Abstract:Malaria parasites evade immune detection by growth and replication within erythrocytes. After erythrocyte invasion, the intracellular pathogen must increase host cell uptake of nutrients from plasma. Here, we report that the parasite-encoded RhopH complex contributes to both invasion and channel-mediated nutrient uptake. As rhoph2 and rhoph3 gene knockouts were not viable in the human P. falciparum pathogen, we used conditional knockdowns to determine that the encoded proteins are essential and to identify the… Show more
“…In agreement with these transcriptional data, analysis of RhopH3 protein expression over time by immunofluorescence microscopy confirmed significantly increased levels in sexually committed cells (AP2-G+) compared to AP2-G- cells and control conditions (
Figure 4d). RhopH3 is present in rhoptries and required for merozoite invasion into red blood cells (RBC), and at the infected RBC surface as part of a nutrient channel
27 . The protein is initially localized to rhoptries and upon invasion it is translocated into the host cell cytosol and surface membrane.…”
Section: Resultsmentioning
confidence: 99%
“…RhopH3 was used as previously published
27 , namely using a 1:1 ratio of acetone:methanol, followed by blocking in 3% milk powder. The RhopH3 rabbit antibody was diluted 1:500 and incubated for 40 min at room temperature.…”
Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in
Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission.
Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells.
Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions.
Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.
“…In agreement with these transcriptional data, analysis of RhopH3 protein expression over time by immunofluorescence microscopy confirmed significantly increased levels in sexually committed cells (AP2-G+) compared to AP2-G- cells and control conditions (
Figure 4d). RhopH3 is present in rhoptries and required for merozoite invasion into red blood cells (RBC), and at the infected RBC surface as part of a nutrient channel
27 . The protein is initially localized to rhoptries and upon invasion it is translocated into the host cell cytosol and surface membrane.…”
Section: Resultsmentioning
confidence: 99%
“…RhopH3 was used as previously published
27 , namely using a 1:1 ratio of acetone:methanol, followed by blocking in 3% milk powder. The RhopH3 rabbit antibody was diluted 1:500 and incubated for 40 min at room temperature.…”
Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in
Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission.
Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells.
Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions.
Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.
“…Interestingly cytoadherence-linked asexual genes (CLAG) proteins, are also targets of PfAP2-I. Although the CLAGs are members of the RhopH protein family, they are not involved in invasion but their expression matches that of invasion-related genes (Counihan et al, 2017; Ito et al, 2017; Sherling et al, 2017). …”
Summary
Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor PfAP2-I, belonging to the Apicomplexan AP2 (ApiAP2) family, that is responsible for regulating the expression of genes involved in RBC invasion. Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of target genes. Although PfAP2-I contains three AP2 DNA-binding domains, only one is required for binding of the target genes during blood stage development. Furthermore, we find that PfAP2-I associates with several chromatin-associated proteins, including the Plasmodium bromodomain protein PfBDP1, and that complex formation is associated with transcriptional regulation. As a key regulator of red blood cell invasion, PfAP2-I represents a potential new antimalarial therapeutic target.
“…A manuscript utilizing the same strategy to deplete RhopH2 in Plasmodium falciparum was also published by Ito and colleagues (Ito et al, 2017). These authors derive very similar conclusions about the role of RhopH2 in nutrient uptake.10.7554/eLife.23217.017Figure 10.Scheme illustrating how RhopH2 knockdown effects blood stage development.( a ) Knockdown of RhopH2 expression in cycle one appears to impair uptake of plasma nutrients in cycle two which delays development and replication in cycles 2 and 3.…”
Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival.DOI:
http://dx.doi.org/10.7554/eLife.23217.001
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