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Analysis of mutant parasites that have a defect in adhésion
- 1. ANALYSIS OF MUTANT PARASITES THAT HAVE A DEFECT IN ADHESION Denise MATTEI Cours International « Atelier Paludisme » 10 Mars au 18 Avril 1008 – Institut Pasteur de Madagascar Mar08
- 2. TRAFFICKING OF VIRULENCE FACTORS IN INFECTED RED BLOOD CELLS SEARCH FOR TRAFFICKING MUTANT PARASITES Mar08
- 3. other PfEMP1 molecules? Band 3 rifin Glycophorin C Ankyrin Adductin 4.1 Spectrin 4.2 4.9 Tropomyosin and Actin Tropomodulin Knob-associated proteins PfHRP-1 PfEMP-2 PfEMP-3 knobs Mar08
- 4. OBSERVATIONS CLAG = cytoadherence linked asexual gene Identified through the study of parasite lines that no longer bound to C32 melanoma cells = subtelomeric deletion in chr 9 (Biggs, BA et al. PNAS,1989) . Genetic knock-out (Trenholme, KR et al. PNAS, 2000) or antisense RNA inhibition Gardiner, DL et al. MBP, 2000) => loss of the CD36 binding phenotype . CLAG9 may be involved in cytoadhesion by binding directly to CD36 or indirectly, through a role in PfEMP1 transport Mar08
- 5. WORKING HYPOTHESIS Is CLAG9 another CD36 binding molecule? EXPERIMENTAL APPROACH • P. falciparum lines selected to bind to CD36 or to CSA. Adhesion to these receptors occurs in a mutually exclusive manner Receptor «panning » ⇒ possibility to study the role of CLAG9 in cytoadherent parasites that do not bind to CD36 CHO CSA CD36 monomorphic parasites Panned FCR3 FCR3 CSA FCR3 CD36 • Specific antibodies Scherf et al., 1998, EMBO J. 17, 5418 => to analyse the location of clag9 during the asexual blood stage development Mar08
- 6. Transcription of the clag9 gene The clag9 gene is transcribed in the schizont stages of both CD36 and CSA-selected parasites 3D7 - 9.49 - 7.46 - 4.40 hrs 2 6 10 14 18 22 26 30 34 38 42 46 Mar08
- 7. Clag9 is part of the RhopH complex in the rhoptries of merozoites r anti-clag9 mab7H8/50 nuclei merged FITC TRICT DAPI and is transferred into the ring-stage parasite r anti-clag9 mAb 4E10 merge DAPI TRICT Oregon green Ling et al 2004. Mol.Microbiol.52:107 Mar08
- 8. Localisation of CLAG9 to the rhoptries Clag9 RhopH2 Ling et al.2004. Mol.Microbiol. Mar08
- 9. CLAG9 • clag9 gene is transcribed and expressed in late asexual blood stages in CD36 and non-CD36 binders • Clag9 is present in merozoites as part of the RhopH complex in the rhoptries • Anti-CLAG9 antibodies do not detect it at the erythrocyte membrane of parasitized RBC (trophozoites/schizonts) Mar08
- 10. Analysis of clag9 natural mutant parasites T9-96 and D10 Mar08
- 11. T9-96 and D10 do not cytoadhere to any known adhesion receptor FCR3CD36 D10 C32 cells CD36 >> ICAM-1 >>CSA Mar08
- 12. OBSERVATIONS • T9-96 and D10 do not cytoadhere • The complex RhopH is detected in the ring stages HYPOTHESIS clag9 is necessary to modify the parasite’s and/or parasitophorous vacuole membrane EXPERIMENTAL APPROACH Analysis by immunofluorescence of clag9neg lines and FCR3CD36 Mar08
- 13. RESULTS The images were similar independently of the genotype FCR3CD36 D10 T9.96 PfEMP1 Pf332 DAPI merge Alexa TRICT ‘PfEMP1’ is located in association with the Maurer’s clefts Mar08
- 14. FCR3CD36 D10 T9.96 Mab89 DAPI merge Alexa CONCLUSION clag9neg parasites can export polypeptides to the different cellular compartments Mar08
- 15. OBSERVATIONS • ‘ PfEMP1’-like molecule is exported in association with Maurer’s clefts • ‘PfEMP1’ ? • Is it exposed on the red blood cell surface ? EXPERIMENTAL APPROACH • Labelling of red blood cell surface of clag9neg strains • Trypsin resistance Mar08
- 16. CLAG9neg lines express a PfEMP1 surface protein 3 CD36 FCR 6 B D10 A 100 µg Trypsin T9.9 0 10 •High molecular weight PfEMP1 •TX100-insol/SDS soluble PfEMP1 •Trypsin sensitive - 200kDa - T9.96 Mar08
- 17. OBSERVATIONS • The isolates T9.96 and D10 express PfEMP1 on the surface but do not cytoadhere to any known receptor HYPOTHESIS • Adhesion negative parasites express a FUNCTIONAL var gene having unique adhesive properties BUT can not switch expression to classical var genes • Adhesion negative parasites display a NON FUNCTIONAL conformation * Post-translational modification to establish a functional state (protease, kinase, etc…) EXPERIMENTAL APPROACH • Northern blot analysis • Cloning and expression of the CIDR domain BINDING ASSAYS Mar08
- 18. PfEMP1 is transcribed in the clag9neg parasite lines T9.96 and D10 D10 FCR3CD36 D10 FCR3CD36 R S R S T Kb R S R S T 8Kb - 9.49 - - 7.46 - - 4.40 - - 2.57 - - 1.35 - D10cl9 ATS Mar08
- 19. Functionality studies of the PfEMP1- CIDR domain in CD36 binding of T9-96 and D10 Mar08
- 20. A classical var gene is transcribed and expressed in mutant parasites Semi-conserved Tandem head structure association NTS DBL1α α CIDR1α α DBL2δ δ CIDR2β β TM ATS CR1 CD36 CD31 blood gr A CD31 heparin IgM hep sulfate ATS: acidic terminal segment; CIDR (α,β,γ): cisteine-rich interdomain region; DBL (α,β,γ,δ,ε): Duffy-binding-like domain; NTS: N-terminal segment; TM: transmembrane domain; Mar08
- 21. The CIDR domain from D10 is predicted to bind to CD36 M1 M2 M3 C9 C12 D10cl9 C L K NNKKTCGKKKCNRDCKCYE RW VKRKKEEFKKIKDHFGKQKD MQPYID PDMTLKILLNYVFLQ DMKDANG NPQHIAKIQELLE KKKVELEDNLNKNTIIDYMFEDDLEEINKC Robinson et al. 2003.Mol.Microbiol. 47:1265-78 al. 2003.Mol.Microbiol. 47:1265- Mar08
- 22. CD36 Binding Assays recombinant CIDR domains MC Cl4 PGEx M2 Cl6 MC Cl1 10 PGEX 2 CSA Cl15 M2 Cl6 2 MC Cl4 2 8 MC Cl1 2 6 4 2 0 100 50 25 12,5 6,25 3,125 1,56 0,781 g/ml Mar08
- 23. Adhesion on different purified receptors FCR3 CD36 on differents receptors CD36 ICAM qC1QR CSA CSC D10 on differents receptors CD36 ICAM qC1QR CSA CSC Mar08
- 24. Binding assays with Dynal beads Anti-CD36 labelled beads CD36 protein M2 domain on the surface CIDR domain (M2 minimum CD36 binding site)- pDisplay vector transfected in COS-7 cells Mar08
- 25. CD36 Binding Properties of D10 CIDR1 Transfected anti-tag COS7 cells merge HA D10 MC + control CSA (-) control Mar08
- 26. Conclusions • Cytoadhesion mutants express a PfEMP1 molecule at the surface of the parasitized red blood cell • The mutant parasites can not bind to the common adhesion phenotypes (CD36, ICAM-1, CSA, Rosetting, etc.) • Panning on endothelial cells does not restore adhesion phenotype Non-functional PfEMP1 surface expression ! M
- 27. Mar08
- 28. ATOMIC FORCE MICROSCOPY Mar08
- 29. RBC infected by Plasmodium falciparum Mar08
- 30. Control Mar08
- 31. 3D7 Parasitised red blood cell (PRBC) Knobs sizes ø 50-60 nm
- 32. FCR3 PRBC Knobs sizes ø 50-60 nm
- 33. D10 PRBC Amplitude error Height 1.5 µm Knobs sizes range from ø 20 nm to 100 nm Mar08
- 34. PFI1705w PFI1710w BPORF PFI1715w Centromere PFI1720w gig PFI1725w in mutant parasites Chromosome truncation PFI1730w clag9 PFI1735c rex1 PFI1740c rex2 PFI1745c PFI1750c PFI1755c rex3 PFI1760w rex4 PFI1765c PFI1770w PFI1775w PFI1780w PFI1785w PFI1790w PFI1795c PFI1800w PFI1805w rifin chromosome 9 deletion PFI1810w rifin D10 and T9.96 carry a large PFI1815c rifin PFI1820w PfEMP1 PFI1825w rifin PFI1830c PfEMP1 Telomere Mar08
- 35. HYPOTHESIS • Adhesion negative parasites display a NON FUNCTIONAL conformation * Post-translational modification to establish a functional state (protease, kinase, etc…) PERSPECTIVES * Clag 9 KO => clag9 functional role * other KO => chr9 region => genotype and phenotype analysis * SYSTEMS BIOLOGY => RNAs transcribed/translated ptn-ptn interactions cellular location Mar08
- 36. National Institute for BIHP (Institut Pasteur, Paris) Medical Research (Mill Hill, London, UK) Artur Scherf Tony Holder Suwanna Chaorattanakawe Irene T. Ling José Manuel L. Nogueira Caroline Davis Ehime University Christine Scheidig-Benatar Yvon Sterkers School of Medicine (Ehime, Japan) Emeric Roux Osamu Kaneko Parasitologie Expérimentale (Université de la Méditerranée, Marseille) Cellular Microbiology and Jürg Gysin Catherine Lepolard Infectious Pathogeny (Institute Pasteur Lille) Frank Lafont Sebastien Janel
- 37. ANALYSIS OF MUTANT PARASITES THAT HAVE A DEFECT IN ADHESION Denise MATTEI Cours International « Atelier Paludisme » 10 Mars au 18 Avril 1008 – Institut Pasteur de Madagascar Mar08
- 39. THE PfRhopH COMPLEX Clag 2 Clag 3.1 RhopH1/Clag Clag 3.2 PFI1730w - 155kDa Clag 8 Clag 9 RhopH Complex approx. 480 kDa RhopH2 PFI1445w - 140 kDa RhopH3 PFI0265c - 110 kDa Kaneko et al. 2005.MBP
- 40. Why the PfEMP1expressed by the ‘clagneg’ isolates is non functional ? HYPOTHESIS • wrong conformation ? • post-translational modification ? how can it be tested? apr07
- 41. Perspectives • A NEW var GENE HAVING UNIQUE ASPECTS? (non CD36, non CSA binding) AND/OR • A NEW HOST ENDOTHELIAL ADHESION RECEPTOR ? OR •A KNOWN PfEMP1 WHICH DISPLAYS A NON FUNCTIONAL CONFORMATION • clag9 KNOCK-OUT
- 43. Analysis of surface-exposed PfEMP1 on PRBC by trypsin cleavage N N R FCR3 CD36 R D10 kDa B B C C Trypsin g 1000 100 0 1000 100 0 175 83 ATS 62 HRP HRP HSP 70 47.5 HSP 70 32.5
- 44. Analysis of surface-exposed PfEMP1 on PRBC by trypsin and chymotrypsin cleavage kDa D10 N FCR3 CD36 N R R B B Trypsin C C Chymotrypsin g 0 100 100 0 100 100 175 83 62 ATS HRP HRP 47.5 HSP 70 HSP 70 32.5
- 45. Identification of natural clag9 mutant parasite lines A 36 CS CD 6 C 7 6 3 3 9-9 D7 NRB 3D T9-9 FCR FCR T 3 kDa T9-96 3D7 r anti-clag9 175 clag9 chr9 83 62 r PI 47.5 32.5 clag9 probe Mar08
- 46. Localisation of CLAG9 Clag9-GST 100 aa pep1 pep2 r anti-clag9 mab7H8/50 nuclei merged FITC TRICT DAPI Clag9 is encoded by members of the clag multigene family Ling,I et al. 2004. Mol.Microbiol. 52:107-118 Mar08
- 47. Rabbit anti-CLAG9 reacts with ring-infected erythrocytes r anti-clag9 mAb 4E10 merge DAPI TRICT Oregon green Mar08
- 48. Perspectives • A NEW var GENE HAVING UNIQUE ASPECTS? (non CD36, non CSA binding) AND/OR • A NEW HOST ENDOTHELIAL ADHESION RECEPTOR ? OR • A KNOWN PfEMP1 WHICH DISPLAYS A NON FUNCTIONAL CONFORMATION • clag9 KNOCK-OUT Mar08
- 49. Why PfEMP1 expressed by the ‘clag9neg’ isolates are non functional ? HYPOTHESIS * wrong conformation * post-translational modification Mar08