomain-like domain of Plasmodium berghei HDP1, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis were performed. Recombinant homeodomain-like domain fused with glutathione S-transferase were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using glutathione-sepharose resin. The obtained DNA fragments were sequenced via the next generation sequencing.
Project description:Variant antigens that are encoded by large multigene families play an important role in the adaptation and immune evasion of a wide range of pathogens. However, the study of their biological function is significantly hampered by the difficulty in controlling their expression in its cellular setting. The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for their survival. However, with the exception of the P. falciparum var genes very little is known about the biological roles of any of the other multigene families. Here we report a highly efficient genetic system to study variant antigens in Plasmodium spp. using the Selection Linked Integration method; we are able to activate the expression of a single member of a multigene of our choice using its endogenous promoter.
Project description:To investigate the DNA-binding property of two tandem AP2 domains of PbSIP2, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis were performed. Recombinant AP2 domains fused with maltose-binding protein (MBP) were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using amylose resin. The obtained DNA fragments were sequenced via the next generation sequencing.
Project description:Background: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels. Methods: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals. Results: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.
Project description:The objective of this study is to provide a novel method to study multigene proteins in Plasmodium spp. The method is based on selection linked integration (SLI), which allows positive selection of genomic integration events. By targeting specific members of multigene families, parasites are being selected for not only genomic integration but also expression of the targeted gene under its endogenous promoter
Project description:Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Yet, the underlying mechanisms driving the pathogenesis of malaria-associated hypoglycemia remain poorly understood. Here we report that labile heme, an alarmin generated as a byproduct of hemolysis during the blood stage of Plasmodium spp. infection, plays a central role in the development of malaria-associated hypoglycemia. Labile heme recapitulated the hypometabolic response to Plasmodium (chabaudi chabaudi; Pcc) infection in mice, including the development of anorexia, transcriptional repression of hepatic glucose production (HGP) and reduction of glycemia, energy expenditure (EE) as well as core body temperature. While this hypometabolic response is protective against immune-mediated liver damage and anemia, when sustained over time it can lead to hypoglycemia and compromise EE as well as thermoregulation. I response, asexual stages of Plasmodium spp. activate a transcriptional program that reduces virulence in favor of sexual commitment and presumably malaria transmission. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic defense strategy against Plasmodium infection.
Project description:Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Yet, the underlying mechanisms driving the pathogenesis of malaria-associated hypoglycemia remain poorly understood. Here we report that labile heme, an alarmin generated as a byproduct of hemolysis during the blood stage of Plasmodium spp. infection, plays a central role in the development of malaria-associated hypoglycemia. Labile heme recapitulated the hypometabolic response to Plasmodium (chabaudi chabaudi; Pcc) infection in mice, including the development of anorexia, transcriptional repression of hepatic glucose production (HGP) and reduction of glycemia, energy expenditure (EE) as well as core body temperature. While this hypometabolic response is protective against immune-mediated liver damage and anemia, when sustained over time it can lead to hypoglycemia and compromise EE as well as thermoregulation. In response, asexual stages of Plasmodium spp. activate a transcriptional program that reduces virulence in favor of sexual commitment and presumably malaria transmission. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic defense strategy against Plasmodium infection.
Project description:In malaria infection, Plasmodium spp. parasites accumulate in the bone marrow near sites of erythroid development. While it has been observed that Plasmodium falciparum infection of late-stage erythroblasts can delay terminal erythroid differentiation and enucleation, the mechanism(s) underlying this phenomenon are unknown. Here, we apply RNA-seq after fluorescence-activated cell sorting (FACS) of infected erythroblasts to identify transcriptional responses to direct and indirect interaction with P. falciparum.