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Abstract Background The ability to transfect and create transgenic cultured malaria parasites has transformed the study of Plasmodium falciparum 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, P. falciparum 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. Results 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. Conclusion 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.

Over the last few decades, novel methods have been developed to study how chromosome positioning within the nucleus may play a role in gene regulation. Adaptation of these methods in the human malaria parasite, Plasmodium falciparum , has recently led to the discovery that the three-dimensional structure of chromatin within the nucleus may be critical in controlling expression of virulence genes ( var genes). Recent work has implicated an unusual, highly conserved var gene called var2csa in contributing to coordinated transcriptional switching, however how this gene functions in this capacity is unknown. To further understand how var2csa influences var gene switching, targeted DNA double-strand breaks (DSBs) within the sub-telomeric region of chromosome 12 were used to delete the gene and the surrounding chromosomal region. To characterize the changes in chromatin architecture stemming from this deletion and how these changes could affect var gene expression, we used a combination of RNA-seq, Chip-seq and Hi-C to pinpoint epigenetic and chromatin structural modifications in regions of differential gene expression. We observed a net gain of interactions in sub-telomeric regions and internal var gene regions following var2csa knockout, indicating an increase of tightly controlled heterochromatin structures. Our results suggest that disruption of var2csa results not only in changes in var gene transcriptional regulation but also a significant tightening of heterochromatin clusters thereby disrupting coordinated activation of var genes throughout the genome. Altogether our result confirms a strong link between the var2csa locus, chromatin structure and var gene expression.

Endemic Burkitt's 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, the most important causative agent of human malaria, undergoes antigenic variation as a means of prolonging infection and ensuring transmission between hosts. Clonal variation is observed in the surface adhesins expressed on infected erythrocytes: primarily in the PfEMP1 adhesin encoded by the large var gene family. The sirtuin PfSIR2A was the first protein discovered to have a major influence on antigenic variation in P. falciparum. In the absence of PfSIR2A, normal silencing of the variantly-expressed var gene family is partially deregulated. To thoroughly investigate the role of PfSIR2A in controlling antigenic variation, multiple independent clones of wildtype and PfSIR2A-knockout (DeltaSir2a) parasites were generated. var gene expression was then measured qualitatively, quantitatively and longitudinally over extended periods in culture. DeltaSir2a parasites were found to activate about 10 specific var genes in every independent clone analyzed. The activated genes were biased towards the upsA, upsBA and upsEvar gene subclasses. The total var transcript level was two to three-fold higher in DeltaSir2a parasites than in wildtype parasites and at least one transcript - encoding the pregnancy malaria adhesin VAR2CSA - was successfully translated and expressed on the infected cell surface. In the absence of PfSIR2A, antigenic switching over time was also diminished, although not abolished. This work expands our understanding of clonal antigenic variation in this important human pathogen and demonstrates a central role for PfSIR2A in regulating both the variant expression of specific var gene subsets and the overall quantity of var gene expression., (Copyright 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

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.

Antigenic variation in Plasmodium falciparum malaria is mediated by transcriptional switches between different members of the multicopy var gene family. Each var gene encodes a member of a group of heterogeneous surface proteins collectively referred to as PfEMP1. Mutually exclusive expression ensures that an individual parasite only transcribes a single var gene at a time. In this work we studied var gene switching to determine if transcriptional switches favour expression of particular subgroups of var genes and if var gene activation within a clonal population of parasites follows a predetermined order. We show that in clonal parasite populations, expression of var genes located in the central regions of chromosomes is remarkably stable and that they rarely undergo transcriptional switches in the absence of selection. In contrast, parasites expressing subtelomerically located var genes readily switched to alternative var loci. We confirmed these observations by generating transgenic parasites carrying drug selectable markers in subtelomeric and central var loci and monitoring switching after release from selection. Our data show that different var genes have different intrinsic switching rates that correlate with chromosomal location, and that there is no predetermined order of expression.

The intra-erythrocytic stages of Plasmodium falciparum assemble a unique protein trafficking system that targets parasite proteins to the red cell cytoplasm and cell surface. It is through this trafficking pathway that the primary virulence determinants of P. falciparum infections are targeted to the erythrocyte surface to mediate adhesion to host endothelial cells. A recent study has shown that SBP-1, a parasite protein associated with Maurer's clefts in the infected red cell cytosol, is essential for transport of the virulence factor PfEMP-1. This discovery sheds new light on the little-understood mechanisms that regulate protein trafficking in infected cells.

Pathogens of the genus Plasmodium are unicellular parasites that infect a variety of animals, including reptiles, birds and mammals. All Plasmodium species target host erythrocytes and replicate asexually within this niche. In humans, proliferation within erythrocytes causes disease symptoms ranging from asymtomatic infection to severe disease, including mild to severe febrile and respiratory symptoms, profound anaemia and obstruction of blood flow. The most serious form of human malaria is caused by Plasmodium falciparum, a pathogen that is responsible for several million deaths annually throughout the developing world. Malaria parasites succeed in evading the host immune response to establish long-term, persistent infections, thus increasing the efficiency by which they are transmitted to the mosquito vector. The ability to evade the host immune system, in particular the avoidance of antibody-mediated immunity against parasite-encoded surface proteins, is the result of amplification of extensive repertoires of multicopy, hypervariable gene families that encode infected erythrocyte or merozoite surface proteins. Via switching between antigenically diverse genes within these large families, populations of parasites have the capacity for rapid variation in antigenicity and virulence over the course of an infection. Here we review the amplification and generation of antigenic diversity within the Plasmodium variant gene families, as well as discuss the mechanisms underlying their tightly controlled gene expression and antigenic switching.

The human malaria parasite, Plasmodium falciparum, maintains a persistent infection altering the proteins expressed on the surface of the infected red blood cells, thus avoiding the host immune response. The primary surface antigen, a protein called PfEMP1, is encoded by a multicopy gene family called var. Each individual parasite only expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. Previous work using reporter genes in transiently transfected plasmid constructs implicated a conserved intron found in all var genes in the silencing process. Here we have utilized episomal recombination within stably transformed parasites to generate different var promoter and intron arrangements and show that loss of the intron results in var promoter activation. Further, in multicopy plasmid concatamers, each intron could only silence a single promoter, suggesting a one-to-one pairing requirement for silencing. Transcriptionally active, "unpaired" promoters remained active after integration into a chromosome; however, they were not recognized by the pathway that maintains mutually exclusive var gene expression. The data indicate that intron/promoter pairing is responsible for silencing each individual var gene and that disruption of silencing of one gene does not affect the transcriptional activity of neighboring var promoters. This suggests that silencing is regulated at the level of individual genes rather than by assembly of silent chromatin throughout a chromosomal region, thus providing a possible explanation of how a var gene can be maintained in a silent state while the immediately adjacent var gene is transcriptionally active.

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.

Antigenic variation of surface membrane proteins by protozoan parasites enables these pathogenic organisms to avoid host immune responses and thus perpetuate long-term infections. Babesia bovis, the causative agent of severe babesiosis in cattle, was previously shown to undergo antigenic variation through modifications to its primary surface antigen, a protein called VESA1. In this issue, Al-Khedery and Allred provide a detailed description of the genes that encode VESA1 and present convincing evidence for progressive, segmental gene conversion in the generation of variant forms of this surface antigen.

Malaria remains one of the most important infectious diseases in the world today, infecting 300 to 500 million people yearly and resulting in 1 to 2 million deaths, primarily of young African children. The most severe form of this disease is caused by infection with the mosquito borne protozoan parasite Plasmodium falciparum. This parasite lives by invading and multiplying within the red blood cells of its host, causing disease through anemia resulting from red cell destruction, and also through modifications made to the surface of infected red cells. These modifications make infected cells cytoadherent or "sticky", allowing them to adhere to the walls of blood vessels, leading to obstruction of blood flow and such clinical manifestations as the often fatal syndrome of cerebral malaria. In addition, parasites are capable of undergoing antigenic variation, a process of continually changing the identity of proteins on the surface of infected cells and thus avoiding the immune response mounted by the host. This process promotes a long term, persistent infection that is difficult to clear.

Each Plasmodium falciparum malaria parasite carries about 50 var genes from a diverse family that encode variable adhesion proteins on the infected red blood cells of the host, but individual parasites single out just one var gene for expression and silence all the others. Here we show that this silencing is established during the DNA-synthesis phase (S phase) of the cell cycle and that it depends on the cooperative interaction between two elements in separate control regions of each var gene (the 5'-flanking region and the intron). This finding should help to clarify the mechanisms by which parasites coordinate the silencing and activation of var genes that are responsible for antigenic variation in malaria.

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Antigenic variation and immune evasion by Plasmodium falciparum parasitized erythrocytes are mediated by expression switches among members of the multicopy var gene family. Here we describe a cluster of var genes on chromosome 12 that showed spontaneous recombination and switches in the transcription of individual genes. The transcription switches were not associated with sequence changes in promoter regions. Transfected episomes containing a luciferase reporter under control of a var promoter were expressed regardless of the transcriptional status of the endogenous promoter. The results suggest epigenetic regulation of P. falciparum var gene transcription that depends upon the local structure of chromatin and its associated proteins.

In response to a blood meal, the fat body of the female mosquito, Aedes aegypti, begins massive production of several yolk proteins which are subsequently stored in the developing oocytes. Although 20-hydroxyecdysone (20E) is important for initiation and maintenance of expression of the yolk protein genes encoding vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP), the exact nature of 20E action has not been clearly defined. A primary question is whether this hormone directly stimulates expression of the genes for Vg and VCP or if it acts indirectly through a hormone response cascade. We have demonstrated that 10(-4) M cycloheximide (Chx) reversibly inhibits > 98% of protein synthesis in in vitro fat body culture. 10(-5) M 20E stimulates high levels of the mRNAs for Vg and VCP in previtellogenic fat bodies cultured in vitro, but initiation of this expression is eliminated by Chx. Thus, our results indicate that protein synthesis is required in response to 20E before increased levels of yolk protein mRNAs can be measured. We therefore conclude that the action of 20E is indirect.

Vitellogenic carboxypeptidase is a 53 kDa yolk protein produced by the fat body of the female mosquito, Aedes aegypti, in response to a blood meal. Its expression is sex-, stage- and tissue-specific and is identical to that of the major yolk protein, vitellogenin. The gene is intronless and two alleles have been cloned and sequenced, including more than 1.5 kb on both sides of the coding region. A capsite consensus recently identified as an arthropod initiator is present at the start site of transcription. Upstream of this capsite is a 16 bp imperfect palindrome repeated four times showing strong homology to defined hormone-response elements. In addition, a region that closely resembles the fat body enhancer and double sex binding site from the Drosophila yolk protein genes and several potential fat body-specific regulatory protein binding sites were found.