The human malaria parasite Plasmodium falciparum undergoes antigenic variation (AV) to avoid immune clearance in the human host. The molecular process of AV is mediated through the mutually exclusive expression of variant surface proteins encoded by the multi-copy var gene family. My laboratory works on different aspects of AV such as trafficking of the variant adhesion to the erythrocyte surface and the genetic and epigenetic processes that determine the expression of a single member of the gene family (monoallelic expression). More recently, we have identified small molecule histone methyltransferase inhibitors that display rapid antimalarial activity against all blood stage forms in P. falciparum. The potential as new anti malarial drugs for various stages will be further explored. Another focus is the molecular understanding of host and parasite factors contributing to malaria pathogenesis.
Molecular mechanisms of antigenic variation
Antigenic Variation is a strategy employed by malaria species to outmanoeuvre the host defence mechanisms long enough for their progeny to spread. Mutually exclusive expression of a single member of a multigene family (called var gene family) leads to the successive expression of variant molecules on the surface of infected erythrocytes. Subtelomeric non-coding elements are critical DNA regions involved in cluster formation between chromosome ends and recruit a number of chromatin silencing factors such as PfSir2. These Perinuclear Repressive Centers are crucial for the control of the silent state of antigenic variation genes and we identified several histone modifications that are linked to epigenetic memory of active and silent var genes. Spatial tethering of members of the gene family to the perinuclear region involves var introns. Another epigenetic factor associated with var gene activation is the relocation of a silent var gene into a particular perinuclear region compatible with transcription. We showed recently that perinuclear actin polymerisation is involved in this process. We identified a specific DNA-binding protein (a member of AP2 family) that binds to var introns. Mutant parasites for this AP2 member are under investigation to study its role in monoallelic expression. We are developing new approaches such as random mutagenesis to identify factors that are not predicted by bioinformatics methods to contribute to mutually exclusive expression (team JJ Lopez-Rubio). More recently, we have studied the role of non-coding RNA (ncRNA) produced within or adjacent to var gene loci. To this end, we developed strand-specific RNA sequencing protocols for AT-rich regions. Our study reveals for the first time a mechanism of transcriptional variation of ncRNA from repeated DNA elements indicating another level of phenotypic plasticity in malaria parasites.
Small molecule inhibitors that target the epigenetic machinery
Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated P. falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115 inhibited P. falciparum 3D7 parasites in culture with IC50 values of approximately 100 nM, values at least 22-fold more potent than their apparent IC50 toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intra-erythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-hour incubation with 1 μM BIX-01294 and resulted in complete parasite killing after a 12-hour incubation. Additionally, mice with patent P. berghei ANKA strain infection treated with a single dose (40mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration- and exposure time-dependent manner. Our data positions histone lysine methyltransferases as a novel target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life cycle stages.
Characterization of new chromatin factors that binds DNA and RNA: the Alba gene family
Perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Recently, we identified a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. The perinuclear PfAlba location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.
Cytoadhesion and malaria pathogenesis
Parasite-encoded adhesion molecules are inserted into the erythrocyte membrane during intracellular blood stage development. The D. Mattei team has investigated mutant parasites that have a defect in infected erythrocyte (IE) adhesion to endothelial cells. The analysis of mutant parasite lines that have deleted a chromosome 9 subtelomeric region including the clag9 gene, revealed that parasites express a member of the var gene family at the surface of IE but these IEs are unable to adhere to common adhesion receptors (CD36, ICAM-1, CSA etc.). Gene knock out of clag9 demonstrates that this gene is not essential to assemble the adhesive complex at the IE surface. Gene complementation studies are ongoing in order to identify novel proteins that are able to restore the adhesion phenotype.
Pathogenesis of experimental cerebral malaria (ECM)
The group of S. Mecheri (E4 attached to BIHP since 2008) is interested in identifying host and Plasmodium parasite factors that are reminiscent of the inflammatory allergic response and are critical for the development of severe forms of malaria disease during blood stage development, in particular ECM using a well established murine model (C57BL/6 mice/P berghei ANKA). The following lines of research linked to malaria pathogenesis have been investigated: i) Beside the histamine signaling pathway in the vertebrate host, they recently made the unprecedented finding that a new population of neutrophils expressing the FceRI/IgE complex was critical for disease expression and severity. This cell population apparently is highly enriched in the brain in mice developing ECM. This mechanism of pathogenesis is further investigated. ii) The relevance of the inflammatory cascade was recently implemented by the finding that a Plasmodium gene product, the “Translationally Controlled Tumor Protein”, which is endowed with a histamine releasing activity, was found to be associated with disease severity. iii) A new candidate parasite gene is now under study, the “High Mobility Group Box” (HMGB2) protein, which when released into the extracellular milieu, acts as a proinflammatory cytokine. This molecule plays a central role in the pathogenesis of many immune mediated inflammatory diseases including malaria. These topics will be further explored by the Mecheri team.