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ANALYSE DE L’EXPRESSION DE GÈNES STADE-SPÉCIFIQUES CHEZ LE PARASITE PROTOZOAIRE LEISHMANIA Barbara Papadop

42ème Réunion du Groupement des Protistologues de Langue Française. ANALYSE DE L’EXPRESSION DE GÈNES STADE-SPÉCIFIQUES CHEZ LE PARASITE PROTOZOAIRE LEISHMANIA Barbara Papadopoulou, Ph.D.

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ANALYSE DE L’EXPRESSION DE GÈNES STADE-SPÉCIFIQUES CHEZ LE PARASITE PROTOZOAIRE LEISHMANIA Barbara Papadop

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  1. 42ème Réunion du Groupement des Protistologues de Langue Française ANALYSE DE L’EXPRESSION DE GÈNES STADE-SPÉCIFIQUES CHEZ LE PARASITE PROTOZOAIRE LEISHMANIA Barbara Papadopoulou, Ph.D. François McNicoll, Maxime Laverdière, Annie Rochette, Michaela Müller, Serge Cloutier Centre de Recherche en Maladies Infectieuses du Centre Hospitalier de l’Université Laval, CHUQ, Québec, Canada

  2. Goals: -IDENTIFY STAGE-SPECIFIC GENES/PROTEINS AND CIS-ACTING REGULATORY ELEMENTS IN LEISHMANIA -Comparative proteomics -In silico screening -STUDY THE MECHANISMS CONTROLLING STAGE-SPECIFIC REGULATION OF GENE EXPRESSION -STUDY THE MECHANISMS OF TRANSLATIONAL CONTROL IN RESPONSE TO INTRACELLULAR STRESS

  3. REGULATION OF GENE EXPRESSION IN LEISHMANIA

  4. The Leishmania genome is predicted to encode ~8,000 ORFs DNA microarray analysis: ~2% of genes are differentially expressed (Akopyants et al., MBP 2001; Saxena et al., MBP 2003) Proteomics data:~ 8% of the proteins are differentially expressed in amastigotes (L. infantum,El-Fakhry et al., 2002, Proteomics;L. mexicana, Nugent et al., MBP 2004; L. donovani, Bente et al., Proteomics 2003) Sequences in the 3'UTRs determine mRNA abundance, stability, and translation rates The molecular mechanisms of stage-specific translational control in Leishmania are still poorly understood STAGE-REGULATED GENE EXPRESSION IN LEISHMANIA

  5. The Leishmania genome is predicted to encode ~8,000 ORFs DNA microarray analysis: ~2% of genes are differentially expressed (Akopyants et al., MBP 2001; Saxena et al., MBP 2003) Proteomics data: 8%-10% of the proteins are differentially expressed in amastigotes (L. infantum,El-Fakhry et al., 2002, Proteomics; L. mexicana, Nugent et al., MBP 2004; L. donovani, Bente et al., Proteomics 2003) Sequences in the 3'UTRs determine mRNA abundance, stability, and translation rates The molecular mechanisms of stage-specific gene expression in Leishmania are still poorly understood STAGE-REGULATED GENE EXPRESSION IN LEISHMANIA

  6. Salting out fractionation % ammonium sulfate crude extract 0 40 50 60 70 80 kDa 250 160 105 75 50 35 30 25 15 10 R.K.Scopes, 1982. Protein Purification: Principles and Practice

  7. pH 4.0 pH 4.0 7.0 7.0 97 - 66 - 45 - 31 - 21 - 14 - Growth stage-dependent protein expression PRO AMA specific 50-60% Ammonium sulfate fraction altered expression level

  8. Differentially expressed proteins in Leishmaniainfantum amastigotes HSP83 Only ama Ama>pro 21x HSP90 Only ama HSP70 Only ama Ama>pro 16x DNAK-type molecular chaperone Only ama Hsc70 heat-shock protein Only ama Heat-shock protein putative Only ama Chaperonin 60.2 Ama>pro 11x Chaperonin 60 Only ama Peroxidoxin 1 Ama>pro 18x Peroxidoxin 3 Only ama Tryparedoxin peroxidase Only ama Thiol-specific antioxidant protein (5 copies) Only ama Iron Superoxide dismutase Only ama b-tubulin Ama>pro 4.3x Only ama -tubulin Only ama Ama>pro 4.2x Isocitrate dehydrogenase Ama>pro 3.5x Triosephosphate isomerase Only ama Phosphomannomutase Only ama Electron transfer flavoprotein a-subunit Only ama Prostaglandin F-synthase Only ama Enolase Only ama Spermidine synthase Ama>pro 19X Elongation factor 1- Only ama Elongation factor 1bOnly ama Possible DEAD/DEAH box helicase Ama>pro 15X 28 kDa guide RNA binding protein Only ama Rab-14 Only ama eIF4E Only ama Leishmanolysin Only ama Hypothetical protein CHR36_tmp.570 Only ama Hypothetical protein CHR11_tmp.310 Only ama 10 Hypothetical proteins Only ama

  9. 100 70 100 95 60 100 100 87 83 88 90 100 100 69 78 77 62 93 73 94 100 100 81 100 59 65 64 55 100 54 G W 100 74 68 T 100 OUT C C 100 86 65 100 94 100 100 99 100 100 80 67 74 68 55 N C IN AMASTIN GENE FAMILY IN LEISHMANIA Amastins are only found in Trypanosomatidae 1member in T. brucei 1 member in T. cruzi ~ 45 members in Leishmania spp. Surface proteins Stage-specific Species-specific Unknown function Putative functional domain: CXTLWGXXXXC

  10. P A P A P A P A P A P A 3 4 5 7 3 4 5 7 3 4 5 7 3 4 5 7 4 5 6 7 4 5 6 7 5 6 4 5 6 Ldi-1804 Ldi-3393orf4 Ldi-3607 Ldi-3487 Ldi-2012 Ldi-1234 18S rRNA 18S rRNA 18S rRNA 18S rRNA 18S rRNA P A P A P A P A 2 3 4 5 7 3 4 5 7 P A P A 3 4 5 5 7 2 3 4 5 7 3 4 5 7 Ldi-3651 Ldi-3133 Ldi-3651 orf2 Ldi-3491 Ldi-3425 Ldi-849 18S rRNA 18S rRNA 18S rRNA 18S rRNA Amastin gene homologues are differentially expressed in the intracellular stage of L. infantum

  11. P A P A P A P A P A P A P A LmjF34.0960 LmjF34.1080 LmjF36.1270 LmjF34.1580 LmjF31.0450 LmjF34.1840 LmjF30.0870 LmjF24.1270 P A P A P A P A P A P A P A LmjF34.1580 LmjF36.1270 LmjF24.1260/ Ldi-2012orf2 LmjF34.0960/ Ldi-849 LmjF30.0870/ Ldi-2812 LmjF34.1840/ Ldi-3491 LmjF31.0450/ Ldi-3670 18S rRNA 18S rRNA 18S rRNA 18S rRNA 18S rRNA 18S rRNA 18S rRNA Amastin gene homologues can be regulated in a species-specific manner L. major L. infantum

  12. Amastin gene % of identity with 450 nt 150 nt 330 nt LmjF08.0720 45% 66% * 68% 45% homologues the amastin LmjF08.0670 45% 66% * 68% 42% 3’UTR CHR34_tmp.0960 83% 76% 88% 45% 34 - 175 53% 74% 63% 46% CHR34_tmp.0500 84% 77% 88% 40% LmjF08.0700 50% 72% 52% 45% LmjF31.0450 55% 74% 70% 44% LmjF08.0710 50% 72% 57% 45% CHR34_tmp.1660 57% 69% * 62% 43% 32 - 445 54% 69% * 62% 45% LmjF36.1270 54% 69% * 62% 43% CHR34_tmp.1680 54% 70% 62% 43% 34 - 85 57% 69% 40% 38% LmjF24.127 0 42% 41% 47% 100% LmjF24.1260 38% 39% 40% 100% LmjF24.1250 40% 41% 44% 41% CHR34_tmp.0970 37% 36% 46% 41% Conserved elements are found within the 3’UTRs of the amastin gene homologues

  13. BLAST search (450 nt 3’UTR element) -3’ end of ~100 L. major ORFs (65-79% identity) 450-nt amastin 3'UTR 5' amastin ORF -Known Leishmania amastigote-specific genes:A2, 5’ A2rel, HSP100, Histone H1 -Surface proteins: Several members of the amastin surface proteins ABC transporters Glycoproteins -Metabolism: Fatty acid beta-oxidation Oxidative pentose phosphate pathway (Glycolysis) -Signaling: cAMP-dependent protein kinase A subunit DAG kinase G-protein coupled receptor -Regulation of gene expression: Spliceosome assembly Translation initiation (eIF-4E) -Chaperones -Motor proteins -Proteins involved in cell adhesion Boucher et al., JBC 2003

  14. BLAST search(150 nt 3’UTR element) 3’UTR of ~20 L. major ORFs (65-83% identity) 5' 3'UTR amastin 150 nt -Hypothetical proteins -Cell wall surface anchor family -Several amastin gene homologues

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