Project description:Cyclic nucleotides are important signalling molecules across evolution, but their roles in malaria parasites are poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another protein with putative cyclic nucleotide binding sites, PfEpac, does not play an essential role in cyclic nucleotide signalling in blood stages. We identify over 2,000 sites whose phosphorylation is dependent on cAMP signalling and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) controls erythrocyte invasion.
Project description:Malaria remains a global health issue requiring the identification of novel therapeutic targets to combat drug resistance. Metabolic serine hydrolases are druggable enzymes playing essential roles in lipid metabolism. However, very few have been investigated in malaria-causing parasites. Here, we used fluorophosphonate broad-spectrum activity-based probes and quantitative chemical proteomics to annotate and profile the activity of more than half of predicted serine hydrolases in P. falciparum across the erythrocytic cycle. Using conditional genetics, we show that the activities of four serine hydrolases, previously annotated as essential (or important) in genetic screens, are actually dispensable for parasite replication. Importantly, we also identified eight human serine hydrolases that are specifically activated at different developmental stages. Chemical inhibition of two of them blocks parasite replication. This strongly suggests that parasites co-opt the activity of host enzymes and opens a new drug development strategy against which the parasite is less likely to develop resistance.
Project description:The purpose of this project was to identify targets and substrates of Protein kinase PfPK2 in malaria parasite P. falciparum. These studies will help understand the mechanisms via which PfPK2, which is indispensable for the parasite survival, regulates parasite development. For this purpose, PfPK2-loxP parasite line was generated for conditional knock down of PfPK2 using rapamycin (RAP), which was used to induce the dimerization of active Cre-recombinase resulting in the depletion of PfPK2.
Project description:ChIP-seq experiments were performed for the putative telomere repeat-binding factor (PfTRF) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRF at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRF was enriched at seven additional, intra-chromosomal sites and called in the PfTRF-HA ChIP-seq only. Plasmodium falciparum 3D7 parasites were generated with -GFP or -3xHA C-terminal tagged TRF (PF3D7_1209300). Nuclei were isolated from formaldehyde cross-linked schizont-stage transgenic parasites and used to prepare chromatin. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001) or rat anti-HA 3F10 (Roche Diagnostics, #12158167001). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.
Project description:In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG). A major function of PKG is to control calcium signals essential for the parasite to exit red blood cells or for transmission to the mosquito vector. However, how PKG controls these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins remains a mystery. Here we identify a tightly-associated PKG partner protein in Plasmodium falciparum asexual blood stages. Named ICM1, the partner is a polytopic membrane protein with homology to transporters and calcium channels, raising the possibility of a direct functional link between PKG and calcium homeostasis
Project description:To identify malaria protein interactions systematically we combined blue native polyacrylamide electrophoresis fractionation with quantitative mass spectrometry and machine learning. Our integrative approach identified over twenty thousand protein interactions in Plasmodium schizonts, organized into 600 complexes, the majority of which are novel, and generated a high-confidence Plasmodium protein interaction network.
Project description:Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1,YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. Indeed, we show that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central M-bM-^@M-^\switch on/offM-bM-^@M-^] proteins to coordinate the regulation of C. albicans morphogenesis. ChIP was performed in 2 independently grown C. albicans sfl1 or sfl2 homozygous mutant strains expressing (sfl1-CaEXP-SFL1-HA or sfl2-CaEXP-SFL2-HA, respectively) or not (sfl1-CaEXP or sfl2-CaEXP, respectively) SFL1-HA or SFL2-HA (-HA, 3'-triple-HA-tagged alleles of SFL1 or SFL2) under the control of a methionine-repressible promoter (Total samples = 8; 2xCaEXP-SFL1-HA, 2xCaEXP-SFL2-HA, 2xCaEXP control for SFL1-HA ChIP and 2xCaEXP control for SFL2-HA ChIP).
Project description:The effects of constitutively active Hypoxia Inducible Factor (HIF) and inactivated von Hippel-Lindau tumor suppressor gene product (pVHL) were examined in a mouse model. Conditionally expressed, constitutively active HIF-1a and HIF-2a were compared with inactivated pVHL.
Project description:Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane then seals and pinches off behind the parasite through an unknown mechanism, enclosing it within the RBC. During invasion, several merozoite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null merozoites with RBCs leads to either successful entry but developmental arrest, or abortive invasion with rapid RBC lysis. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for the correct functioning of merozoite surface protein substrates and for host RBC membrane sealing following parasite internalisation.
Project description:RiboTag-based translatome analysis of pooled oAβ and PBS-challenged brain homogenates of Cx3cr1ccre:Lyve1ncre (n=8 mice) and Cx3cr1ccreSall1ncre:RiboTag-mice (n=4 mice).