Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production

The primary virulence determinant of Plasmodium falciparum malaria parasite–infected cells is a family of heterogeneous surface receptors collectively referred to as PfEMP1. These proteins are encoded by a large, polymorphic gene family called var. The family contains approximately 60 individual genes, which are subject to strict, mutually exclusive expression, with the single expressed var gene determining the antigenic, cytoadherent, and virulence phenotype of the infected cell. The mutually exclusive expression pattern of var genes is imperative for the parasite's ability to evade the host's immune response and is similar to the process of “allelic exclusion” described for mammalian Ig and odorant receptor genes. In mammalian systems, mutually exclusive expression is ensured by negative feedback inhibition mediated by production of a functional protein. To investigate how expression of the var gene family is regulated, we have created transgenic lines of parasites in which expression of individual var loci can be manipulated. Here we show that no such negative feedback system exists in P. falciparum and that this process is dependent solely on the transcriptional regulatory elements immediately adjacent to each gene. Transgenic parasites that are selected to express a var gene in which the PfEMP1 coding region has been replaced by a drug-selectable marker silence all other var genes in the genome, thus effectively knocking out all PfEMP1 expression and indicating that the modified gene is still recognized as a member of the var gene family. Mutually exclusive expression in P. falciparum is therefore regulated exclusively at the level of transcription, and a functional PfEMP1 protein is not necessary for viability or for proper gene regulation in cultured parasites

Pregnancy-associated Malaria and the Prospects for Syndrome-specific Antimalaria Vaccines

Aided by the Plasmodium falciparum genome project, recent discoveries regarding the molecular basis of malaria pathogenesis have led to a better understanding of the interactions between host and parasite. Although vaccines that prevent infection by malaria parasites remain only hopes for the future, there are now more immediate prospects for vaccines that protect against specific disease syndromes. Here, we discuss the latest advances in the development of a vaccine that specifically targets pregnancy-associated malaria (PAM)

A regulatable transgene expression system for cultured Plasmodium falciparum parasites

<p>Abstract</p> <p>Background</p> <p>The ability to transfect and create transgenic cultured malaria parasites has transformed the study of <it>Plasmodium falciparum </it>over the last decade. With the completion of the annotated genome sequence, the process of gene discovery now routinely includes gene knockouts, over-expression and complementation analysis. However, while this technology has proven extremely valuable, significant limitations exist. In particular, <it>P. falciparum </it>DNA is often unstable and difficult to clone because of its AT-rich, repetitive nature. As a result, transgene expression constructs can be difficult to assemble due to the need to include two expression cassettes on a single plasmid, one to drive expression of the transgene of interest and a second for expression of the selectable marker. In addition, transgene expression levels are usually not regulatable, making it difficult to assess phenotypes that are sensitive to the amount of protein expressed.</p> <p>Results</p> <p>A plasmid based system for transgene expression is described that uses a single, bidirectional promoter to drive expression of both the transgene and the selectable marker, thus greatly reducing the size of the construct and enhancing stability. Further, by altering the concentration of drug used for selection, it is possible to modulate the copy number of the concatameric episomes and thereby regulate the expression level of the transgene through a range greater than 10 fold.</p> <p>Conclusion</p> <p>The transgene expression system described here should prove useful for both routine protein over-expression and complementation experiments as well as for experiments in which precisely manipulating the expression level of candidate proteins is desirable. This should provide an additional level of precision to the tools used to study the molecular biology of malaria parasites.</p

Horizontal gene transfer of epigenetic machinery and evolution of parasitism in the malaria parasite Plasmodium falciparum and other apicomplexans

Background The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology. Results In this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available. Conclusions Animal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in the evolution to parasitism in this important protozoan lineage

Malaria Epigenetics

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

A multifactorial role for P. falciparum malaria in endemic Burkitt\u27s lymphoma pathogenesis

Endemic Burkitt\u27s lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation

Gametocyte Development and Carriage in Ghanaian Individuals with Uncomplicated Plasmodium falciparum Malaria.

Plasmodium falciparum gametocytes develop over 9-12 days while sequestered in deep tissues. On emergence into the bloodstream, they circulate for varied amounts of time during which certain host factors might influence their further development. We aimed to evaluate the potential association of patient clinical parameters with gametocyte development and carriage via in vivo methods. Seventy-two patients were enrolled from three hospitals in the Volta region of Ghana in 2016. Clinical parameters were documented for all patients, and gametocyte prevalence by microscopy was estimated at 12.5%. By measuring RNA transcripts representing two distinct gametocyte developmental stages using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we obtained a more precise estimate of gametocyte carriage while also inferring gametocyte maturation. Fifty-three percent of the study participants harbored parasites expressing transcripts of the immature gametocyte-specific gene (PF3D7_1477700), whereas 36% harbored PF3D7_1438800 RNA-positive parasites, which is enriched in mid and mature gametocytes, suggesting the presence of more immature stages. Linear logistic regression showed that patients older than 5 years but less than 16 years were more likely to carry gametocytes expressing both PF3D7_1477700 and PF3D7_1438800 compared with younger participants, and gametocytemia was more likely in mildly anemic individuals compared with those with severe/moderate anemia. These data provide further evidence that a greater number of malaria patients harbor gametocytes than typically estimated by microscopy and suggest a possible association between age, fever, anemia, and gametocytemia

The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II–IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates

Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum . Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P . falciparum , suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes