a immune-response molecule which limits trypanosome infection of tsetse midguts. mike lehane...
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A immune-response molecule A immune-response molecule which limits trypanosome which limits trypanosome
infection of tsetse midguts.infection of tsetse midguts.
Mike LehaneMike Lehane
Liverpool School of Tropical MedicineLiverpool School of Tropical Medicine
MetacyclogenesMetacyclogenesisis
Midgut Midgut establishmenestablishmentt
1.
Complex differentiation to Complex differentiation to asymmetric dividing asymmetric dividing epimastigoteepimastigote + migration+ migration
2.
3.
T. bruceiT. brucei development in the development in the tsetse flytsetse fly
Trypanosome numbers (Log10) in the fly
0
1
2
3
4
5
6
7
1 3 5 7 9 11 13 15
Days
Log
Successful infection
Midgut Cardia Salivary Gland
Trypanosome numbers (Log10) in the fly
0
1
2
3
4
5
6
7
1 3 5 7 9 11 13 15
Days
Log
Infection fails in the midgut
Log trypanosome numbers in the fly
0
1
2
3
4
5
6
7
1 3 5 7 9 11 13 15
Days
Log
Prevalence of Trypanosome Infection vs Infective Bloodmeal
0
10
20
30
40
50
60
70
% t
ryp
ano
som
e p
reva
len
ce
1st 2nd 3rd 4th
Mean +/- S.E.M.
70
10
>90% of laboratory midgut
Infections fail
Midgut infection success rates
30+
Blood meal
The fly immune system affects trypanosome
establishment in the midgut
0
10
20
30
40
50
60
1 PBS LPS E.coli
Try
pano
som
e pr
eval
ence
NS NS NS
M.luteus PTG Laminarin
P<.0.01 P< 0.05
GlossinaGlossina gene discovery gene discovery (EST programs)(EST programs)
TissueTissue ReadsReads Cluster/Cluster/SingletonSingleton
MidgutMidgut 21,42721,427 7,0157,015
Salivary Salivary glandgland
27,42627,426 7,4937,493
Fat BodyFat Body 20,25720,257 6,3726,372
TOTALSTOTALS 69,11069,110 ~ 10,000~ 10,000
LSTM, Sanger, Yale and TIGRLSTM, Sanger, Yale and TIGR
Head library and fat body library will soon be available within GeneDB making the EST much more useful to the research community
Development of suitable tools Development of suitable tools (RNAi)(RNAi)
Injection of : Injection of :
1. TsetseEP dsRNA1. TsetseEP dsRNA2. NFW2. NFW3. non-injected3. non-injected
1 2 3
TsetseEP
GAPDH Gmm2412Gmm2412
GAPDHGAPDH
1 2 3 1 2 3
Injection of : Injection of :
1. Gmm2412 dsRNA1. Gmm2412 dsRNA2. Ampicillin dsRNA2. Ampicillin dsRNA3. PBS3. PBS
Gmm2412Gmm2412
GAPDHGAPDH
Injection of : Injection of :
1. Gmm2412 dsRNA1. Gmm2412 dsRNA2. NFW2. NFW
1 2 1 2
Trypanosoma brucei EP procyclin
TsetseEP
3.21
2.78
6.826.56
1.34
0
10
20
30
40
50
60
70
80
Infective Blood meal
Infe
ctio
n p
reva
len
ce (
%)
0
1
2
3
4
5
6
7
8
1 3 4 5 10
Fold
differen
ce
\
Trypanosome prevalence following knockdown (RNAi) of tsetseEP
Where is TsetseEP produced?
Is TsetseEP immune Is TsetseEP immune responsive?responsive?
2D gels of midgut protein:
A. 3 days post injection with PBS
B. 3 days post injection with live E. coli K12RM148
savannah riverine forest
TsetseEP is present in all tsetse species investigated and is
unique to Glossina.
Infected at indicated blood meal
Number of biological replicates Mean percentage infected following dsTsetseEPinjection (number of flies)
Mean percentage infectedfollowing dsAmp controlinjection (number of flies)
1 1 30.00 (30) 6.67 (15)
G. palpalis
SummarySummary The tsetse fly immune response The tsetse fly immune response
molecule TsetseEP affects the ability molecule TsetseEP affects the ability of trypanosomes to become of trypanosomes to become established in the midgut of tsetse established in the midgut of tsetse flies.flies.
LSTMLee HainesStella LehaneDeidre Walshe
UVICTerry Pearson
Bristol Wendy GibsonLori Peacock
YaleSerap Aksoy
SangerMatt Berriman
TIGR / LiverpoolNeil Hall
Candidate GenesCandidate GenesMidgut lectinsMidgut lectins
21 genes with carbohydrate recognition domains in EST libraries. 21 genes with carbohydrate recognition domains in EST libraries.
Gmm2412 cType signal sequenceGmm2412 cType signal sequence
Gmm3236Gmm3236 cType signal sequence cType signal sequence
Gmm9056Gmm9056 galectin no-signal sequence galectin no-signal sequence
TsetseEPTsetseEP M. Chandra, M. Liniger, L. Tetley, I. Roditi, J. D. Barry, M. Chandra, M. Liniger, L. Tetley, I. Roditi, J. D. Barry, Insect Insect
Biochemistry And Molecular BiologyBiochemistry And Molecular Biology 34, 1163 (Nov, 2004). 34, 1163 (Nov, 2004).
Trypanosome prevalence following knockdown (RNAi) of Lectins
Injection Infected at
indicated blood meal
Number of biological replicates
Total number of flies
Overall percentage prevalence of infection
± S.E.
P value
dsAmpicillin control
3 7 149 10.58 ± 3.00
dsGmm2412 3 6 91 9.29 ± 2.74 NS
dsGmm3236 3 4 65 8.24 ± 5.17 NS
dsGmm9056 3 3 85 10.15 ± 5.08 NS
Gmm2412 no effectGmm2412 no effect
Gmm3236 no effectGmm3236 no effect
Gmm9056 no effectGmm9056 no effect
Trypanosome prevalence following knockdown (RNAi) of Lectins
TsetseEP strong effectTsetseEP strong effect
Infected at indicated blood meal
Number of biological replicates Mean percentage infected following dsTsetseEPinjection (total number of flies)
Mean percentage infectedfollowing dsAmp controlinjection (total number of flies)
P value
1♦ 3 72.0 (100)
54.3 (108)
0.042
3 3 15.1 (180)
5.6 (113)
0.020
4 2 37.1 0.4(62)
12.03 2.28(60)
NA
5 2 46.6 21.60(50)
10.65 8.15(56)
NA
10 1 18.8(32)
2.9(35)
NA
Trypanosoma brucei EP procyclin
MTTTMSRVLHLMTVTLLCARVGMGQASDDDDCGGQSIPQKVEEVQTMCDVVQQLRALETASQSAVASVVAFAREASEAKERAEKAVERAKSKKRGVDAATEAAARATAAAQRAETVVSDARKHAADLTAASKDAIETADESLRLLATCEKADEPIRTAAEKCTGAAAEVTSKSLESAFDALAELLPDGADEIREHGAVFVVGLKSLEDDV RTAGEAKSEAEKAEGDANDAADGARAVLTGVCVLLLLAALH
Trypanosoma congolense GARP procyclin
TsetseEP
Binding to the trypanosome surface?
X
Antibody
Does TsetseEP knockdown Does TsetseEP knockdown affect the prevalence of affect the prevalence of T. T. congolense?congolense?
Infected atblood meal
Mean percentage infected following dsTsetseEPinjection (number of flies)
Mean percentage infectedfollowing dsAmp controlinjection (number of flies)
3 33.33 (36) 9.50 (21)