Mechanisms underlying mutually exclusive expression of virulence genes by malaria parasites

A fundamental yet poorly understood aspect of gene regulation in eukaryotic organisms is the mechanisms that control allelic exclusion and mutually exclusive gene expression. In the malaria parasite Plasmodium falciparum, this process regulates expression of the var gene family--a large, hypervariable repertoire of genes that are responsible for the ability of the parasite to evade the host immune system and for pathogenesis of the disease. A central problem in understanding this process concerns the mechanisms that limit expression to a single gene at a time. Here, we describe results that provide information on the mechanisms that control silencing and single gene expression and differentiate between several models that have recently been proposed. The results provide the first evidence, to our knowledge, supporting the existence of a postulated var-specific, subnuclear expression site and also reinforce the conclusion that var gene regulation is based on cooperative interactions between the two promoters of each var gene.     

Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion

The process of erythrocyte invasion by merozoites of Plasmodium falciparum involves multiple steps, including the formation of a moving junction between parasite and host cell, and it is characterised by the redundancy of many of the receptor-ligand interactions involved. Several parasite proteins that interact with erythrocyte receptors or participate in other steps of invasion are encoded by small subtelomerically located gene families of four to seven members. We report here that members of the eba, rhoph1/clag, acbp, and pfRh multigene families exist in either an active or a silenced state. In the case of two members of the rhoph1/clag family, clag3.1 and clag3.2, expression was mutually exclusive. Silencing was clonally transmitted and occurred in the absence of detectable DNA alterations, suggesting that it is epigenetic. This was demonstrated for eba-140. Our data demonstrate that variant or mutually exclusive expression and epigenetic silencing in Plasmodium are not unique to genes such as var, which encode proteins that are exported to the surface of the erythrocyte, but also occur for genes involved in host cell invasion. Clonal variant expression of invasion-related ligands increases the flexibility of the parasite to adapt to its human host.     

Malaria: Could its unusual epigenome be the weak spot?

The epigenetic contribution to the regulation and maintenance of gene expression patterns by histone modifications is well established in eukaryotes. In Plasmodium falciparum, the mechanisms and factors regulating gene expression during progression through its infected red blood cell cycle (iRBC) and underlying mutually exclusive expression of antigenic variation genes involved in immune evasion are far from understood. Recently, the first comprehensive analyses of the P. falciparum chromatin landscape at different iRBC stages have been performed. These studies uncovered the existence of well-defined heterochromatic regions within a generally euchromatic epigenome. Notably, silencing of genes encoding for virulence determinants such as var genes, appears to be orchestrated by the concerted action of the Sir2 and HP1 orthologs and the presence of the histone mark, H3K9me3. Epigenetic speciation could make the parasite exquisitely vulnerable to epigenetic drug treatment, unless this deadly parasite still has a number of tricks up his sleeves.     

Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR)

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.     

Molecular mechanisms of Plasmodium falciparum placental adhesion

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.     

MHC genes and oxidative stress in sticklebacks: an immuno-ecological approach

Individual variation in the susceptibility to infection may result from the varying ability of hosts to specifically recognize different parasite strains. Alternatively, there could be individual host differences in fitness costs of immune defence. Although, these two explanations are not mutually exclusive, they have so far been treated in separate experimental approaches. To analyse potential relationships, we studied body condition and oxidative stress, which may reflect costs of immunity, in three-spined sticklebacks that had been experimentally exposed to three species of naturally occurring parasite. These sticklebacks differed in a trait, which is crucial to specific parasite defence, i.e. individual genetic diversity at major histocompatibility complex (MHC) class IIB loci. Oxidative stress was quantified as tissue acrolein, a technique that has been applied to questions of immuno-ecology for the first time. We measured gene expression at the MHC and other estimates of immune activation. We found that fish with high levels of MHC expression had poor condition and elevated oxidative stress. These results indicate that MHC-based specific immunity is connected with oxidative stress. They could, thus, also be relevant in the broader context of the evolution of sexually selected signals that are based on carotenoids and are, thus supposed to reflect oxidative stress resistance.     

Concurrent expression of recombination activating genes 1 and 2 in zebrafish olfactory sensory neurons

Each olfactory sensory neuron (OSN) expresses a single odorant receptor (OR) from a large repertoire of clustered OR genes. It has been hypothesized that OR gene regulation may involve stochastic DNA rearrangement, which in lymphocytes requires the recombination activating genes, rag1 and rag2. We have recently demonstrated that rag1 is expressed in zebrafish OSNs. Here we report that rag2, the obligate partner for rag1 function, is also expressed in OSNs and that its expression pattern mimics that of rag1. The onset of rag1 and rag2 expression preceded that of known zebrafish ORs and the number of rag1-positive OSNs corresponded with the number expressing the olfactory cyclic nucleotide-gated cation channel, an OSN marker. Zebrafish OSNs are the first example of concurrent rag expression in a nonlymphoid tissue. The expression of rag1 and rag2 in OSNs adds to the list of similarities between the olfactory and immune systems that includes monoallelic and mutually exclusive gene expression.