Plasmodium Gene Expression Research Articles

Cerebral Malaria Is Regulated by Host-Mediated Changes in Plasmodium Gene Expression.

Cerebral malaria (CM), the deadliest complication of Plasmodium infection, is a complex and unpredictable disease. However, our understanding of the host and parasite factors that cause CM is limited. Using a mouse model of CM, experimental CM (ECM), we performed a three-way comparison between ECM-susceptible C57BL/6 mice infected with ECM-causing Plasmodium ANKA parasites [ANKA(C57BL/6)], ECM-resistant BALB/c mice infected with Plasmodium ANKA [ANKA(BALB/c)], and C57BL/6 mice infected with Plasmodium NK65 that does not cause ECM [NK65(C57BL/6)]. All ANKA(C57BL/6) mice developed CM. In contrast, in ANKA(BALB/c) and NK65(C57BL/6), infections do not result in CM and proceed similarly in terms of parasite growth, disease course, and host immune response. However, parasite gene expression in ANKA(BALB/c) was remarkably different than that in ANKA(C57BL/6) but similar to the gene expression in NK65(C57BL/6). Thus, Plasmodium ANKA has an ECM-specific gene expression profile that is activated only in susceptible hosts, providing evidence that the host has a critical influence on the outcome of infection. IMPORTANCE Hundreds of thousands of lives are lost each year due to the brain damage caused by malaria disease. The overwhelming majority of these deaths occur in young children living in sub-Saharan Africa. Thus far, there are no vaccines against this deadly disease, and we still do not know why fatal brain damage occurs in some children while others have milder, self-limiting disease progression. Our research provides an important clue to this problem. Here, we showed that the genetic background of the host has an important role in determining the course and the outcome of the disease. Our research also identified parasite molecules that can potentially be targeted in vaccination and therapy approaches.

Long read single cell RNA sequencing reveals the isoform diversity of Plasmodium vivax transcripts.

Plasmodium vivax infections often consist of heterogenous populations of parasites at different developmental stages and with distinct transcriptional profiles, which complicates gene expression analyses. The advent of single cell RNA sequencing (scRNA-seq) enabled disentangling this complexity and has provided robust and stage-specific characterization of Plasmodium gene expression. However, scRNA-seq information is typically derived from the end of each mRNA molecule (usually the 3'-end) and therefore fails to capture the diversity in transcript isoforms documented in bulk RNA-seq data. Here, we describe the sequencing of scRNA-seq libraries using Pacific Biosciences (PacBio) chemistry to characterize full-length Plasmodium vivax transcripts from single cell parasites. Our results show that many P. vivax genes are transcribed into multiple isoforms, primarily through variations in untranslated region (UTR) length or splicing, and that the expression of many isoforms is developmentally regulated. Our findings demonstrate that long read sequencing can be used to characterize mRNA molecules at the single cell level and provides an additional resource to better understand the regulation of gene expression throughout the Plasmodium life cycle.

A toolbox for conditional control of gene expression in apicomplexan parasites.

Apicomplexan parasites encompass diverse pathogens for humans and animals, including the causative agents of malaria and toxoplasmosis, Plasmodium spp. and Toxoplasma gondii. Genetic manipulation of these parasites has become central to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies. Tremendous progress has been achieved over the past years with the advent of next generation sequencing and powerful genome editing methods. In particular, various methods for conditional gene expression have been developed in both Plasmodium and Toxoplasma to knockout or knockdown essential genes, or for inducible expression of master developmental regulators or mutant versions of proteins. Conditional gene expression can be achieved at three distinct levels. At the DNA level, inducible site‐specific recombinases allow conditional genome editing. At the RNA level, regulation can be achieved during transcription, using stage‐specific or regulatable promoters, or post‐transcriptionally through alteration of mRNA stability or translation. At the protein level, several systems have been developed for inducible degradation or displacement of a protein of interest. In this review, we provide an overview of current systems for conditional control of gene expression in Plasmodium and Toxoplasma parasites, highlighting the advantages and limitations of each approach.

Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites.

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite’s AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria.

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

Effect of Dihydroartemisinin on Plasmodium NADH-Dependent Glutamate Synthase: The Implication in Malaria Management.

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum (P.falciparum) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from . 3D7 invitro but also from Plasmodium berghei (P.berghei) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P.falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the "feedback pattern" is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

Human and Rodent Malaria Parasites Differ in Their Histone Methylation Machinery - a Bioinformatic Study

Gene expression in Plasmodium can be regulated epigenetically similarly to the other eukaryotic species. Methylation is one of the histone modifications that affect chromatin structure and influence gene expression. Recently it was reported that rodent Plasmodium species lack orthologs to [PlasmoDB:PF3D7_0809900], a putative histone demethylase. We take advantage of progress in genomics and perform a whole genome analysis.Based on sequence and phylogenetic analysis of putative enzymes involved in histone methylation in Plasmodium, we show that rodent malaria parasites lost not only a putative histone demethylase but also a putative histone methylase. In silico prediction showed that these enzymes were likely to target the same lysine in histone H3, and suggested the whole histone-modifying module was lost in rodent Plasmodium species. The evolutionarily old histone modification machinery differs in rodent and primate Plasmodium species, suggesting that change of the histone code during malaria evolution took place early in Plasmodium speciation.

Histone lysine methyltransferase structure activity relationships that allow for segregation of G9a inhibition and anti-Plasmodium activity.

Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti-Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon 'Nle mimic' at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.

The role of chromatin in Plasmodium gene expression

The malaria parasite Plasmodium falciparum dynamically regulates transcription of the majority of its genes during its intraerythrocytic developmental cycle. Chromatin is an important contributor to this tight regulation of gene expression. P. falciparum appears to utilize most of the mechanisms of chromatin creation and modification found in other eukaryotes, although it occasionally uses them in surprising ways. Much of the P. falciparum genome is maintained in a euchromatic state, potentially permissive for transcription and heterochromatin appears to have a specialized role limited to silencing islands of genes involved in redundant host-parasite interactions. P. falciparum histones share canonical modifications with other eukaryotes but also have unique modifications of unknown function including hyperacetylations of two alternative histones possibly involved in gene regulation. Much of our knowledge of chromatin regulation of gene expression in P. falciparum derives from the study of virulence genes that are subject to chromatin regulatory mechanisms ranging from histone modifications and nucleosomal occupancy to non-protein-coding RNAs and subnuclear architecture. These mechanisms will be discussed along with other characterized components of P. falciparum chromatin.

Regulation of gene expression in protozoa parasites.

Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

Whole‐Transcriptome Analysis ofPlasmodium falciparumField Isolates: Identification of New Pathogenicity Factors

Severe malaria and one of its most important pathogenic processes, cerebral malaria, involves the sequestration of parasitized red blood cells (pRBCs) in brain postcapillary venules. Although the pathogenic mechanisms underlying malaria remain poorly characterized, it has been established that adhesion of pRBCs to endothelial cells (ECs) can result in cell apoptosis, which in turn may lead to disruption of the blood-brain barrier. The nature of the parasite molecules involved in the pathogenesis of severe malaria remains elusive. Whole-transcriptome profiling of nonapoptogenic versus apoptogenic parasite field isolates obtained from Gabonese children was performed with pan-genomic Plasmodium falciparum DNA microarrays; radiolabeled instead of fluorescent cDNAs were used to improve the sensitivity of signal detection. Our methods allowed the identification of 59 genes putatively associated with the induction of EC apoptosis. Silencing of Plasmodium gene expression with specific double-stranded RNA was performed on 8 selected genes; 5 of these, named "Plasmodium apoptosis-linked pathogenicity factors" (PALPFs), were found to be linked to parasite apoptogenicity. Of these genes, 2 might act via parasite cytoadherence. This is the first attempt to identify genes involved in parasite pathogenic mechanisms against human ECs. The finding of PALPFs illuminates perspectives for novel therapeutic strategies against cerebral complications of malaria.

Effect of chloroquine on gene expression of Plasmodium yoelii nigeriensis during its sporogonic development in the mosquito vector

BackgroundThe anti-malarial chloroquine can modulate the outcome of infection during the Plasmodium sporogonic development, interfering with Plasmodium gene expression and subsequently, with transmission. The present study sets to identify Plasmodium genes that might be regulated by chloroquine in the mosquito vector.MethodsDifferential display RT-PCR (DDRT-PCR) was used to identify genes expressed during the sporogonic cycle that are regulated by exposure to chloroquine. Anopheles stephensi mosquitoes were fed on Plasmodium yoelii nigeriensis-infected mice. Three days post-infection, mosquitoes were fed a non-infectious blood meal from mice treated orally with 50 mg/kg chloroquine. Two differentially expressed Plasmodium transcripts (Pyn_chl091 and Pyn_chl055) were further characterized by DNA sequencing and real-time PCR analysis.ResultsBoth transcripts were represented in Plasmodium EST databases, but displayed no homology with any known genes. Pyn_chl091 was upregulated by day 18 post infection when the mosquito had a second blood meal. However, when the effect of chloroquine on that transcript was investigated during the erythrocytic cycle, no significant differences were observed. Although slightly upregulated by chloroquine exposure the expression of Pyn_chl055 was more affected by development, increasing towards the end of the sporogonic cycle. Transcript abundance of Pyn_chl055 was reduced when erythrocytic stages were treated with chloroquine.ConclusionChloroquine increased parasite load in mosquito salivary glands and interferes with the expression of at least two Plasmodium genes. The transcripts identified contain putative signal peptides and transmembrane domains suggesting that these proteins, due to their location, are targets of chloroquine (not as an antimalarial) probably through cell trafficking and recycling.

Characterization and Functional Analysis of HMG Proteins in Plasmodium falciparum

Little is known about regulation of gene expression in Plasmodium. Indeed, only few regulatory elements and no clearly defined transcription factors have been reported, so far. In order to evaluate the interplay between cis‐ and trans‐acting elements controlling the regulation of transcription, we annotated several High Mobility Group proteins of the HMGB superfamily containing one or two HMG‐box(es) of 80 aa consisting of three α‐helices arranged in L‐shaped configuration. These computational analyses suggest that the Plasmodium proteins (PfHMG1‐4) might function as architectural factors through their ability to bind DNA in a structure‐specific manner and to induce bending of DNA. PfHMG1 (97 aa) was cloned from the genomic DNA of the 3D7 clone. The protein was expressed in E. coli and antibodies were raised in mice. A messenger of 1.3 kb was characterised by Northern blotting of 3D7 total RNA. Temporal expression of the transcript and protein was examined in two Plasmodium clones, 3D7 and F12 its gametocyte‐less derivative. The transcript appeared to be developmentally regulated throughout the erythrocytic cycle of Plasmodium as well as the protein present in the nuclear extracts. The functional analysis of the recombinant PfHMG1 via EMSA with synthetic fourway DNA junctions and ligase‐mediated circularisation assays showed that the factor is a genuine architectural protein. In addition, the functional analyses of two other factors PfHMG3 and PfHMG4 are underway. The sequences of PfHMG1 and PfHMG3 are quite similar; however, pfhmg1 transcript is mainly expressed during erythrocytic asexual cycle in contrast to pfhmg3 preferentially expressed in the gametocytes.

Proteomic studies of Plasmodium knowlesi SICA variant antigens demonstrate their relationship with P. falciparum EMP1

Malaria variant antigens are encoded by large multigene families and expressed on the surface of infected erythrocytes. The Plasmodium knowlesi Schizont-infected cell agglutination (SICA) antigens are encoded by the SICAvar multigene family, and the P. falciparum erythrocyte membrane protein-1 (PfEMP1) antigens are encoded by the var gene family. Although these variant antigens share many fundamental features, P. knowlesi and P. falciparum are phylogenetically distantly related, and so far a significant level of sequence identity has not been observed in alignments of either the SICAvar and var gene families or their encoded proteins. In support of their orthologous relationship, however, here we demonstrate through proteomic analysis that the P. knowlesi SICA variant antigens share significant common sequences with P. falciparum EMP1 molecules. As many as forty P. knowlesi SICA peptides show identity with a particular P. falciparum EMP1, mapping throughout all characterized domains, including the externally exposed cysteine-rich domains that are characteristic of both proteins. These findings provide further validation of the classical in vivo P. knowlesi-rhesus monkey model system for advancing our understanding of the immunobiology of antigenic variation and variant antigen gene expression in Plasmodium.

Conservation and Developmental Control of Alternative Splicing in maebl Among Malaria Parasites

Genes of malaria parasites and other unicellular organisms have larger exons with fewer and smaller introns than metaozoans. Such differences in gene structure are perceived to extend to simpler mechanisms for transcriptional control and mRNA processing. Instead, we discovered a surprisingly complex level of post-transcriptional mRNA processing in analysis of maebl transcripts in several Plasmodium species. Mechanisms for internal alternative cis-splicing and exon skipping were active in multiple life cycle stages to change exon structure in the deduced coding sequence (CDS). The major alternatively spliced transcript utilized a less favorable acceptor splice site, which shifted codon triplet usage to a different CDS with a hydrophilic C terminus, changing the canonical type I membrane MAEBL product to a predicted soluble isoform. We found that developmental control of the alternative splicing pattern was distinct from the canonical splicing pattern. Western blot analysis indicated that MAEBL expression was better correlated with the appearance of the canonical ORF1 transcript. Together these data reveal that RNA metabolism in unicellular eukaryotes like Plasmodium is more sophisticated than believed and may have a significant role regulating gene expression in Plasmodium.

State-Specific Expressions of the Calmodulin Gene in Plasmodium falciparum1

We studied the synthesis of calmodulin mRNA and of calmodulin during the asexual cell cycle of Plasmodium falciparum. The expression of the gene was low during the early stages of the erythrocytic asexual cycle. When the parasites were 28 h old the level of mRNA increased steadily to reach a peak at the age of 40 h. During the last 8 h of the cycle a constant decrease in the amount of calmodulin mRNA was observed. The synthesis of the protein followed the expression pattern of the mRNA, but without the final decrease. The average amounts of calmodulin measured were 2.5 +/- 0.9 ng/10(8) 14-h-old rings, 4.38 +/- 0.35 ng/10(8) 36-h-old trophozoites, 11.8 +/- 1.45 ng/10(8) 44-h-old trophozoites, and 19.6 +/- 1.9 ng/10(8) 48-h-old schizonts. The in vivo stability of mRNA in different developmental stages was evaluated by pulse and chase experiments. The RNA of very mature trophozoites was quickly degraded, in contrast with the high stability shown by the RNA in other stages. The results here suggest that the calmodulin gene expression in Plasmodium does not follow a housekeeping pattern of expression such as in other eukaryotic cells, but that it is regulated at the level of transcription and RNA degradation.