Malaria is caused by
parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and ...transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of
prevents sexual conversion in proliferating parasites. In this study, we identified
gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing
Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a
antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.
Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, ...polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA-DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (
) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G-C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.
Summary
Background The adipokine chemerin modulates the function of innate immune cells and may link obesity and inflammation, and therefore, a possible relation of chemerin to inflammatory proteins ...in obesity and type 2 diabetes (T2D) was analysed. As visceral fat contributes to systemic inflammation, chemerin was measured in portal venous (PVS), hepatic venous (HVS) and systemic venous (SVS) blood of patients with liver cirrhosis.
Patients and methods Systemic chemerin was determined by ELISA in the serum of normal‐weight, overweight and T2D males, in the serum of T2D patients of both sexes, and in PVS, HVS and SVS of patients with liver cirrhosis.
Results Circulating chemerin was similar in T2D and obese individuals but was significantly elevated in both cohorts compared to normal‐weight individuals. Chemerin positively correlated with leptin, resistin and C‐reactive protein (CRP). In T2D, chemerin was similar in male and female patients and increased in patients with elevated CRP. Chemerin was similar in PVS and SVS, indicating that visceral fat is not a major site of chemerin synthesis. Higher levels of chemerin in HVS demonstrate that chemerin is also released by the liver.
Conclusions Visceral fat is not a major site of chemerin release, and elevated systemic levels of chemerin in obesity and T2D seem to be associated with inflammation rather than body mass index.
Background:
Chemerin is an adipokine that stimulates chemotaxis of cells of the innate immune system. Inflammatory bowel disease (IBD) is linked to an impaired immune response and, therefore, we ...hypothesized that systemic chemerin may be altered in IBD patients.
Methods:
Serum was collected from patients with Crohn's disease (CD, 230 patients), ulcerative colitis (UC, 80 patients), and healthy controls (HC, 80 probands). Chemerin and adiponectin, which has already been measured in the serum of similar cohorts by others, were determined by enzyme‐linked immunosorbent assay (ELISA).
Results:
Chemerin was elevated in IBD compared to HC and was higher in male CD than UC patients. Female and male CD patients had lower adiponectin levels compared to UC, and adiponectin was lower in female CD patients compared to female HC. Adiponectin tended to be higher in female and male UC patients compared to HC and this difference became significant in the whole study group. Correlations with disease activity were only found in males. Here, chemerin was higher in CD patients on remission but was reduced in UC with nonactive disease. Adiponectin was higher in UC with inactive disease. Treatment with corticosteroids was linked to elevated adiponectin in male CD patients and higher chemerin in female UC patients. Unlike adiponectin, which was elevated in female serum in all cohorts, chemerin was only higher in female UC patients.
Conclusions:
These findings further indicate potential regulatory functions of adipokines in intestinal inflammation that are partly gender‐dependent and that may even be associated with the distinct immunopathogenesis of UC and CD. (Inflamm Bowel Dis 2009;)
Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein ...essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.
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•Plasmodium falciparum CyRPA binds to carbohydrates with terminal 2-6-linked Neu5Ac•Lectin activity of Plasmodium falciparum CyRPA contributes to erythrocyte invasion•Targeting lectin activity with drugs and antibodies is a promising anti-malarial strategy
Day et al. show that the essential Plasmodium falciparum invasion protein PfCyRPA is a lectin targeting 2-6-linked Neu5Ac. Molecular modeling, mutagenesis, and transgenic parasite studies show that PfCyRPA lectin activity is required for erythrocyte invasion. Drug and antibody inhibitors validate this activity as a therapeutic target to prevent and treat malaria.
Transmission of the malaria parasite
from the human to the mosquito is mediated by the intraerythrocytic gametocytes, which, once taken up during a blood meal, become activated to initiate sexual ...reproduction. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they are crucial for spreading the tropical disease. During gametocyte maturation, different repertoires of genes are switched on and off in a well-coordinated sequence, pointing to regulatory mechanisms of gene expression. While epigenetic gene control has been studied during erythrocytic schizogony of
, little is known about this process during human-to-mosquito transmission of the parasite. To unveil the potential role of histone acetylation during gene expression in gametocytes, we carried out a microarray-based transcriptome analysis on gametocytes treated with the histone deacetylase inhibitor trichostatin A (TSA). TSA-treatment impaired gametocyte maturation and lead to histone hyper-acetylation in these stages. Comparative transcriptomics identified 294 transcripts, which were more than 2-fold up-regulated during gametocytogenesis following TSA-treatment. In activated gametocytes, which were less sensitive to TSA, the transcript levels of 48 genes were increased. TSA-treatment further led to repression of ~145 genes in immature and mature gametocytes and 7 genes in activated gametocytes. Up-regulated genes are mainly associated with functions in invasion, cytoadherence, and protein export, while down-regulated genes could particularly be assigned to transcription and translation. Chromatin immunoprecipitation demonstrated a link between gene activation and histone acetylation for selected genes. Among the genes up-regulated in TSA-treated mature gametocytes was a gene encoding the ring finger (RING)-domain protein PfRNF1, a putative E3 ligase of the ubiquitin-mediated signaling pathway. Immunochemistry demonstrated PfRNF1 expression mainly in the sexual stages of
with peak expression in stage II gametocytes, where the protein localized to the nucleus and cytoplasm.
promoter and coding regions associated with acetylated histones, and TSA-treatment resulted in increased PfRNF1 levels. Our combined data point to an essential role of histone acetylation for gene regulation in gametocytes, which can be exploited for malaria transmission-blocking interventions.
Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in ...humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.
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•Multi-gene families are common targets of heterochromatin in malaria parasites•Conserved heterochromatic genes are rare and tend to have regulatory function•Heterochromatin is stable during asexual replication but variable between strains•Gametocyte differentiation is linked to changes in the heterochromatin landscape
Fraschka, Filarsky et al. performed a genome-wide characterization of heterochromatin organization across multiple species, strains, and life cycle stages of malaria parasites. This revealed that heterochromatic gene silencing is a conserved strategy to drive clonal variation of surface antigens and to control life cycle stage transitions and cell differentiation.
The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of ...daughter cells and plays an important role in motility and invasion during different life cycle stages of these single‐celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity‐dependent biotin identification (BioID)‐based proteomics approach, using the established IMC marker protein Photosensitized INA‐Labelled protein 1 (PhIL1) as bait in asexual blood‐stage parasites. Subsequent mass spectrometry‐based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP‐tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood‐stage parasites.
Proximity‐dependent biotin identification (BioID) and subsequent localization studies identified three apical annuli proteins (AAP) and six novel inner membrane complex (IMC) as PhIL1 interacting candidates (PICs) of the malaria parasite Plasmodium falciparum. Co‐localization of the IMC with the Kelch13 compartment revealed their spatial association.
Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate ...interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins.
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•We provide for the first time a crystal structure of a P. falciparum rhoptry protein.•We identify a PfARO interacting protein (PfAIP) and provide an in-depth phylogenetic analysis.•Structure-based mutagenesis, high-resolution microscopy and proximity-based protein identification.
The DNase I accessibility and chromatin organization of genes within the nucleus do correlate to their transcriptional activity. Here, we show that both serum starvation and overexpression of Tip5, a ...key regulator of ribosomal RNA gene (rDNA) repression, dictate DNase I accessibility, facilitate the association of rDNA with the nuclear matrix and thus regulate large-scale rDNA chromatin organization. Tip5 contains four AT-hooks and a TAM (Tip5/ARBP/MBD) domain, which were proposed to bind matrix-attachment regions (MARs) of the genome. Remarkably, the TAM domain of Tip5 functions as nucleolar localization and nuclear matrix targeting module, whereas AT-hooks do not mediate association with the nuclear matrix, but they are required for nucleolar targeting. These findings suggest a dual role for Tip5's AT-hooks and TAM domain, targeting the nucleolus and anchoring to the nuclear matrix, and suggest a function for Tip5 in the regulation of higher-order rDNA chromatin structure.
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