1. 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 42ème Réunion du Groupement des Protistologues de Langue Française

2. -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 Goals:

3. REGULATION OF GENE EXPRESSION IN LEISHMANIA

4. STAGE-REGULATED GENE EXPRESSION IN LEISHMANIA 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 The Leishmania genome is predicted to encode ~8,000 ORFs

5. STAGE-REGULATED GENE EXPRESSION IN LEISHMANIA 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 The Leishmania genome is predicted to encode ~8,000 ORFs

6. Salting out fractionation R.K.Scopes, 1982. Protein Purification: Principles and Practice % ammonium sulfate 04060708050 crude extract 250 160 105 75 50 35 30 25 10 15 kDa

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

8. HSP83Only ama Ama>pro21x HSP90Only ama HSP70Only ama Ama>pro16x DNAK-type molecular chaperoneOnly ama Hsc70 heat-shock proteinOnly ama Heat-shock protein putativeOnly ama Chaperonin 60.2Ama>pro11x Chaperonin 60 Only ama Peroxidoxin 1Ama>pro18x Peroxidoxin 3Only ama Tryparedoxin peroxidaseOnly ama Thiol-specific antioxidant protein (5 copies)Only ama Iron Superoxide dismutase Only ama  tubulinAma>pro4.3x Only ama  tubulin Only ama Ama>pro4.2x Isocitrate dehydrogenase Ama>pro3.5x Triosephosphate isomerase Only ama Phosphomannomutase Only ama Electron transfer flavoprotein a-subunitOnly ama Prostaglandin F-synthaseOnly ama EnolaseOnly ama Spermidine synthaseAma>pro19X Elongation factor 1 -  Only ama Elongation factor 1 bOnly ama Possible DEAD/DEAH box helicaseAma>pro15X 28 kDa guide RNA binding proteinOnly ama Rab-14Only ama eIF4EOnly ama LeishmanolysinOnly ama Hypothetical protein CHR36_tmp.570Only ama Hypothetical protein CHR11_tmp.310Only ama 10 Hypothetical proteinsOnly ama Differentially expressed proteins in Leishmania infantum amastigotes

9. AMASTIN GENE FAMILY IN LEISHMANIA 100 70 95 60 100 87 83 88 100 90 100 78 77 62 73 69 93 100 94 100 81 59 65 55 64 100 54 74 68 100 94 65 99 100 80 74 67 68 55 86 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 OUT IN C N C C T W G Putative functional domain: CXTLWGXXXXC

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

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

12. Conserved elements are found within the 3’UTRs of the amastin gene homologues

13. - 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 5' 450-nt amastin ORF amastin 3'UTR -3’ end of ~100 L. major ORFs (65-79% identity) BLAST search (450 nt 3’UTR element) Boucher et al., JBC 2003

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