Peter Rainer Preiser

Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance

Mechanisms propelling drug resistance If it were to spread, resistance to the drug artemisinin would seriously derail the recent gains of global malaria control programs (see the Perspective by Sibley). Mutations in a region called the K13-propeller are predictive for artemisinin resistance in Southeast Asia. Mok et al. looked at the patterns of gene expression in parasites isolated from more than 1000 patients sampled in Africa, Bangladesh, and the Mekong region. A range of mutations that alter protein repair pathways and the timing of the parasites developmental cycle were only found in parasites from the Mekong region. Straimer et al. genetically engineered the K13 region of parasites obtained from recent clinical isolates. Mutations in this region were indeed responsible for the resistance phenotypes. Science, this issue p. 431, p. 428; see also p. 373 Resistance to the primary antimalarial drug lies in mutations in protein repair and developmental pathways. [Also see Perspective by Sibley] Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain–carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.

Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum

Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that ∼59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.

Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription.

BackgroundArtemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.ResultsIn the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.ConclusionsThe decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes

Bacterial plasmids introduced into the human malaria parasite Plasmodium falciparum replicate well but are poorly segregated during mitosis. In this paper, we screened a random P.falciparum genomic library in order to identify sequences that overcome this segregation defect. Using this approach, we selected for parasites that harbor a unique 21 bp repeat sequence known as Rep20. Rep20 is one of six different repeats found in the subtelomeric regions of all P.falciparum chromosomes but which is not found in other eukaryotes or in other plasmodia. Using a number of approaches, we demonstrate that Rep20 sequences lead to dramatically improved episomal maintenance by promoting plasmid segregation between daughter merozoites. We show that Rep20+, but not Rep20−, plasmids co‐localize with terminal chromosomal clusters, indicating that Rep20 mediates plasmid tethering to chromosomes, a mechanism that explains the improved segregation phenotype. This study implicates a direct role for Rep20 in the physical association of chromosome ends, which is a process that facilitates the generation of diversity in the terminally located P.falciparum virulence genes.

Transcriptional variation in the malaria parasite Plasmodium falciparum

Malaria genetic variation has been extensively characterized, but the level of epigenetic plasticity remains largely unexplored. Here we provide a comprehensive characterization of transcriptional variation in the most lethal malaria parasite, Plasmodium falciparum, based on highly accurate transcriptional analysis of isogenic parasite lines grown under homogeneous conditions. This analysis revealed extensive transcriptional heterogeneity within genetically homogeneous clonal parasite populations. We show that clonally variant expression controlled at the epigenetic level is an intrinsic property of specific genes and gene families, the majority of which participate in host-parasite interactions. Intrinsic transcriptional variability is not restricted to genes involved in immune evasion, but also affects genes linked to lipid metabolism, protein folding, erythrocyte remodeling, or transcriptional regulation, among others, indicating that epigenetic variation results in both antigenic and functional variation. We observed a general association between heterochromatin marks and clonally variant expression, extending previous observations for specific genes to essentially all variantly expressed gene families. These results suggest that phenotypic variation of functionally unrelated P. falciparum gene families is mediated by a common mechanism based on reversible formation of H3K9me3-based heterochromatin. In changing environments, diversity confers fitness to a population. Our results support the idea that P. falciparum uses a bet-hedging strategy, as an alternative to directed transcriptional responses, to adapt to common fluctuations in its environment. Consistent with this idea, we found that transcriptionally different isogenic parasite lines markedly differed in their survival to heat-shock mimicking febrile episodes and adapted to periodic heat-shock with a pattern consistent with natural selection of pre-existing parasites.

The Plasmodium falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte.

Modifications of the Plasmodium falciparum–infected red blood cell (iRBC) surface have been linked to parasite-associated pathology. Such modifications enable the parasite to establish long-lasting chronic infection by evading antibody mediate immune recognition and splenic clearance. With the exception of the well-demonstrated roles of var-encoded PfEMP1 in virulence and immune evasion, the biological significance of other variant surface antigens (rif and stevor) is largely unknown. While PfEMP1 and RIFIN have been located on the iRBC surface, recent studies have located STEVOR at the iRBC membrane where it may be exposed on the erythrocyte surface. To investigate the role of STEVOR in more detail, we have developed antibodies against two putative STEVOR proteins and used a combination of indirect immunofluorescence assays (IFA), live IFA, flow cytometry, as well as agglutination assays, which enable us to demonstrate that STEVOR is clonally variant at the surface of schizont stage parasites. Crucially, expression of different STEVOR on the surface of the iRBC changes the antigenic property of the parasite. Taken together, our data for the first time demonstrate that STEVOR plays a role in creating antigenic diversity of schizont stage parasites, thereby adding additional complexity to the immunogenic properties of the iRBC. Furthermore, it clearly demonstrates that to obtain a complete understanding of how parasite-induced pathology is linked to variation on the surface of the iRBC, focusing the interactions of multiple multigene families needs to be considered.

Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites

BackgroundMalaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized.ResultsHere, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins - including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins - are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation.ConclusionsTogether, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum.

Quantitative time-course profiling of parasite and host cell proteins in the human malaria parasite Plasmodium falciparum

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy.

Distinct Trafficking and Localization of STEVOR Proteins in Three Stages of the Plasmodium falciparum Life Cycle

ABSTRACT The genome of Plasmodium falciparum harbors three extensive multigene families, var, rif, and stevor (for subtelomeric variable open reading frame), located mainly in the subtelomeric regions of the parasites 14 chromosomes. STEVOR variants are known to be expressed in asexual parasites, but no function has as yet been ascribed to this protein family. We have examined the expression of STEVOR proteins in intraerythrocytic sexual stages, gametocytes, and extracellular sporozoites isolated from infected Anopheles mosquitoes. In gametocytes, stevor transcripts appear transiently early in development but STEVOR proteins persist for several days and are transported out of the parasite, travel through the host cell cytoplasm, and localize to the erythrocyte plasma membrane. In contrast to asexual parasites, gametocytes move STEVOR to the periphery via a trafficking pathway independent of Maurers clefts. In sporozoites, STEVOR appear dispersed throughout the cytoplasm in vesicle-like structures. The pattern of STEVOR localization we have observed in gametocytes and sporozoites differs significantly from that in asexual parasite stages. STEVOR variants are therefore likely to perform different functions in each stage of the parasites life cycle in which they occur.

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.