ArticleLiterature Review

Mechanisms of gene regulation in Plasmodium

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Abstract

Recent investigations into gene expression in malarial systems suggest that locus-specific, promoter-based transcriptional control is not the dominant mode of regulation in Plasmodium. Although global transcript and protein profiles across the life cycle of Plasmodium imply significant control of developmental progression,1,2 the exact mechanisms underlying this pattern are largely unknown, and appear to be unconventional compared with those of most model organisms. As such, our understanding of transcriptional, post-transcriptional, and epigenetic mechanisms in Plasmodium, as well as their relative contributions to gene regulation, lags far behind that of other eukaryotic systems. We seek a comprehensive characterization of the mechanisms that Plasmodium species have evolved to control gene expression during their complex life cycles. To address these concerns, a panel of experts recently convened for an initial consultation at the Broad Institute (Cambridge MA) in September 2006. Both the integration of existing data in a manner that will be most useful to the malarial community and the types of information currently lacking in the field were discussed. Preliminary recommendations include further delineation of the parasite's genomic structure, as well as elucidation of chromatin structure and epigenetic features; a systematic comparison of global expression data from nuclear run-on, RNA expression microarrays, and proteomic based assays; and the re-evaluation and functional testing of putative regulatory nucleic acid motifs, including characterization of their binding proteins. Detailed examination of regulation on a whole-genome scale, as well as among specific loci, will ultimately make way for control measures aimed at interfering with processes crucial for parasite survival, such as differentiation, antigenic variation, and development of drug resistance. Overview. Plasmodium, the causal agent of malaria, afflicts more than half a billion people worldwide. Despite global efforts to curb malaria, the spread of drug and insecticide resistance and the continuing lack of an effective vaccine contribute to its persistence as a major health burden. A fundamental understanding of how parasite genes governing transmission success, immune evasion, and drug resistance are regulated is critical to developing novel therapeutic strategies against these processes. Although focused studies on candidate genes have provided some insights, we remain largely ignorant of the mechanisms underlying gene control, and the relative role of transcriptional and post-transcriptional/translational regulation in the parasite. For example, although genes appear to be monocistronically transcribed, no clear canonical promoter has been defined to date and the handful of functional cis-acting elements uncovered in Plasmodium3-9 is unique to this system. The dearth of annotated transcription factors in the genome of Plasmodium falciparum10,11 in conjunction with phased expression of stage-specific transcripts1 suggests that post-transcriptional control may be a major means of regulating gene expression, as supported by a recent study documenting the significant role of translation repression in sexual differentiation.12 The successful application of high-throughput approaches in malarial systems now provides the opportunity to fundamentally transform the pace of post-genomic research on gene regulation in Plasmodium. For example, both microarray and proteomics-based assays document significant regulation of transcript and protein expression profiles across asexual and sexual stages of development.1,2,13,14 Such technologies have opened the door to bioinformatically investigating the co-regulation of genes, with the potential to define sequence elements that may be behind this control. Variation in global rates of RNA decay during the intraerythrocytic developmental cycle contributes to mounting evidence supporting a more prominent role for post-transcriptional control in the specific regulation of transcript subsets, as do more recent experiments uncovering global control of transcriptional activity. This provocative combination follows a growing emphasis on chromatin remodeling15,16 and gene-silencing, 4,15,17 and suggests a complex, multi-layered regulatory network in the parasite. Collectively, these studies underscore the need for a comprehensive, concerted effort to gain the same level of understanding in Plasmodium as has been achieved in the model eukaryotic systems for which these gene regulatory mechanisms were first elucidated. Objectives/Conclusions. We first describe the state of the field in Plasmodium, with specific emphasis on what is known about the regulation of steady-state RNA levels during the life cycle and exposure to stress. Although microarray studies clearly demonstrate a "hard-wired" pattern of expression tied to developmental staging, perturbation with small molecules has rendered conflicting results, leading to difficulties in interpretation of this data. We next describe efforts to bioinformatically mine these steady-state RNA data sets to uncover civ-acting sequence motifs that may control expression of gene clusters. Preliminary evidence that more global mechanisms of regulating transcriptional activity may dominate this paradigm is also presented. The single exception to this model characterized to date, the antigenic var gene family, and its regulatory features are subsequently detailed. The growing body of evidence for post-transcriptional regulation in Plasmodium species is then discussed, including function-dependent changes in mRNA decay rate across the life cycle, and translational repression and proteomic regulation in P. berghei gametocytes. Finally, we propose the following recommendations for the next steps to be taken in our multilateral investigation: 1) determination of chromatin organization and structure; 2) comparison and further characterization of global expression profiles from nuclear run-on, microarray, and proteomic-based assays; and 3) identification, functional testing, and re-evaluation of potentially regulatory nucleic acid binding proteins and their binding sites. In conclusion, we articulate a way forward in the field of malarial gene expression, and propose experiments that are needed to pursue these goals systematically. This complex topic demands that experts from the malaria field as well as authorities on transcriptional, post-transcriptional, and epigenetic gene regulation in other systems be brought together. With this initial consultation, we have taken the first step for forming and funding such a consortium of scientists. Copyright © 2007 by The American Society of Tropical Medicine and Hygiene.

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... Following the publication of the parasite's genome in 2002 [1], rapid technical advances in the application of high throughput microarray and proteomic analyses provided evidence of an extensive programme of temporal gene expression throughout the parasite's life cycle [2][3][4]. Subsequent investigations profiling changes in gene expression in drug-treated parasites suggested an additional facet to this programme of gene expression -that it appeared to be "hard-wired" [2,5]. mRNA and protein accumulation profiles of drug-treated parasites provided little consistent evidence for a change in this tight programme of control, with the exception of drugs that target apicoplast function [5][6][7][8][9]. ...
... Subsequent investigations profiling changes in gene expression in drug-treated parasites suggested an additional facet to this programme of gene expression -that it appeared to be "hard-wired" [2,5]. mRNA and protein accumulation profiles of drug-treated parasites provided little consistent evidence for a change in this tight programme of control, with the exception of drugs that target apicoplast function [5][6][7][8][9]. The changes observed tended to suggest either a switch to gametogenesis, an upregulation of variant gene families located at chromosome ends and/or cell cycle arrest at an ill-defined point of development. ...
... This apparent inflexibility in the "hard-wired" control of gene expression, however, was not observed when conditions of stress that more closely mimic the challenges the parasite faces in vivo were applied. These include, for example, glucose starvation and heat shock, both of which result in rapid and profound changes in mRNA profiles [5,[10][11][12]. These data seem to suggest that the parasite is able alter its global programme of gene expression when exposed to external challenges. ...
Article
Ten years ago this journal published a review with an almost identical title detailing how the then recent introduction of transfection technology had advanced our understanding of the molecular control of transcriptional processes in Plasmodium falciparum, particularly in terms of promoter structure and function. In the succeeding years, sequencing of several Plasmodium spp. genomes and application of high throughput global postgenomic technologies have proven as significant, if not more, as has the ability to genetically manipulate these parasites in dissecting the molecular control of gene expression. Here we aim to review our current understanding of the control of gene expression in P. falciparum, including evidence available from other Plasmodium spp. and apicomplexan parasites. Specifically, however, we will address the current polarised debate regarding the level at which control is mediated, and attempt to identify some of the challenges this field faces in the next 10 years.
... Fluorescence labeling with dUTP-Cy3 or dUTP-Cy5 was performed by reverse transcription using 30 g of total RNA with both oligo(dT) [12][13][14][15][16][17][18][19][20] and random hexamers according to the manufacturer's instructions (Amersham). Hybridization to microarray slides and data transformation were performed as described previously (16,28). ...
... In some cases, the response to chemical perturbation is profound: chemical carcinogens could evoke significant changes in transcript level for more than one-third of the 6,200 genes in budding yeast (34). This is in sharp contrast to what is found for the malaria parasite, where attempts to relate global gene expression changes to perturbation with antimalarial drugs often detect either subtle changes in steady-state RNA levels or non-function-related changes as the result of altered growth rates of the treated parasites (18,30,31), which led to the speculation that the malaria parasite transcription regulatory network is probably "hardwired." However, some studies did find consistent impact of drug treatments on specific metabolic pathways of the parasite, suggesting that the parasite has some capacity to respond at the transcript level to environmental perturbations (17,18). ...
... This is in sharp contrast to what is found for the malaria parasite, where attempts to relate global gene expression changes to perturbation with antimalarial drugs often detect either subtle changes in steady-state RNA levels or non-function-related changes as the result of altered growth rates of the treated parasites (18,30,31), which led to the speculation that the malaria parasite transcription regulatory network is probably "hardwired." However, some studies did find consistent impact of drug treatments on specific metabolic pathways of the parasite, suggesting that the parasite has some capacity to respond at the transcript level to environmental perturbations (17,18). In this study, we found that AA treatment at a sub-IC 50 s for a short period of time resulted in twofold or greater changes in 5% of the parasite genes in late trophozoites. ...
Article
Full-text available
To better understand the role of histone lysine acetylation in transcription in Plasmodium falciparum, we sought to attenuate histone acetyltransferase (HAT) activity using anacardic acid (AA). We showed that AA reversibly and noncompetitively inhibited the HAT activity of recombinant PfGCN5. To a lesser extent, AA inhibited the PfGCN5 activity in parasite nuclear extracts but did not affect histone deacetylase activity. AA blocked the growth of both chloroquine-sensitive and -resistant strains, with a 50% inhibitory concentration of approximately 30 microM. Treatment of the parasites with 20 microM of AA for 12 h had no obvious effect on parasite growth or gross morphology but induced hypoacetylation of histone H3 at K9 and K14, but not H4 at K5, K8, K12, and K16, suggesting inhibition of the PfGCN5 HAT. Microarray analysis showed that this AA treatment resulted in twofold or greater change in the expression of 271 (approximately 5%) parasite genes in late trophozoites, among which 207 genes were downregulated. Cluster analysis of gene expression indicated that AA mostly downregulated active genes, and this gene pool significantly overlapped with that enriched for H3K9 acetylation. We further demonstrated by chromatin immunoprecipitation and real-time PCR that AA treatment reduced acetylation near the putative promoters of a set of downregulated genes. This study suggests that the parasiticidal effect of AA is at least partially associated with its inhibition of PfGCN5 HAT, resulting in the disturbance of the transcription program in the parasites.
... Distinct expression patterns can be observed for the various life stages as described by transcriptome and proteome analysis (Kooij, et al., 2006;Le Roch, et al., 2004;Le Roch, et al., 2003;Llinas and del Portillo, 2005;Wilson, 2004). Despite the apparent well-coordinated expression patterns, detailed mechanisms of gene regulation behind the expression patterns seem rather complex and remain elusive (Deitsch, et al., 2007;Gunasekera, et al., 2007;Horrocks, et al., 1998;Le Roch, et al., 2004). In particular, the extent of transcriptional regulation in Plasmodium is still unclear, and few regulatory elements have been discovered (Deitsch, et al., 2007;Gunasekera, et al., 2007;Horrocks, et al., 1998;Le Roch, et al., 2004). ...
... Despite the apparent well-coordinated expression patterns, detailed mechanisms of gene regulation behind the expression patterns seem rather complex and remain elusive (Deitsch, et al., 2007;Gunasekera, et al., 2007;Horrocks, et al., 1998;Le Roch, et al., 2004). In particular, the extent of transcriptional regulation in Plasmodium is still unclear, and few regulatory elements have been discovered (Deitsch, et al., 2007;Gunasekera, et al., 2007;Horrocks, et al., 1998;Le Roch, et al., 2004). Fully understanding these regulatory mechanisms will be essential for deciphering the complex life cycle of Plasmodium, and may lead to possible avenues for therapeutic development. ...
... So far, we have focused on motif discovery in regions upstream of genes. It is well known that sequences downstream of genes also play important roles in gene regulation, commonly act via posttranscriptional mechanisms (a feature suggested for Plasmodium gene regulation; (Deitsch, et al., 2007)). We have also sought to discover motifs that occur downstream of genes. ...
Article
Understanding gene regulation in Plasmodium, the causative agent of malaria, is an important step in deciphering its complex life cycle as well as leading to possible new targets for therapeutic applications. Very little is known about gene regulation in Plasmodium, and in particular, few regulatory elements have been identified. Such discovery has been significantly hampered by the high A-T content of some of the genomes of Plasmodium species, as well as the challenge in associating discovered regulatory elements to gene regulatory cascades due to Plasmodium's complex life cycle. We report a new method of using comparative genomics to systematically discover motifs in Plasmodium without requiring any functional data. Different from previous methods, our method does not depend on sequence alignments, and thus is particularly suitable for highly divergent genomes. We applied our method to discovering regulatory motifs between the human parasite, P.falciparum, and its rodent-infectious relative, P.yoelii. We also tested our procedure against comparisons between P.falciparum and the primate-infectious, P.knowlesi. Our computational effort leads to an initial catalog of 38 distinct motifs, corresponding to over 16 200 sites in the Plasmodium genome. The functionality of these motifs was further supported by their defined distribution within the genome as well as a correlation with gene expression patterns. This initial map provides a systematic view of gene regulation in Plasmodium, which can be refined as additional genomes become available. The new algorithm, named motif discovery using orthologous sequences (MDOS), is available at http://www.ics.uci.edu/ approximately xhx/project/mdos/.
... However, this response does not appear to primarily occur at the transcriptional level. For many years, whether or not malaria parasites are able to produce directed protective transcriptional responses in reaction to fluctuating conditions within the same host has remained controversial, and some authors proposed that P. falciparum blood stages have a hard-wired transcriptome unable to respond to changes in their environment [116][117][118]. Other authors described transcriptional responses following some challenges [116,[119][120][121][122][123][124][125][126][127][128], although in most cases they were of low magnitude compared to the responses commonly observed in other microorganisms. ...
... For many years, whether or not malaria parasites are able to produce directed protective transcriptional responses in reaction to fluctuating conditions within the same host has remained controversial, and some authors proposed that P. falciparum blood stages have a hard-wired transcriptome unable to respond to changes in their environment [116][117][118]. Other authors described transcriptional responses following some challenges [116,[119][120][121][122][123][124][125][126][127][128], although in most cases they were of low magnitude compared to the responses commonly observed in other microorganisms. Additionally, the link between the transcriptional alteration and protection often remained unclear, and in some cases the transcriptional changes observed may reflect parasite death or delayed life cycle progression rather than a protective response. ...
Article
Full-text available
Transcriptional differences enable the generation of alternative phenotypes from the same genome. In malaria parasites, transcriptional plasticity plays a major role in the process of adaptation to fluctuations in the environment. Multiple studies with culture-adapted parasites and field isolates are starting to unravel the different transcriptional alternatives available to Plasmodium falciparum and the underlying molecular mechanisms. Here we discuss how epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival. Some transcriptional changes are driven by stochastic events. These changes can occur spontaneously, resulting in heterogeneity within parasite populations that provides the grounds for adaptation by dynamic natural selection. However, transcriptional changes can also occur in response to external cues. A better understanding of the mechanisms that the parasite has evolved to alter its transcriptome may ultimately contribute to the design of strategies to combat malaria to which the parasite cannot adapt.
... In sub-Saharan Africa for instance, up to a staggering 90% of the malaria mortality recorded occurred in children population under the age of 5 years. [3]- [5] predicted that the incidence of malaria may increase by 50% within 20 years unless some new methods of eradication and control are devised In the post-genomic era, the ability to predict the behaviour, the function, or the structure of biological entities (such as genes and proteins), as well as interactions among them, play a major role in the discovery of information to help biologists explain biological mechanisms [5]- [8]. ...
... In sub-Saharan Africa for instance, up to a staggering 90% of the malaria mortality recorded occurred in children population under the age of 5 years. [3]- [5] predicted that the incidence of malaria may increase by 50% within 20 years unless some new methods of eradication and control are devised In the post-genomic era, the ability to predict the behaviour, the function, or the structure of biological entities (such as genes and proteins), as well as interactions among them, play a major role in the discovery of information to help biologists explain biological mechanisms [5]- [8]. ...
Article
Motif discovery is one of the fundamental problems that have important applications in identifying drug targets and regulatory sites. Regulatory sites on DNA sequence normally correspond to shared conservative sequence patterns among the regulatory regions of correlated genes. These conserved sequence patterns are called motifs. Identifying motifs and corresponding instances is very important, so biologists can investigate the interactions between DNA and proteins, gene regulation, cell development and cell reaction under physiological and pathological conditions. In this work, we developed a motif finding algorithm based on a multi-objective genetic algorithm technique and incorporated the hypergeometric scoring function to enable it discover gapped motifs from organisms with challenging genomic structure such as the malaria parasite. The runtime performance of our resulting algorithm, EMOGAMOD (Extended Multi Objective Genetic Algorithm MOtif Discovery) was evaluated with that of some common motif discovery algorithms and the result was remarkable.
... The human malarial parasite Plasmodium falciparum adopts numerous morphologically distinct forms as it completes its complex life cycle in the human host and mosquito vector. As the parasite invades, colonises and multiplies within these diverse host environments a complex programme of developmentally-linked gene expression, utilising a diverse range of molecular mechanisms to exert control, has been described; for reviews see [1][2][3]. These are perhaps best exemplified during asexual intraerythrocytic development, where morphological transition from the newly invaded ring form progresses over a 48 hour period, through trophozoites and schizonts, to produce merozoites ready to reinitiate invasion in a new host erythrocyte. ...
... Analyses of the molecular mechanisms that govern this developmentally-linked gene expression suggest that this "hard-wiring" is likely the result of globally-acting regulatory mechanisms, specifically; stage-specific variations in nucleosome positioning, processivity of the RNA polymerase II complex and stage-specific variations in the stability of the mRNA transcript [12][13][14][15][16][17][18][19]. Hypotheses that considered regulation of stage-specific gene expression exerted at the level of individual promoters, through specific transcription factor biding to cis-regulatory DNA motifs, fell out of favour in the early 2000′s due to the apparent absence of transcription factors in the P. falciparum genome [1,20,21]. In 2008, however, a restricted number of specific transcription factors, sharing the apetela 2 (AP2) DNA binding motif, were found in P. falciparum, with homologues quickly identified throughout all apicomplexans, leading to their designation as ApiAP2 transcription factors [22][23][24][25]. ...
Article
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Background Over 2700 genes are subject to stage-specific regulation during the intraerythrocytic development of the human malaria parasite Plasmodium falciparum. Bioinformatic analyses have identified a large number of over-represented motifs in the 5¿ flanking regions of these genes that may act as cis-acting factors in the promoter-based control of temporal expression. Triaging these lists to provide candidates most likely to play a role in regulating temporal expression is challenging, but important if we are to effectively design in vitro studies to validate this role.Methods We report here the application of a repeated search of variations of 5¿ flanking sequences from P. falciparum using the Finding Informative Regulatory Elements (FIRE) algorithm.ResultsOur approach repeatedly found a short-list of high scoring DNA motifs, for which cognate specific transcription factors were available, that appear to be typically associated with upregulation of mRNA accumulation during the first half of intraerythrocytic development.Conclusions We propose these cis-trans interactions may provide a combinatorial promoter-based control of gene expression to complement more global mechanisms of gene regulation that can account for temporal control during the second half of intraerythrocytic development.
... This malarial parasite has a lifecycle that alternates between a human host and mosquito vector, requiring multiple morphological and biological adaptations to successfully invade, colonise and divide within diverse cellular environments. Progression of parasites through this complex life cycle and the manifestation of virulence within the human host are both tightly linked to the temporal and spatial control of gene expression [2][3][4][5][6][7][8][9]. Over recent years we have garnered a greater appreciation of the interplay between the molecular mechanisms operating at the genetic and epigenetic levels in regulating developmentally-linked gene expression [4][5][6]8]. ...
... Progression of parasites through this complex life cycle and the manifestation of virulence within the human host are both tightly linked to the temporal and spatial control of gene expression [2][3][4][5][6][7][8][9]. Over recent years we have garnered a greater appreciation of the interplay between the molecular mechanisms operating at the genetic and epigenetic levels in regulating developmentally-linked gene expression [4][5][6]8]. These insights have been provided by global analyses of the temporal programme of steady-state transcript accumulation [10][11][12], mRNA stability and RNA polymerase II complex activity [13][14][15][16]. ...
Article
Full-text available
Background The ability of the human malarial parasite Plasmodium falciparum to invade, colonise and multiply within diverse host environments, as well as to manifest its virulence within the human host, are activities tightly linked to the temporal and spatial control of gene expression. Yet, despite the wealth of high throughput transcriptomic data available for this organism there is very little information regarding the location of key transcriptional landmarks or their associated cis-acting regulatory elements. Here we provide a systematic exploration of the size and organisation of transcripts within intergenic regions to yield surrogate information regarding transcriptional landmarks, and to also explore the spatial and temporal organisation of transcripts over these poorly characterised genomic regions. Results Utilising the transcript data for a cohort of 105 genes we demonstrate that the untranscribed regions of mRNA are large and apportioned predominantly to the 5′ end of the open reading frame. Given the relatively compact size of the P. falciparum genome, we suggest that whilst transcriptional units are likely to spatially overlap, temporal co-transcription of adjacent transcriptional units is actually limited. Critically, the size of intergenic regions is directly dependent on the orientation of the two transcriptional units arrayed over them, an observation we extend to an analysis of the complete sequences of twelve additional organisms that share moderately compact genomes. Conclusions Our study provides a theoretical framework that extends our current understanding of the transcriptional landscape across the P. falciparum genome. Demonstration of a consensus gene-spacing rule that is shared between P. falciparum and ten other moderately compact genomes of apicomplexan parasites reveals the potential for our findings to have a wider impact across a phylum that contains many organisms important to human and veterinary health.
... The importance of this work cannot be overemphasized. The 'Functional Genomics Workshop Group' meeting in Harvard, 2006, whose report was published in Deitsch et al., 2007, identified the resolved of a number of challenges as key to bringing about the understanding of the biology of the deadly malaria parasite, P.f. Key among these challenges is: identification of P.f proteins involved in gene regulatory mechanisms. ...
Article
In this work, we re-implemented the most effective algorithm for mining simple motifs (consist of single words) GEMS (Gene Enrichment Motif Searching) on the suffix tree, STGEMS(Suffix Tree Gene Enrichment Motif Searching). This leads to a significant speed up in the run time of the algorithm. We then extend this to mine structured motifs (consist of several words with well-defined gaps). The resulting algorithm runs in linear time.
... The nuclear periphery of P. falciparum includes regions of condensed heterochromatin marked by the histone modification H3K9me3. This histone modification appears to be an epigenetic marker specifically devoted to gene families that undergo clonally variant transcription that localize to the nuclear periphery (3,22). We performed dSTORM imaging and found that while it surrounds the nucleus, PfSUN1-GFP does not co-localize with either the heterochromatic marker H3K9me3 or the euchromatin marker H3K9ac (Fig. 1E & F). ...
Preprint
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The protozoan parasite Plasmodium falciparum , which is responsible for the deadliest form of human malaria, accounts for over half a million deaths a year. These parasites proliferate in human red blood cells by consecutive rounds of closed mitoses called schizogony. Their virulence is attributed to their ability to modify the infected red cells to adhere to the vascular endothelium and to evade immunity through antigenic switches. Spatial dynamics at the nuclear periphery were associated with regulation of processes that enable the parasites to establish long-term infection. However, our knowledge of components of the nuclear envelope (NE) in Plasmodium remains limited. One of the major protein complexes at the NE is the LINC complex that forms a connecting bridge between the cytoplasm and the nucleus through the interaction of SUN and KASH domain proteins. Here we have identified two SUN-domain proteins as components of the LINC complex of P. falciparum and show that their proper expression is essential for the parasite’s proliferation in human red blood cells and that their depletion leads to the formation of membranous whorls and morphological changes of the NE. In addition, we found that PfSUN2 is associated with heterochromatin and that PfSUN1 expression is essential for activation of the DNA damage response. Our data provide indications for the involvement of the LINC complex in crucial biological processes in the intraerythrocytic development cycle of malaria parasites.
... The NPC carries out selective bidirectional nucleocytoplasmic transport of RNA and proteins while possessing numerous non-transport functions, including gene regulation. There appears to be considerable genetic diversity in the Plasmodium NPC make-up compared to other eukaryotes; this has so far prevented identification by amino acid sequence but also structural homology through bioinformatics even from relatively closely related species such as Toxoplasma gondii, a fellow member of the Apicomplexa (4,(8)(9)(10)(11)(12)(13). To date, a mere 4 FG-nucleoporins (Nups) and Sec13 proteins are identified across the genus (12,(14)(15)(16)(17)(18), while P. falciparum Nup116 exhibits a low degree of conservation in rodent malaria parasites (19). The Plasmodium NPC remains, therefore, poorly defined. ...
Article
Full-text available
The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches.
... Studies comparing transcriptional and translational profiles in the IDC reveal poor correlation and delay of up to 18 h in transcript and protein levels (Le Roch et al, 2004;Foth et al, 2008Foth et al, , 2011Bunnik et al, 2013), which suggests important roles for post-transcriptional and translational regulation of gene expression in the IDC of P. falciparum (Vembar et al, 2016). Proposed mechanisms for post-transcriptional control in P. falciparum include mRNA processing and degradation (Brengues et al, 2005;Newbury, 2006;Deitsch et al, 2007;Parker & Sheth, 2007;Sims et al, 2007;Yamasaki & Anderson, 2008;Horrocks et al, 2009), translational repression (Hall et al, 2005;Mair et al, 2006;Parker & Sheth, 2007;Shaw et al, 2007;Abaza & Gebauer, 2008), translational regulation by untranslated regions (UTR) in mRNA (Hasenkamp et al, 2013;Brancucci et al, 2014;Cui et al, 2015), and natural (endogenous) anti-sense transcripts (Patankar et al, 2001;Kyes et al, 2002;Gunasekera et al, 2004;Militello et al, 2005Militello et al, , 2008Lu et al, 2007). However, these gene-specific mechanisms which have local effects do not explain a more global systematic scale mechanism involved in the control of Plasmodium gene expression at the level of translation. ...
Article
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Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.
... While the development of P. falciparum through the different stages of its life is thought to be driven by coordinated changes in gene expression, the relative paucity of transcription factors points to unusual gene regulatory mechanisms. Meanwhile, the relative abundance of proteins related to chromatin structures, mRNA decay, and translation rates suggest alternative mechanisms of gene regulation at the epigenetic and post-translational levels [3][4][5][6][7]. Thus an improved understanding of the P. falciparum genome architecture, at both local and global scales, will provide clues for developing new therapies. ...
Article
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The development of new ways to probe samples for the three-dimensional (3D) structure of DNA paves the way for in depth and systematic analyses of the genome architecture. 3C-like methods coupled with high-throughput sequencing can now assess physical interactions between pairs of loci in a genome-wide fashion, thus enabling the creation of genome-by-genome contact maps. The spreading of such protocols creates many new opportunities for methodological development: how can we infer 3D models from these contact maps? Can such models help us gain insights into biological processes? Several recent studies applied such protocols to P. falciparum (the deadliest of the five human malaria parasites), assessing its genome organization at different moments of its life cycle. With its small genomic size, fairly simple (yet changing) genomic organization during its lifecyle and strong correlation between chromatin folding and gene expression, this parasite is the ideal case study for applying and developing methods to infer 3D models and use them for downstream analysis. Here, I review a set of methods used to build and analyse three-dimensional models from contact maps data with a special highlight on P. falciparum ’s genome organization.
... Several functional and structural properties and also evolutionary mechanisms, can be predicted either by the comparison of new elements with already classified elements, or by the comparison of elements with a similar structure or function and using it to infer the common mechanism that is at the basis of the observed similar behavior. Such elements are commonly called motifs [11]. ...
Article
Full-text available
One of the major challenges in bioinformatics is the development of efficient computational algorithms for biological sequence motif discovery. In the post-genomic era, the ability to predict the behavior, the function, or the structure of biological entities or motifs such as genes and proteins, as well as interactions among them, play a fundamental role in the discovery of information to help explain biological mechanisms. This necessitated the development of computational methods for identifying these entities. Consequently, a large number of motif finding algorithms have been implemented and applied to various organisms over the past decade. This paper presents a comparative analysis of the latest developments in motif finding algorithms and proposed an algorithm for motif discovery based on a combinatorial approach of pattern driven and statistical based approach. The proposed algorithm, Suffix Tree Gene Enrichment Motif Searching (STGEMS) as reported in [30], proved effective in identifying motifs from organisms with peculiarity in their genomic structure such as the AT-rich sequence of the malaria parasite, P. falciparum . The empirical time analysis of seven motif discovery algorithms was evaluated using four sets of genes from the intraerythrocytic development cycle of P. falciparum . The result shows that algorithms based on a combinatorial approach are more desirable.
... There exists a multitude of ways to control gene expression at different levels, for example transcriptional and post-transcriptional regulation, translational regulation and mRNA degradation (reviewed in Deitsch et al., 2007). One of the most important post-transcriptional mechanisms is pre-mRNA splicing. ...
Thesis
Besides HIV and tuberculosis, malaria still is one of the most devastating infectious diseases especially in developing countries, with Plasmodium falciparum being responsible for the frequently lethal form of malaria tropica. It is a major cause of mortality as well as morbidity, whereby pregnant women and children under the age of five years are most severely affected. Rapidly emerging drug resistances and the lack of an effective and safe vaccine hamper the combat against malaria by chemical and pharmacological regimens, and moreover the poor socio-economic and healthcare conditions in malaria-endemic countries are compromising the extermination of this deadly tropical disease to a large extent. Malaria research is still questing for druggable targets in the parasitic protozoan which pledge to be refractory against evolving resistance-mediating mutations and yet constitute affordable and compliant antimalarial chemotherapeutics. The parasite kinome consists of members that represent most eukaryotic protein kinase groups, but also contains several groups that can not be assigned to conservative ePK groups. Moreover, given the remarkable divergence of plasmodial kinases in respect to the human host kinome and the fact that several plasmodial kinases have been identified that are essential for the intraerythrocytic developmental cycle, these parasite enzymes represent auspicious targets for antimalarial regimens. Despite elaborate investigations on several other ePK groups, merely scant research has been conducted regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family, PfCLK-1-4. In other eukaryotes, CLKs are involved in mRNA processing and splicing by means of phosphorylation of serine/arginine-rich (SR) proteins, which are crucial components of the splicing machinery in the alternative splicing pathway. All four PfCLKs are abundantly expressed in asexual parasites and gametocytes, and stage-specific expression profiles of PfCLK-1 and PfCLK-2 exhibited nucleus-associated localization and an association with phosphorylation activity. In the course of this study, PfCLK-3 and PfCLK-4 were functionally characterized by indirect immunofluorescence, Western blot analysis and kinase activity assays. These data confirm that the two kinases are primarily expressed in the nucleus of trophozoites and both kinases possess in vitro phosphorylation activity on physiological substrates. Likewise PfCLK-1 and PfCLK-2, reverse genetic studies exhibited the indispensability of both PfCLKs on the asexual life cycle of P. falciparum, rendering them as potential candidates for antiplasmodial strategies. Moreover, this study was conducted to identify putative SR proteins as substrates of all four PfCLKs. Previous alignments revealed a significant homology of the parasite CLKs to yeast SR protein kinase Sky1p. Kinase activity assays showed in vitro phosphorylation of the yeast Sky1p substrate and SR protein Npl3p by precipitated PfCLKs. In addition, four homologous plasmodial SR proteins were identified that are phosphorylated by PfCLKs in vitro: PfASF-1, PFSRSF12, PfSFRS4 and PfSR-1. All four parasite SR splicing factors are predominantly expressed in the nuclei of trophozoites. For PfCLK-1, a co-localization with the SR proteins was verified. Finally, a library of human and microbial CLK inhibitors and the antiseptic chlorhexidine (CHX) was screened to determine their inhibitory effect on different parasite life cycle stages and on the PfCLKs specifically. Five inhibitors out of 63 compounds from the investigated library were selected that show a moderate inhibition on asexual life cycle stages with IC50 values ranging between approximately 4 and 8 µM. Noteworthy, these inhibitors belong to the substance classes of aminopyrimidines or oxo-β-carbolines. Actually, the antibiotic compound CHX demonstrated an IC50 in the low nanomolar range. Stage-of-inhibition assays revealed that CHX severely affects the formation of schizonts. All of the selected CLKs inhibitors also affect gametocytogenesis as well as gametogenesis, as scrutinized in gametocyte toxicity assays and exflagellation assays, respectively. Kinase activity assays confirm a specific inhibition of CLK-mediated phosphorylation of all four kinases, when the CLK inhibitors are applied on immunoprecipitated PfCLKs. These findings on PfCLK-inhibiting compounds are initial attempts to determine putative antimalarial compounds targeting the PfCLKs. Moreover, these results provide an effective means to generate chemical kinase KOs in order to phenotypically study the role of the PfCLKs especially in splicing events and mRNA metabolism. This approach of functionally characterizing the CLKs in P. falciparum is of particular interest since the malarial spliceosome is still poorly understood and will gain further insight into the parasite splicing machinery.
... Regulation of Plasmodium transcription may instead be 'hard-wired' with the only short-term changes in transcription being those required for differentiation within and between parasite life stages. Several studies looking specifically for changes in transcription were able to find no short-term change in response to metabolic inhibitors [51][52][53]. ...
Article
The malarial parasite Plasmodium falciparum is exposed to substantial redox challenges during its complex life cycle. In intraerythrocytic parasites, haemoglobin breakdown is a major source of reactive oxygen species. Deficiencies in human glucose 6-phosphate dehydrogenase (G6PD), the initial enzyme in the pentose phosphate pathway (PPP), lead to a disturbed redox equilibrium in infected erythrocytes and partial protection against severe malaria. In P. falciparum, the first two reactions of the PPP are catalysed by the bifunctional enzyme glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). This enzyme differs structurally from its human counterparts and represents a potential target for drugs. In the present study we used epitope tagging of endogenous PfGluPho to verify that the enzyme localises to the parasite cytosol. Furthermore, attempted double cross-over disruption of the PfGluPho gene indicates that the enzyme is essential for the growth of blood stage parasites. As a further step towards targeting PfGluPho pharmacologically, ellagic acid was characterised as potent PfGluPho inhibitor with an IC50 of 76 nM. Interestingly, pro-oxidative drugs or treatment of the parasites with H2 O2 only slightly altered PfGluPho expression or activity under the conditions tested. Furthermore, metabolic profiling suggested that pro-oxidative drugs do not significantly perturb the abundance of PPP intermediates. These data indicate that PfGluPho is essential in asexual parasites, but that the oxidative arm of the pentose phosphate pathway is not strongly regulated in response to oxidative challenge. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
... The development of P. falciparum through the different stages of its life cycle is thought to be driven by coordinated changes in gene expression. Over the last decade, it has become clear that the parasite relies on an unusual combination of regulatory mechanisms for gene expression, and that these mechanisms are largely dependent on epigenetic processes (reviewed in [8][9][10][11][12][13]). In multicellular eukaryotes, gene expression is often mediated by transcription factors that bind to cell-or tissuespecific promoters and give rise to the expression of a subset of genes specific to that cell type or tissue [14]. ...
Article
Plasmodium falciparum is the most deadly human malarial parasite, responsible for an estimated 207 million cases of disease and 627,000 deaths in 2012. Recent studies reveal that the parasite actively regulates a large fraction of its genes throughout its replicative cycle inside human red blood cells and that epigenetics plays an important role in this precise gene regulation. Here, we discuss recent advances in our understanding of three aspects of epigenetic regulation in P. falciparum: changes in histone modifications, nucleosome occupancy and the three-dimensional genome structure. We compare these three aspects of the P. falciparum epigenome to those of other eukaryotes, and show that large-scale compartmentalization is particularly important in determining histone decomposition and gene regulation in P. falciparum. We conclude by presenting a gene regulation model for P. falciparum that combines the described epigenetic factors, and by discussing the implications of this model for the future of malaria research.
... During life cycle progression from humans to mosquitoes, P. falciparum switches between stages with high replication rates and ones arrested in their cell cycle and also passes through a phase of sexual reproduction. These rapid transformations require fine-tuned mechanisms of gene expression, and the importance of post-transcriptional regulation of gene expression in Plasmodium parasites has previously been highlighted [2]. These include the alternative splicing (AS) of pre-mRNA, enabling the parasite to express functionally different protein isoforms. ...
Article
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Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-β-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.
... Although the function of rifin is not well understood, the regulation of rifin expression upon chemical stress could be the survival mechanism of P. falciparum. It has also been proposed that cell cycle arrest and stimulation of malaria parasite sexual development (gametogenesis) are initiated upon chemical stress (Deitsch et al, 2007). Indeed, some of the upregulated gene has been found to be specifically expressed in gametocyte stages. ...
Article
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Investigations on the fundamental of malaria parasite biology, such as invasion, growth cycle, metabolism and cell signalling have uncovered a number of potential antimalarial drug targets, including choline kinase, a key enzyme involved in the synthesis of phosphatidylcholine, an important component in parasite membrane compartment. The effect on gene expression of Plasmodium falciparum K1 strain following 72 hours exposure to 2 µM (IC50 concentration) of the choline kinase inhibitor, hexadecyltrimethylammonium bromide (HDTAB) was evaluated by DNA microarray analysis. Genes important in P. falciparum intraerythrocytic life cycle, such as invasion, cytoadherance and growth were among those affected by at least 2-fold changes in their expression levels compared with non HDTAB-treated control
... Developmentally linked and mutually exclusive transcriptional programmes are essential for the malarial parasite Plasmodium falciparum to successfully invade, colonise and subsequently propagate within the diverse host environments it encounters throughout its life cycle (Chookajorn et al., 2007;Cui and Miao, 2010;Deitsch et al., 2007;Horrocks et al., 2009;Hughes et al., 2010;Liu et al., 2011;Llinas et al., 2008;Scherf et al., 2008). Over recent years, various studies employing global transcriptomic technologies have provided significant insights into the interplay of the various molecular mechanisms that contribute to the steady-state production of transcripts (Bozdech et al., 2003;Gopalakrishnan et al., 2009;Le Roch et al., 2003;Llinas et al., 2006;Otto et al., 2010;Shock et al., 2007;Sims et al., 2007). ...
... The use of reporter assay systems in the human malarial parasite Plasmodium falciparum have proven an invaluable tool in the functional characterization of cis-acting sequences that govern the promoter and terminator activities of gene flanking sequences [1][2][3]. The two most commonly used reporter systems utilize either a bacterial chloramphenicol acetyltransferase (cat) gene or a Photinus pyralis luciferase (luc) gene [4,5]. ...
Article
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This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore absolute and temporal gene expression activity.
... Focused studies on certain AP2domain transcription factors expressed in sexual stages will shed light on the functions of these proteins in gametocytogenesis. In addition, integration of researches in the malaria field with those in model organisms for eukaryotic gene expression is needed to make further advancement in the field of gene regulation in malaria parasites [185]. ...
Article
Malaria parasites have evolved a complicated life cycle alternating between two hosts. Gametocytes are produced in the vertebrate hosts and are obligatory for natural transmission of the parasites through mosquito vectors. The mechanism of sexual development in Plasmodium has been the focus of extensive studies. In the postgenomic era, the advent of genome-wide analytical tools and genetic manipulation technology has enabled rapid advancement of our knowledge in this area. Patterns of gene expression during sexual development, molecular distinction of the two sexes, and mechanisms underlying subsequent formation of gametes and their fertilization have been progressively elucidated. However, the triggers and mechanism of sexual development remain largely unknown. This article provides an update of our understanding of the molecular and cellular events associated with the decision for commitment to sexual development and regulation of gene expression during gametocytogenesis. Insights into the molecular mechanisms of gametocyte development are essential for designing proper control strategies for interruption of malaria transmission and ultimate elimination.
... The only HMM that appears overrepresented in this study, the CCCH-type zinc finger, is most commonly associated with mRNA stability, supporting the hypothesis that complete control of gene expression in Plasmodium involves a large post-transcriptional component. This idea is further supported by the delay between maximal mRNA levels and maximal protein levels observed for a subset of genes (Le Roch et al., 2004), changes in mRNA half-life and bulk transcriptional activity across the asexual life cycle (Deitsch et al., 2007; Shock et al., 2007) and the unusual abundance of antisense transcripts in the parasite (Gunasekera et al., 2004). A discussion of the relative significance of these aspects of regulation is beyond the scope of this review, but the recent computational discovery of the first expanded family of transcription factors in Plasmodium species (Balaji et al., 2005) means that the critical roles of canonical transcription factors, which had been previously discounted relative to post-transcriptional mechanisms, require re-evaluation. ...
Article
Infection with the apicomplexan parasite Plasmodium falciparum is associated with a high burden of morbidity and mortality across the developing world, yet the mechanisms of transcriptional control in this organism are poorly understood. While P. falciparum possesses many of the characteristics common to eukaryotic transcription, including much of the canonical machinery, it also demonstrates unique patterns of gene expression and possesses unusually AT-rich intergenic sequences. Importantly, several biological processes that are critical to parasite virulence involve highly regulated patterns of gene expression and silencing. The relative scarcity of transcription-associated proteins and specific cis-regulatory motifs recognized in the P. falciparum genome have been thought to reflect a reduced role for transcription factors in transcriptional control in these parasites. New approaches and technologies, however, have led to the discovery of many more of these elements, including an expanded family of DNA-binding proteins, and a re-assessment of this hypothesis is required. We review the current understanding of transcriptional control in P. falciparum, specifically highlighting promoter-driven and epigenetic mechanisms involved in the control of transcription initiation.
... In other eukaryotes, CLKs are major regulators of mRNA splicing by phosphorylation of Serine/Arginine-rich (SR) proteins, which function in the RNA processing pathway [reviewed in Huang and Steitz, 2005;Sanford et al., 2005;Godin and Varani, 2007]. Thus, CLKs participate in the control of gene expression, and may be particularly important in malaria parasites, in view of the importance of post-transcriptional regulation of gene expression in these protozoa [Deitsch et al., 2007]. Four kinases cluster within the CLK family, which we call PfCLK-1 (PF14_0431), PfCLK-2 (PF14_0408), PfCLK-3 (PF11_0156), and PfCLK-4 (PFC0105w). ...
Article
The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Despite extensive research on most plasmodial enzymes, little information is available regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family. In other eukaryotes, CLKs regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Here, we investigate two of the PfCLKs, the Lammer kinase homolog PfCLK-1, and PfCLK-2. Both PfCLKs show homology with the yeast Serine/Arginine protein kinase Sky1p and are transcribed throughout the asexual blood stages and in gametocytes. PfCLK-1/Lammer possesses two nuclear localization signal sites and PfCLK-2 possesses one of these signal sites upstream of the C-terminal catalytic domains. Indirect immunofluorescence, Western blot, and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and PfCLK-2 is further present in the cytoplasm. The two kinases are important for completion of the asexual replication cycle of P. falciparum, as demonstrated by reverse genetics approaches. In vitro kinase assays show substrate phosphorylation by the PfCLKs, including the Sky1p substrate, splicing factor Npl3p, and the plasmodial alternative splicing factor PfASF-1. Mass spectrometric analysis of co-immunoprecipitated proteins indicates assembly of the two PfCLKs with proteins with predicted nuclease, phosphatase, or helicase functions. Our data indicate a crucial role of PfCLKs for malaria blood stage parasites, presumably by participating in gene regulation through the post-transcriptional modification of mRNA.
... In the yeast, S. cerevisiae, MYST family histone acetyltransferase (HAT), Esa1, is recruited to RP gene promoters to regulate transcription [27]. Apetela 2 (AP2) transcription factors have been identified in Plasmodia, T. gondii and plants [28]–[38], but are not present in animals. In Plasmodium, a global yeast two hybrid analysis suggested that ApiAP2 proteins interact with chromatin remodeling factors and each other [39]. ...
Article
Full-text available
Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.
... Evidence for the rescue of PfSpdSyn inhibition by exogenous polyamines is contradictory and seems to be dependent on the inhibitor used: reversibility was found with the putrescine analogue dicyclohexylamine [16] but not with the more potent inhibitors, trans-4-methylcyclohex- ylamine (4 MCHA) or 5-amino-1-pentene (APE) [12]. The modes of gene regulation in P. falciparum is currently controversial, with evidence supporting the dominant role of post-transcriptional control on the one hand18192021 and evidence mounting for the presence of transcriptional control, particularly in response to external perturbations on the parasite, on the other [9,222324252627. A recent functional genomics study of co-inhibited PfAdoMetDC/ODC demonstrated that perturbation-specific compensatory transcriptional responses are induced within the parasite to alleviate the effects of polyamine depletion [9] , similar to a previous study of ODC-inhibited malaria parasites [28]. ...
Article
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Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels. This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
... Nevertheless, each stage of erythrocytic development requires a highly coordinated, time-dependant mechanism to control the expression of mRNA of distinct sets of genes as determined by various transcriptome and proteome reports. Recently, in addition to the steady state transcript analysis a nuclear run on study was undertaken throughout erythrocytic cycle that showed that transcriptional and post transcriptional regulation including the transcript stability [56] participate in the regulation of gene expression [44,57]. At the moment it is impossible to attest the supposedly low contribution of STFs in Plasmodium since this concept was based on a poor annotation of STFs, as well as of their DNA motif counterparts. ...
Article
Full-text available
Malaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription. A comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes), and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed. This is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.
... It should be remembered that a moderately high positive correlation was shown to exist between mRNA and semiquantitative protein abundance for each stage observed [49]. However, it is not unusual that not all mRNA levels and the respective protein levels correlate given that it was shown that post-transcriptional regulatory mechanisms control gene expression in the parasite [52,53]. On the other hand it was shown that protein abundance of several HSPs, including HSP60, HSP70 and HSP90 showed a bell-shaped curve peaking at trophozoite stage [14,49], thus corresponding to the mRNA expression pattern shown here. ...
Article
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Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.
... The stage specificity of steady-state RNA expression supports previous observations that the morphological changes of the IDC correspond to many less-apparent changes in physiology, such as protein and nucleic acid metabolism (10,19) and sensitivity to small molecules (14,22,61). The Plasmodium repertoire of gene regulatory mechanisms is comparable to those of other eukaryotes (7,23), implying that stage-specific gene expression may be regulated at many levels, including chromatin structure, transcriptional activation, and posttranscriptional modulation of translation and stability (11,39,41,59). However, the relationship between such mechanisms during the IDC, specifically whether regula-tion of primary transcriptional activity determines the steadystate RNA level, remains uncertain for the vast majority of genes. ...
Article
Full-text available
The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data. For half of the genes analyzed, sense-strand transcriptional activity peaked at the same time point as total activity. The antisense strands of several genes were substantially transcribed. Comparison of the transcriptional activity of the sense strand of each gene to its steady-state RNA abundance across the time points assayed revealed both correlations and discrepancies, implying transcriptional and posttranscriptional regulation, respectively. Our results demonstrate that such comparisons can effectively indicate gene regulatory mechanisms in P. falciparum and suggest that genes with diverse transcriptional activity levels and patterns combine to produce total transcriptional activity levels tied to parasite development during the IDC.
... This study is so far the only published example of a specific transcriptome response to a drug related to the drug's known target-pathway. No significant changes of the mRNA levels for enzymes involved in the lipid biosynthesis pathway could be evidenced upon exposure for 24–36 hours to the antimalarial choline analog T4, which targets the inhibition of phosphatidyl choline biosynthe- sis [7] . In a recent study of the transcriptome of asynchronous P. falciparum cultures under chloroquine pressure [8], around 600 genes were differentially expressed in presence of the drug, but only 38 of these were observed in two different experiments. ...
Article
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Translation of the genome sequence of Plasmodium sp. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of P. falciparum parasites exposed to antimalarial drugs. The rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole. The high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.
Article
The capacity of malaria parasites to respond to changes in their environment at the transcriptional level has been the subject of debate, but recent evidence has unambiguously demonstrated that Plasmodium spp. can produce adaptive transcriptional responses when exposed to some specific types of stress. These include metabolic conditions and febrile temperature. The Plasmodium falciparum protective response to thermal stress is similar to the response in other organisms, but it is regulated by a transcription factor evolutionarily unrelated to the conserved transcription factor that drives the heat shock (HS) response in most eukaryotes. Of the many genes that change expression during HS, only a subset constitutes an authentic response that contributes to parasite survival.
Article
Organisms with identical genome sequences can show substantial differences in their phenotypes owing to epigenetic changes that result in different use of their genes. Epigenetic regulation of gene expression plays a key role in the control of several fundamental processes in the biology of malaria parasites, including antigenic variation and sexual differentiation. Some of the histone modifications and chromatin-modifying enzymes that control the epigenetic states of malaria genes have been characterized, and their functions are beginning to be unraveled. The fundamental principles of epigenetic regulation of gene expression appear to be conserved between malaria parasites and model eukaryotes, but important peculiarities exist. Here, we review the current knowledge of malaria epigenetics and discuss how it can be exploited for the development of new molecular markers and new types of drugs that may contribute to malaria eradication efforts.
Thesis
Trotz weltweiter Bemühungen, die Tropenkrankheit Malaria zurückzudrängen, erkranken jährlich bis zu einer halben Milliarde Menschen an Malaria mit der Folge von über einer Million Todesopfern. Da zur Zeit eine wirksame Impfung nicht in Sicht ist und sich Resistenzen gegen gängige Medikamente ausbreiten, werden dringend neue Antimalariamittel benötigt. Um die Suche nach neuen Angriffsorten für Medikamente zu unterstützen, untersucht die vorliegende Arbeit mit einem rechnergestützten Ansatz den Stoffwechsel von Plasmodium falciparum, dem tödlichsten Malaria-Erreger. Basierend auf einem aus dem aktuellen Forschungsstand rekonstruierten metabolischen Netzwerk des Parasiten werden metabolische Flüsse für die einzelnen Stadien des Lebenszyklus von P. falciparum berechnet. Dabei wird ein im Rahmen dieser Arbeit entwickelter Fluss-Bilanz-Analyse-Ansatz verwendet, der ausgehend von in den jeweiligen Entwicklungsstadien gemessenen Genexpressionsprofilen entsprechende Flussverteilungen ableitet. Für das so ermittelte stadienspezifische Flussgeschehen ergibt sich eine gute Übereinstimmung mit bekannten Austauschprozessen von Stoffen zwischen Parasit und infiziertem Erythrozyt. Knockout Simulationen, die mit Hilfe einer ähnlichen Vorhersagemethode durchgeführte werden, decken essentielle metabolische Reaktionen im Netzwerk auf. Fast 90% eines Sets von experimentell bestimmten essentiellen Enzymen wird wiedergefunden, wenn die Annahme getroffen wird, dass Nährstoffe nur begrenzt aus der Wirtszelle aufgenommen werden können. Die als essentiell vorhergesagten Enzyme stellen mögliche Angriffsorte für Medikamente dar. Anhand der Flussverteilungen, die für die einzelnen Entwicklungsstadien berechnet wurden, können diese potenziellen Targets nach Relevanz für Malaria Prophylaxe und Therapie sortiert werden, je nachdem, in welchem Stadium die Enzyme als aktiv vorhergesagt wurden. Dies bietet einen vielversprechenden Startpunkt für die Entwicklung von neuen Antimalariamitteln.
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