geographical distribution 14 million cases (2 million new cases each year), distributed
TRANSCRIPT
Geographical Distribution
http://www.who.int/emc-documents/surveillance/docs/whocdscsrisr2001.html
14 million cases (2 million new cases each year), distributed in 88 countries (350 million people at risk).
cutaneous leishmaniasis
visceral leishmaniasis
Clinical syndromes
Cutaneous leishmaniasis – L. braziliensis, L. amazonensis, L.
guyanensisL. major
Diffuse leishmaniasis – L. amazonensis
Mucocutaneous leishmaniasis – L. braziliensis
Visceral leishmaniasis – L. chagasi, L. donovani
New World species Old World species
Clinical picture
AIDS(Persistence)
Chemotherapy
Gene regulation
Virulence
Sacks, David L. (2001) Cellular Microbiology 3 (4), 189-196.
Development in the insect vector
Stage-regulated gene expression
2.1 kb
L S
rRNA
Characterisation of the meta 1 gene in L. major
- 12 kDa, pI 9.7
- expressed in promastigotes, upregulated in
metacyclics
- localises to vacuoles close to the
flagellar pocket
- conserved in all Leishmania
species
4 6 8 10 12 14 16
5
10
15
20
Les
ion
Ind
ex
Weeks
BALB/c
WT ARHyg Hyg3’ HE
A
BALC/c
4 6 8 10 12 14 16
5
10
15
20
25
Les
ion
Ind
ex
Weeks
WT HE
B
Meta 1 overexpressor in vivo
promastigotes
amastigotes
0
100
200
300
400
500
600
PB
S
pVX
OR
F-1
pVX
-mcp3
pV
X-M
1
pV
X-m
cp
3-
M1
Co
nce
ntr
atio
n (
ng
/mL
)
IgG1
IgG2a
**
Meta 1 DNA Vaccine
meta 1 is a strong Th2 inducer
L. major
L. amaz.
L. donovani
META domain: Small domain family found in proteins of of unknown function. Some are secreted and implicated in motility in bacteria. Also occurs in Leishmania spp. as an essential gene. Over-expression in L.amazonensis increases virulence. A pair of cysteine residues show correlated conservation, suggesting that they form a disulphide bond. Taxa: cellular organisms
M1
7,46
9,49
4,40
2,37
Probe B
L S A
1,8
2,2
2,0
Kb
Probe A
L S A
2,37
Probe M2 L S A
1,35
0,24
Kb
Probe K2
L S A Kb
1,8
2,0
Probe K1
L S A 2,1
Probe M1
L S A
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
contig Lm572 L. major (Sanger Centre)
M1
CARD
M2
Acyltransferase Helicase
?Lariat-deb Pseudomonas
?
META2
META2
META1 NOD
Acyltransf U5
VISTAUKHuman Genome Mapping Project Resource CentreFunded by the MRC
EMBOSS (European Molecular Biology Open Software) http://www.hgmp.mrc.ac.uk/
0
20
40
60
80
100
1 3 5 7 9
11 13 15 17 19
-250
-200
-150
-100
-50
0La gaps
Lm gaps
ORF
UTR/IG
Nt Similarity (%)
Gaps
calculated by Point Replacer v.2.2 (www.geocities.com/alvesjp/software)
Conserved synteny and linkage in Leishmania
Conserved synteny and linkage in trypanosomatids
B
A
1 50 100 150 200 250 300 350 400 444
META META META
M2a M2b M2c
M2
m1 -MEMKSLIGKRRVLSVNGRPAPAG--VTVEFKASENSGTIQMHANVANFMNGQLKLGNRK 57 m2a MTTADDVCGTYTLSHCDGKVTPAK--ATLTIHRCG--ETLTAHATVANDLRGTVQYENCH 56 m2b -WKLSDLNGEHAIIAINDQSPNQE--MTIGFTPDG-NGGSFVTANIANSLRGNCQIDAGL 166 m2c -VSQSDLEGEYVLKSFNGAAVPTRNQPSIVFKPVN-TNEVEISIVVANRIRGTAALNQNV 278 m1 LSG--TLVSTMMLGSDDLMNIENALIQGFMDGMTYTVHDGGKLTLKSNTHTIKLVPA 112 m2a IVG--SLHSTGNEASPAQESVEQALSKGFADGFNVVVEIN-QVLLKNANSSFVFERS 110 m2b LRG--DLATTQGKADESSMQVEKVISEGFQQGFHVCTNDS-GILLQSSEANIQLCRI 220 m2c LSSEEPLMSTRMVGTEEESQLEGAFNVGFQYGLETISHGN-ELTLKNQDCEFV---- 330
A B C
Pré
-im
un
eA
nti
- M
BP
An
ti-
M2
/MB
P
L a. WT
L a. M2
L.a. M2/GFP
Phase contrast DAPI -Meta 2 e-GFP
m1/1-117 : 2c/1-117 : 2a/1-117 : 2b/1-117 : calpastati :
* 20 * 40 * 60-MEMKSLIGKRRVLSVNGRPAPAG--VTVEFKASENSGTIQMHANVANFMNGQLKLGNRK-VSQSDLEGEYVLKSFNGAAVPTRNQPSIVFKPVN-TNEVEISIVVANRIRGTAALNQNVMTTADDVCGTYTLSHCDGKVTPAK--ATLTIHRCG--ETLTAHATVANDLRGTVQYENCH-WKLSDLNGEHAIIAINDQSPNQE--MTIGFTPDG-NGGSFVTANIANSLRGNCQIDAGL--------------------------------------------------SG---MDAAL
: 57 : 58 : 56 : 56 : 7
m1/1-117 : 2c/1-117 : 2a/1-117 : 2b/1-117 : calpastati :
* 80 * 100 * LSG--TLVSTMMLGSDDLMNIENALIQGFMDGMTYTVHDGGKLTLKSNTHTIKLVPALSSEEPLMSTRMVGTEEESQLEGAFNVGFQYGLETISHGN-ELTLKNQDCEFV----IVG--SLHSTGNEASPAQESVEQALSKGFADGFNVVVEIN-QVLLKNANSSFVFERSLRG--DLATTQGKADESSMQVEKVISEGFQQGFHVCTNDS-GILLQSSEANIQLCRILDD---LIDT--LGGPEETEEENTTYTGPEVS-------------------------
: 112 : 110 : 110 : 110 : 34
A
C
B INIBIÇÃO
Conclusions
• Meta 1 and meta 2 genes- conserved in Leishmania, synteny with Trypanosoma
• Meta 1 related to virulence; Meta 2 ?
• Meta genes: several copies in Trypanosoma – evaluate function in T. brucei (RNAi)
• Meta 2 protein: Calcium? regulation of calpain activity?
• Meta locus: other stage-regulated genes - virulence ?
Camila S. RamosFernando A. L. FrancoAmanda R. FrancoMariana WajcCarlos H. SerezaniJenicer K. U. Yokoyama-YasunakaSilvia R. B. Uliana
Deborah F. Smith
ICB - USP
Imperial College University of London