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Introduction: The Leitmotif of the BIHP research group can be summarized as follows: “From basic research to new intervention strategies against malaria parasites”. The teams investigate various topics related to blood stage infection such as immune evasion strategies, chronic infection, erythrocyte adhesion and parasite molecules that stimulate inflammation. State-of-the-art molecular, cellular and genetic methods are used to tackle fundamental question on monoallelic gene expression of virulence genes to the functional expression of surface adhesion proteins and host factors (cellular and molecular) contributing to pathogenesis. Although the majority of our research is conducted on the human malaria parasite P. falciparum, we use the rodent mouse model P. berghei to gain insight into the interplay of host and malaria parasite factors in pathogenesis. Clearly, certain parasites genes only reveal their function when studied in the context of the host infection. Conceptual discoveries and advances will be validated in malaria endemic regions using various established collaborations (Institute Pasteur network members in Senegal and Madagascar and other endemic regions).
Molecular mechanism of antigenic variation of P. falciparum
The protozoan pathogen P. falciparum undergoes antigenic variation to establish persistent blood stage infection. Several clonally variant gene families undergo antigenic variation in P. falciparum and are expressed during blood stage infection at the surface of infected erythrocytes. One family encoded by 60 var genes expresses the major virulence adhesion surface molecule (called P. falciparum Erythrocyte Membrane Protein 1, PfEMP1) causing severe malaria (capillary blockages in the brain and other organs mediated by infected erythrocytes). The ‘clogging’ of blood vessel by infected RBC is strongly linked to P. falciparum pathogenesis. One peculiarity of this mechanism is that only one member of the 60 var genes is expressed (monoallelic expression). Switching expression between the 60-member var gene family avoids parasite immune clearance and prolongs the period of infection and transmission to the mosquito (for review see Scherf et al., 2008 Ann. Review Microbiol.; Scherf et al., 2008 Cell).
Distinct histone methylation determines the fate of var gene activity:Lopez-Rubio, JJ., Mancio Silva, L., Zhang, Q., Guizetti, J., Scheidig, C., Claes A.
After having demonstrated the key role of distinct types of histone methylation marks in var gene activation (H3K4me3), poised state (epigenetic memory) (H3K4me2/3) and silencing (H3K9me3)(Lopez-Rubio et al., 2007 Mol. Micro.) we showed for the first time, by using genome-wide ChIP analysis, that clonally variant gene expression share common epigenetic control mechanisms (Lopez-Rubio et al., 2009 Cell Host Microbes). In a very recent collaborative work of the L. Miller (NIH) laboratory, we participated in a study that revealed the key role of another histone methylation mark at position H3K36. Gene knock out of the corresponding SET gene (termed PfSETvs), results in the transcription of virtually all var genes analysed in the single parasite nucleus (Jiang et al., 2013 Nature). H3K9me and H3K36me are crucial for the default silencing of var genes, however, the interplay between the histone methyltransferases involved in this process remains to be determined.
We investigated whether nuclear pores associate with the var gene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore, P. falciparum Nup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Co-localization studies of var transcripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and the var gene expression site in ring stage. Our results indicate that P. falciparum does form compartments of high transcriptional activity at the nuclear periphery, which are, unlike the case in yeast, devoid of nuclear pores (Guizetti et al., 2013 Euk. Cell). We will investigate the biochemical nature of the expression site using new technologies.
New technology development:Lopez-Rubio JJ., Ghorbal M., McPherson C., Guizetti J., Claes A., Scheidig C.
Given the total absence of knowledge about the nature of the var expression site, we are developing in vivo promoter tagging techniques. We have started to develop the TetO elements and TetR fused to GFP to pull down the active var gene to study its molecular chromatin composition and to develop specific markers for this expression site. The very recent CRISPR/Cas9 system developed in the unit by the JJ. Lopez-Rubio team provides a novel powerful tool for genome editing and will be explored for various projects in the unit. A second inducible system to down-regulate mRNA based on a riboswitch element inserted at the 3’ UTR has been tested and appears to be a suitable tool to study essential gene function.
Fig. 2: Transmission electron micrograph of an iRBC (schizont). Nucleus shown in blue.
Nuclear biology and the identification of novel chromatin factors linked to the control of virulence gene expression and genome variability
Only few factors that interact with the DNA of P. falciparum have been identified and the recruitment of specific factors such as the plasmodial Sir2 and Set domain proteins to specific genes or chromosome compartments remains unknown. Gene relocation (active var gene) and spatial chromosome organization are key regulatory factors in gene expression, with however, few mechanistic insight yet.
Role of Sir2 in ribosomal RNAMancio Silva L., Lopez-Rubio JJ., Claes A.
The histone deacetylase PfSir2a is a crucial regulator of facultative heterochromatin and contributes to virulence gene family default silencing. We have observed that this NAD-dependent enzyme has additional important nuclear functions linked to the parasite virulence. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. We demonstrated that PfSir2a fine-tunes ribosomal RNA gene transcription. The synthesis of eukaryotic ribosomal RNA (rRNA) determines ribosome production and the potential for cell growth and proliferation. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide for the first insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence (Mancio Silva et al., 2013 Nature Communication).
Role of Sir2 in genome stability and diversity in P. falciparumLopez-Rubio JJ., Claes, A. Macpherson, C.
We started analysing a potential role of plasmodial sirtuins in subtelomere DNA stability. In PfSir2 knock out parasites, we observed major chromosome rearrangements at subtelomeres using karyotype analysis. Complete DNA re-sequencing of both mutant strains revealed genetic recombination events that are higher than in 3D7 wild type, pointing to a role of PfSir2-dependent heterochromatin in maintaining the integrity of the subtelomeric regions by suppressing their recombination in P. falciparum. Long-term culture follow-up experiments of clones are ongoing to determine the rate and type of DNA rearrangements. This novel association of PfSir2 to genetic diversity does raise the possibility that metabolic changes on NAD+/NADH, or other environmental factors that could modulate PfSir2 activity, may increase genetic diversity in order to enhance genetic parasite variants able to survive better stress situations.
PfAlbas constitute a new eukaryotic DNA/RNA-binding protein familyVembar, S. Claes, A., Scheidig, C.
In P. falciparum, perinuclear subtelomeric chromatin (histone deacetylase and methylase) conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends and recruit effector molecules are poorly described. We used the major subtelomeric DNA element called Telomere-Associated Repetitive Elements 6 (TARE6) to affinity purify novel chromatin components. 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 subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2. 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 (Chene A., Vembar S., et al., 2012 Nucleic Acid Res.). We have now overexpressed different Alba members episomally and observe striking changes in transcript levels, suggesting a role in either transcript stability or directly by controlling gene transcription levels.
An ApiAP2 member controls expression of clonally variant gene families in P. falciparumMartins, R., MacPherson C., Scheidig-Benatar C.
During the intraerythrocytic cycle, P. falciparum expresses on the surface of its host red blood cell different clonally variant gene families such as the var, rif, stevor, Pfmc2TM and surf. The role of the non-var surface molecules in disease is less apparent yet. To further investigate transcriptional regulation of virulence genes, we focused on putative transcriptional regulators (ApiAP2 members), whose binding sites were predicted to be enriched in the 5' UTR of var genes. Antibodies raised against one member of the ApiAP2 gene family called here AP2var14, showed perinuclear labelling, supporting a role in regulation of subtelomeric virulence genes. Transcriptional analysis of AP2var14 gene KO clones by RNA-Seq showed overexpression of genes (more than 4-fold) mainly derived from clonally variant gene families (rif, stevor, and Pfmc2TM). The interaction with promoter regions of these virulence genes with AP2var14 using ChIP analysis is ongoing.
Identification of parasite co-factors that are critical for adhesion of P. falciparum–infected RBC
Mattei D., Nacer A. & Harris, D.
Although PfEMP1/var is studied in many laboratories in the world, a number of key questions remain unknown. By analysing a mutant parasite (D10) with a chromosome 9 deletion and loss of adhesion, we linked the non-cytoadherence phenotype to the loss of 25 subtelomeric genes. However, in contrast to previous publications, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. In addition, we demonstrate for the first time the surface expression of PfEMP1 that has lost binding to receptors but is still recognized strongly by human hyperimmune serum similar to infected RBC that cytoadhere (Nacer et al., 2011 PLoS One). By carefully analysing the genes deleted on chromosome 9 of D10, 12 genes were selected as possible candidates and used for episomal complementation studies of D10. One single gene annotated as an open reading frame with no obvious homology to any known gene was able to restore adhesion. We termed this gene PfVAP1 (Virulence Associated Protein 1). To further validate this finding, an inducible protein knock down of the PfVAP1 gene was obtained for the strongly cytoadherent reference line FCR3. In conditions of reduced expression of PfVAP1, FCR3 parasite adhesion is to at least 60 to 70%. Further analysis of PfVAP1 is ongoing to investigate it’s biological role in this adhesion process.
Targeting epigenetic factors for new intervention strategies
Malmquist, N., Chen P., Ding S.
Our basic research on virulence gene control revealed that epigenetic factors such as histone methylation control antigenic variation but also the developmental progression of malaria parasites during the complex life cycle in the human host (Lopez-Rubio et al., 2009 Cell Host & Microbe). We hypothesized that this epigenetic mechanism could be a novel target that could block the immune evasion process and interfere with parasite development in general, depending on the inhibition of the histone methylation site. We investigated P. falciparum histone lysine methyltransferases (HKMT) as a potential target class for the development of novel antimalarials. In collaboration with Matt Fuchter (medicinal chemist, Imperial College London) who synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds (BIX-01294 and TM2-115) inhibit malaria parasite HKMTs, resulting in rapid and irreversible parasite death. Our data position HKMTs as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages (Malmquist et al., 2012 PNAS). We are now exploring the potential of the BIX compound libraries (>150 compounds) to specifically interfere with var gene repression via H3K9 methylation. More recently, we initiated a collaboration with Prof. Mazier (UPMC/INSERM UMR 945) to exploit the BIX compound on liver dormant stages (hypnozoites). The Mazier laboratory has developed a ground-breaking protocol for long-term in vitro cultivation of P. cynomolgi-infected primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation (Dembele et al., 2014 Nature Medicine).
P. berghei, a rodent malaria model to investigate host-parasite interactions central for malaria pathogenesis
Mecheri, S., Gueirard, P., Demarta-Gatgsi, C., Peronet, R.
During the past year, using a murine model for experimental cerebral malaria (ECM), we pursued our investigation regarding the contribution of the allergic inflammatory response in the pathogenesis of malaria disease. Following the demonstration of the critical role of the IgE/FceRI complex and the identification of neutrophils as key factors for the disease severity, we went on by identifying a parasite gene product, the Histamine Releasing Factor (HRF), secreted by P. berghei parasites. In this work, we showed that development of P. berghei liver stage lacking HRF, but not blood-stage, was delayed and was associated with an early rise in systemic IL-6, a cytokine that apparently suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF was sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. Our data support a role for HRF in the down-regulation of a cytokine with anti-parasite activity. In parallel, we examined the role of another parasite gene which encodes a highly pro-inflammatory protein termed High Mobility Group Box (HMGB), also secreted by P. berghei parasites. The pathogenesis of ECM was suppressed in C57BL/6 mice infected with PbANKA when its hmgb2 gene was deleted by homologous recombination, an effect associated with reduction of histological brain lesions and expression of several pro-inflammatory genes involved in the pathogenesis of ECM, leading to mouse survival. Altogether these data support the concept that the severity of the pathology results primarily from an inflammatory disease.
Updated on 23/06/2014
The unit is a member of the CNRS URA2581 unit and of the European Virtual Institute of Malaria Research, EVIMalaR (European Network of Excellence).
It is also a member of the French Network of Excellence on Parasitology (LabEx) ParaFrap whose scientific directors are Artur Scherf, Stan Tomavo and Frédéric Bringaud.
Biology of Host Parasite Interactions Unit
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