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:
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, 1982. Protein Purification: Principles and Practice
% ammonium sulfate
0 40 60 70 8050
crude extract
250160
105
75
50
35
30
25
10
15
kDa
PRO AMA
97 -66 -
45 -
31 -
21 -
14 -
Growth stage-dependent protein expression
pH 4.0 7.0 pH 4.0 7.0
specificaltered expression level
50-60% Ammonium sulfate fraction
HSP83 Only amaAma>pro 21x
HSP90 Only ama HSP70 Only ama
Ama>pro 16xDNAK-type molecular chaperone Only amaHsc70 heat-shock protein Only amaHeat-shock protein putative Only amaChaperonin 60.2 Ama>pro 11x Chaperonin 60
Only ama
Peroxidoxin 1 Ama>pro 18x Peroxidoxin 3Only ama
Tryparedoxin peroxidase Only amaThiol-specific antioxidant protein (5 copies) Only amaIron Superoxide dismutase Only ama
tubulin Ama>pro 4.3xOnly ama
tubulin Only amaAma>pro 4.2x
Isocitrate dehydrogenase Ama>pro 3.5xTriosephosphate isomerase Only ama Phosphomannomutase
Only amaElectron transfer flavoprotein a-subunit Only amaProstaglandin F-synthase Only amaEnolase Only amaSpermidine synthase Ama>pro 19X
Elongation factor 1-Only ama Elongation factor 1bOnly ama
Possible DEAD/DEAH box helicase Ama>pro 15X28 kDa guide RNA binding protein Only amaRab-14 Only amaeIF4E Only amaLeishmanolysin Only amaHypothetical protein CHR36_tmp.570 Only amaHypothetical protein CHR11_tmp.310 Only ama10 Hypothetical proteins Only ama
Differentially expressed proteins in Leishmania infantum amastigotes
AMASTIN GENE FAMILY IN LEISHMANIA
100
10070
95
60100
10087
83
88
10090
100
7877
6273
69
93
100 94
100
10081
59
65
5564
10054
7468
100
100
94
65
99
100
100
100
100
100
100
8074
6768
55
86
Amastins are only found in
Trypanosomatidae
1member in T. brucei1 member in T. cruzi~ 45 members in Leishmania spp.
Surface proteins
Stage-specific
Species-specific
Unknown function
OUT
IN CN
C CTW G
Putative functional domain: CXTLWGXXXXC
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 18S rRNA
Ldi-3425
18S rRNA
P A
Ldi-3651 orf2
Ldi-1804
18S rRNA
P A
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 A4 5 6 7 4 5 6 7
Ldi-1234
P A
18S rRNA18S rRNA 18S rRNA 18S rRNA
18S rRNA
Ldi-3133
P A P A
Ldi-3651
Amastin gene homologues can be regulated in a species-specific manner
LmjF34.0960LmjF34.1080
LmjF34.1840
P A 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
Amastin gene homologues
% of identity with the amastin
3’UTR 450 nt
150 nt
330 nt
CHR34_tmp.0960 83% 76% 88% 45% CHR34_tmp.0500 84% 77% 88% 40%
LmjF31.0450 55% 74% 70% 44% LmjF08.0720 45% 66% * 68% 45% LmjF08.0670 45% 66% * 68% 42%
34-175 53% 74% 63% 46%
LmjF08.0700 50% 72% 52% 45% 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%
LmjF24.1270 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’UTRsof the amastin gene homologues
-Known Leishmania amastigote-specific genes: A2, 5’ A2rel, HSP100, Histone H1
-Surface proteins: Several members of the amastin surface proteinsABC transportersGlycoproteins
-Metabolism: Fatty acid beta-oxidationOxidative pentose phosphate pathway (Glycolysis)
-Signaling: cAMP-dependent protein kinase A subunitDAG kinaseG-protein coupled receptor
-Regulation of gene expression: Spliceosome assemblyTranslation 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