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

-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:

REGULATION OF GENE EXPRESSION IN LEISHMANIA

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

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

Salting out fractionation R.K.Scopes, Protein Purification: Principles and Practice % ammonium sulfate crude extract kDa

PRO AMA Growth stage-dependent protein expression pH pH specific altered expression level 50-60% Ammonium sulfate fraction

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

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 OUT IN C N C C T W G Putative functional domain: CXTLWGXXXXC

Amastin gene homologues are differentially expressed in the intracellular stage of L. infantum P A P A S rRNA Ldi S rRNA P A Ldi-3651 orf2 Ldi S rRNA P A Ldi-3487 P A Ldi-3491 Ldi Ldi-2012 P A Ldi-3393orf4 P A Ldi-3607 P A Ldi-1234 P A 18S rRNA Ldi-3133 P A Ldi-3651

Amastin gene homologues can be regulated in a species-specific manner LmjF LmjF LmjF P A LmjF P A LmjF P A LmjF P A LmjF P A LmjF P A LmjF / Ldi-2012orf2 P A 18S rRNA LmjF / Ldi-3670 P A 18S rRNA LmjF / Ldi-2812 P A 18S rRNA LmjF P A 18S rRNA LmjF P A 18S rRNA LmjF / Ldi-3491 P A 18S rRNA LmjF / Ldi-849 P A 18S rRNA L. infantum L. major

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

- 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

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