In Silico Gene Regulatory Network of the Maurer's Cleft Pathway in Plasmodium falciparum.
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ABSTRACT:
SUBMITTER: Isewon I
PROVIDER: S-EPMC4620995 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
In Silico Gene Regulatory Network of the Maurer's Cleft Pathway in Plasmodium falciparum.
Isewon Itunuoluwa I Oyelade Jelili J Brors Benedikt B Adebiyi Ezekiel E
Evolutionary bioinformatics online 20151022
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Project description:The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer's clefts. We developed a method for efficient enrichment of Maurer's clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer's cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer's cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer's clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer's clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer's cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer's clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.
| S-EPMC7078486 | biostudies-literature
Project description:Plasmodium falciparum, the causative agent of malaria, completely remodels the infected human erythrocyte to acquire nutrients and to evade the immune system. For this process, the parasite exports more than 10% of all its proteins into the host cell cytosol, including the major virulence factor PfEMP1 (P. falciparum erythrocyte surface protein 1). This unusual protein trafficking system involves long-known parasite-derived membranous structures in the host cell cytosol, called Maurer's clefts. However, the genesis, role, and function of Maurer's clefts remain elusive. Similarly unclear is how proteins are sorted and how they are transported to and from these structures. Recent years have seen a large increase of knowledge but, as yet, no functional model has been established. In this perspective we review the most important findings and conclude with potential possibilities to shed light into the enigma of Maurer's clefts. Understanding the mechanism and function of these structures, as well as their involvement in protein export in P. falciparum, might lead to innovative control strategies and might give us a handle with which to help to eliminate this deadly parasite.
| S-EPMC3864307 | biostudies-literature
Project description:Many of the gene regulatory processes of Plasmodium falciparum, the deadliest malaria parasite, remain poorly understood. To develop a comprehensive guide for exploring this organism's gene regulatory network, we generated a systems-level model of P. falciparum gene regulation using a well-validated, machine-learning approach for predicting interactions between transcription regulators and their targets. The resulting network accurately predicts expression levels of transcriptionally coherent gene regulatory programs in independent transcriptomic data sets from parasites collected by different research groups in diverse laboratory and field settings. Thus, our results indicate that our gene regulatory model has predictive power and utility as a hypothesis-generating tool for illuminating clinically relevant gene regulatory mechanisms within P. falciparum. Using the set of regulatory programs we identified, we also investigated correlates of artemisinin resistance based on gene expression coherence. We report that resistance is associated with incoherent expression across many regulatory programs, including those controlling genes associated with erythrocyte-host engagement. These results suggest that parasite populations with reduced artemisinin sensitivity are more transcriptionally heterogenous. This pattern is consistent with a model where the parasite utilizes bet-hedging strategies to diversify the population, rendering a subpopulation more able to navigate drug treatment.
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Project description:In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.
| S-EPMC8066109 | biostudies-literature
Project description:The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.
| S-EPMC1173160 | biostudies-literature
Project description:BackgroundWith the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (approximately 90% AT) presents significant challenges to in silico cis-regulatory element discovery.ResultsWe have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.ConclusionThis GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.
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Project description:BackgroundGene Regulatory Networks (GRN) produce powerful insights into transcriptional regulation in cells. The power of GRNs has been underutilized in malaria research. The Arboreto library was incorporated into a user-friendly web-based application for malaria researchers ( http://malboost.bi.up.ac.za ). This application will assist researchers with gaining an in depth understanding of transcriptomic datasets.MethodsThe web application for MALBoost was built in Python-Flask with Redis and Celery workers for queue submission handling, which execute the Arboreto suite algorithms. A submission of 5-50 regulators and total expression set of 5200 genes is permitted. The program runs in a point-and-click web user interface built using Bootstrap4 templates. Post-analysis submission, users are redirected to a status page with run time estimates and ultimately a download button upon completion. Result updates or failure updates will be emailed to the users.ResultsA web-based application with an easy-to-use interface is presented with a use case validation of AP2-G and AP2-I. The validation set incorporates cross-referencing with ChIP-seq and transcriptome datasets. For AP2-G, 5 ChIP-seq targets were significantly enriched with seven more targets presenting with strong evidence of validated targets.ConclusionThe MALBoost application provides the first tool for easy interfacing and efficiently allows gene regulatory network construction for Plasmodium. Additionally, access is provided to a pre-compiled network for use as reference framework. Validation for sexually committed ring-stage parasite targets of AP2-G, suggests the algorithm was effective in resolving "traditionally" low-level signatures even in bulk RNA datasets.
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Project description:During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle.
| S-EPMC3502387 | biostudies-literature
Project description:Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.
| S-EPMC1489082 | biostudies-literature
Project description:Underlying the development of malaria parasites within erythrocytes and the resulting pathogenicity is a hardwired program that secures proper timing of gene transcription and production of functionally relevant proteins. How stage-specific gene expression is orchestrated in vivo remains unclear. Here, using the assay for transposase accessible chromatin sequencing (ATAC-seq), we identified ∼4,000 regulatory regions in P. falciparum intraerythrocytic stages. The vast majority of these sites are located within 2 kb upstream of transcribed genes and their chromatin accessibility pattern correlates positively with abundance of the respective mRNA transcript. Importantly, these regions are sufficient to drive stage-specific reporter gene expression and DNA motifs enriched in stage-specific sets of regulatory regions interact with members of the P. falciparum AP2 transcription factor family. Collectively, this study provides initial insights into the in vivo gene regulatory network of P. falciparum intraerythrocytic stages and should serve as a valuable resource for future studies.
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