Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites

Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad‐selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These resultsdemonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.     

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.     

The Plasmodium falciparum RhopH2 promoter and first 24 amino acids are sufficient to target proteins to the rhoptries

The rhoptry secretory organelles of the malaria parasite, Plasmodium falciparum, contain a RhopH complex, which is composed of the proteins RhopH1, RhopH2, and RhopH3. RhopH1 is encoded by the rhoph1/clag multi-gene family, whereas RhopH2 and RhopH3 are encoded by single-copy genes. The precise function of the RhopH complex has not been identified, but it has been shown that the component proteins are involved in erythrocyte binding and perhaps participate in the formation of the parasitophorous vacuolar membrane. In this study, we have isolated pfrhoph2 promoter plus the signal peptide encoding sequence and generated transgene expression constructs to evaluate a trafficking and the RhopH complex formation in transgenic P. falciparum parasite lines. Interestingly, we found that the N-terminal 24 amino acids of RhopH2, including signal peptide sequence, were sufficient to target GFP to the rhoptries under the rhoph2 promoter. Because it was previously shown that the timing of the expression alone could not target proteins to the apical organelles, this targeting is likely mediated via a unique mechanism that is dependent on N-terminal 24 amino acids of RhopH2 early in the secretory pathway. The N-terminal one third of Clag3.1, which contains a distinct conserved domain with Toxoplasma gondii RON2, can not associate the RhopH complex as a GFP chimera, but a c-Myc-Clag3.1 chimera lacking the C-terminus successfully associates the RhopH complex indicating that cooperation of middle region is likely required but the C-terminus is not necessary.     

Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion

The process of erythrocyte invasion by merozoites of Plasmodium falciparum involves multiple steps, including the formation of a moving junction between parasite and host cell, and it is characterised by the redundancy of many of the receptor-ligand interactions involved. Several parasite proteins that interact with erythrocyte receptors or participate in other steps of invasion are encoded by small subtelomerically located gene families of four to seven members. We report here that members of the eba, rhoph1/clag, acbp, and pfRh multigene families exist in either an active or a silenced state. In the case of two members of the rhoph1/clag family, clag3.1 and clag3.2, expression was mutually exclusive. Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic. This was demonstrated for eba-140. Our data demonstrate that variant or mutually exclusive expression and epigenetic silencing in Plasmodium are not unique to genes such as var, which encode proteins that are exported to the surface of the erythrocyte, but also occur for genes involved in host cell invasion. Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.     

Heterochromatin formation in bistable chromatin domains controls the epigenetic repression of clonally variant Plasmodium falciparum genes linked to erythrocyte invasion

Clonally variant gene expression is a common survival strategy used by many pathogens, including the malaria parasite Plasmodium falciparum. Among the genes that show variant expression in this parasite are several members of small gene families linked to erythrocyte invasion, including the clag and eba families. The active or repressed state of these genes is clonally transmitted by epigenetic mechanisms. Here we characterized the promoters of clag3.1, clag3.2 and eba-140, and compared nuclease accessibility and post-translational histone modifications between their active and repressed states. Activity of these promoters in an episomal context is similar between parasite subclones characterized by different patterns of expression of the endogenous genes. Variant expression is controlled by the euchromatic or heterochromatic state of bistable chromatin domains. Repression is mediated by H3K9me3-based heterochromatin, whereas the active state is characterized by H3K9ac. These marks are maintained throughout the asexual blood cycle to transmit the epigenetic memory. Furthermore, eba-140 is organized in two distinct chromatin domains, probably separated by a barrier insulator located within its ORF. The 5' chromatin domain controls expression of the gene, whereas the 3' domain shares the chromatin conformation with the upstream region of the neighbouring phista family gene, which also shows variant expression.     

Clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion

Background

The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.

Methodology/Principal Findings

Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.

Conclusions/Significance

Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.     

长效和短效NO发生剂体内干预疟原虫红内期重要侵袭分子转录水平的观察

为探讨NO对疟原虫红内期侵袭相关分子MSP-1、AMA-1和RhopH complex转录水平的影响。通过雌性BALB／c小鼠腹腔感染1×10^6致死型约氏疟原虫P．yoelii 17XL,体内给予NO长效（NOC18）和短效（NOC5）发生剂进行干预后,纯化疟原虫成熟裂殖体,提取总RNA,通过Real—time PCR相对定量方法检测MSP-1、AMA—1和RhopH complex的转录水平。结果显示和正常感染组相比,NOC5处理后疟原虫侵入的关键分子MSP—1、AMA-1和RhopH complex的转录水平明显下降;而NOC18处理则未见这一现象。本研究结果提示NO抑制疟原虫侵袭关键分子的转录水平,进而可能下调疟原虫相应蛋白的表达,从而影响疟原虫的侵入过程。     

The cytoadherence linked asexual gene family of Plasmodium falciparum: are there roles other than cytoadherence?

The binding of erythrocytes infected with Plasmodium falciparum to the endothelium lining the small blood vessels of the brain and other organs can mediate severe pathology. A region at the right end of chromosome 9 has been implicated in the binding of parasitised erythrocytes to the endothelial receptor CD36. A gene expressed in asexual erythrocytic stage parasites has been identified in this region and termed the cytoadherence linked asexual gene (clag). Antisense RNA production and targeted gene disruption of clag resulted in greatly reduced binding to CD36. Hybridisation to 3D7 chromosomes showed clag to be a part of a gene family of at least nine members. All members analysed so far have a conserved gene structure of at least nine exons, as well as putative transmembrane domains. The possible functions of the gene family are discussed.     

RhopH3, rhoptry gene conserved in the free-living alveolate flagellate Colpodella sp. (Apicomplexa)

In this study, we investigated morphological, immunological and molecular characteristics of Colpodella sp. (American Type Culture Collection 50594) in a diprotist culture containing Bodo caudatus as prey using Plasmodium rhoptry specific antibodies and oligonucleotide primers targeting Plasmodium falciparum rhoptry genes. In culture, Colpodella sp. attached to its prey using the apical end with attachment lasting for approximately 20 min while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella sp. Encystment of Colpodella sp. was observed following feeding. Indirect immunofluorescence assay (IFA) and confocal microscopy using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella sp. Bodo caudatus from diprotist and monoprotist (ATCC 30395) cultures showed weak background reactivity. Giemsa staining permitted differentiation of both protists. Genomic DNA isolated from the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers targeting the P. falciparum rhoptry genes RhopH3, RhopH1/Clag3.2 and RAMA. Primers targeting exon 7 of the P. falciparum RhopH3 gene amplified an approximately 2 kb DNA fragment from the diprotist DNA template. DNA sequence and BLAST search analysis of the amplified product from diprotist DNA identified the RhopH3 gene demonstrating that the RhopH3 gene is conserved in Colpodella sp.     

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events

The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.     

Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites

To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre‐erythrocytic liver‐stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS‐adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5′ and 3′ flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild‐type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre‐erythrocytic forms. Mammalian cells infected by these parasites can be separated from non‐infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages.     

Removal of heterologous sequences from Plasmodium falciparum mutants using FLPe-recombinase

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines.     

Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells

Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.     

Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites

Malaria parasites invade erythrocytes in a process mediated by a series of molecular interactions. Invasion of human erythrocytes by Plasmodium vivax is dependent upon the presence of a single receptor, but P. falciparum, as well as some other species, exhibits the ability to utilize multiple alternative invasion pathways. Conserved cysteine-rich domains play important roles at critical times during this invasion process and at other stages in the life cycle of malaria parasites. Duffy-binding-like (DBL) domains, expressed as a part of the erythrocyte-binding proteins (DBL-EBP), are such essential cysteine-rich ligands that recognize specific host cell surface receptors. DBL-EBP, which are products of the erythrocyte-binding-like (ebl) gene family, act as critical determinants of erythrocyte specificity and are the best-defined ligands from invasive stages of malaria parasites. The ebl genes include the P. falciparum erythrocyte-binding antigen-175 (EBA-175) and P. vivax Duffy-binding protein. DBL domains also mediate cytoadherence as a part of the variant erythrocytic membrane protein-1 (PfEMP-1) antigens expressed from var genes on the surface of P. falciparum-infected erythrocytes. A paralogue of the ebl family is the malarial ligand MAEBL, which has a chimeric structure where the DBL domain is functionally replaced with a distinct cysteine-rich erythrocyte-binding domain with similarity to the apical membrane antigen-1 (AMA-1) ligand domain. The Plasmodium AMA-1 ligand domain, which encompasses the extracellular cysteine domains 1 and 2 and is well conserved in a Toxoplasma gondii AMA-1, has erythrocyte-binding activity distinct from that of MAEBL. These important families of Plasmodium molecules (DBL-EBP, PfEMP-1, MAEBL, AMA-1) are interrelated through the MAEBL. Because MAEBL and the other ebl products have the characteristics expected of homologous ligands involved in equivalent alternative invasion pathways to each other, we sought to better understand their roles during invasion by determining their relative origins in the Plasmodium genome. An analysis of their multiple cysteine-rich domains permitted a unique insight into the evolutionary development of PLASMODIUM: Our data indicate that maebl, ama-1, and ebl genes have ancient origins which predate Plasmodium speciation. The maebl evolved as a single locus, including its unique chimeric structure, in each Plasmodium species, in parallel with the ama-1 and the ebl genes families. The ancient character of maebl, along with its different expression characteristics suggests that MAEBL is unique and does not play an alternative role in invasion to ebl products such as EBA-175. The multiple P. falciparum ebl paralogues that express DBL domains, which have occurred by duplication and diversification, potentially do provide multiple functionally equivalent ligands to EBA-175 for alternative invasion pathways.