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, 1982. Protein Purification: Principles and Practice

% ammonium sulfate

0 40 60 70 8050crude extract

250160

105

75

50

35

30

25

1015

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 3 Only amaTryparedoxin peroxidase Only amaThiol-specific antioxidant protein (5 copies) Only amaIron Superoxide dismutase Only ama

tubulin Ama>pro 4.3x Only amatubulin Only ama

Ama>pro 4.2xIsocitrate 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 1b Only amaPossible 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 67

6855

86

Amastins are only found in Trypanosomatidae1member 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 A2 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

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