A immune-response molecule which limits trypanosome infection of tsetse midguts.

1
A immune-response molecule which limits
trypanosome infection of tsetse midguts.

• Mike Lehane
• Liverpool School of Tropical Medicine

2
T. brucei development in the tsetse fly
3.
Metacyclogenesis
3
Successful infection
4
Infection fails in the midgut
5
gt90 of laboratory midgut Infections fail
Midgut infection success rates
70
10
30
Blood meal
6
The fly immune system affects trypanosome
establishment in the midgut
NS NS NS
Plt.0.01 Plt 0.05
Trypanosome prevalence
PBS
LPS E.coli
M.luteus PTG Laminarin
7
Glossina gene discovery (EST programs)
Tissue Reads Cluster/Singleton
Midgut 21,427 7,015
Salivary gland 27,426 7,493
Fat Body 20,257 6,372
TOTALS 69,110 10,000
Head library and fat body library will soon be
available within GeneDB making the EST much
more useful to the research community
LSTM, Sanger, Yale and TIGR
8
Development of suitable tools (RNAi)

Injection of 1. TsetseEP dsRNA 2. NFW 3.
non-injected
Injection of 1. Gmm2412 dsRNA 2. NFW
1 2
Gmm2412 GAPDH
1 2 3
TsetseEP GAPDH
9
Trypanosoma brucei EP procyclin
TsetseEP
10
Trypanosome prevalence following knockdown (RNAi)
of tsetseEP
11
Where is TsetseEP produced?
12
Is TsetseEP immune responsive?

• 2D gels of midgut protein
• A. 3 days post injection with PBS
• B. 3 days post injection with live E. coli
  K12RM148

13
TsetseEP is present in all tsetse species
investigated and is unique to Glossina.
savannah riverine forest
G. palpalis
Infected at indicated blood meal Number of biological replicates Mean percentage infected following dsTsetseEP injection (number of flies) Mean percentage infected following dsAmp control injection (number of flies)
1 1 30.00 (30) 6.67 (15)
14
Summary

• The tsetse fly immune response molecule TsetseEP
  affects the ability of trypanosomes to become
  established in the midgut of tsetse flies.

15
LSTM Lee Haines Stella Lehane Deidre
Walshe UVIC Terry Pearson Bristol Wendy
Gibson Lori Peacock Yale Serap
Aksoy Sanger Matt Berriman TIGR /
Liverpool Neil Hall

16
Candidate GenesMidgut lectins21 genes with
carbohydrate recognition domains in EST
libraries.

• Gmm2412 cType signal sequence
• Gmm3236 cType signal sequence
• Gmm9056 galectin no-signal sequence

• TsetseEP M. Chandra, M. Liniger, L. Tetley, I.
  Roditi, J. D. Barry, Insect Biochemistry And
  Molecular Biology 34, 1163 (Nov, 2004).

17
Trypanosome prevalence following knockdown (RNAi)
of Lectins
Gmm2412 no effect Gmm3236 no effect Gmm9056
no effect
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
18
Trypanosome prevalence following knockdown (RNAi)
of Lectins
TsetseEP strong effect
Infected at indicated blood meal Number of biological replicates Mean percentage infected following dsTsetseEP injection (total number of flies) Mean percentage infected following dsAmp control injection (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
19
Trypanosoma brucei EP procyclin
Trypanosoma congolense GARP procyclin
MTTTMSRVLHLMTVTLLCARVGMGQASDDDDCGGQSIPQKVEEVQTMCD
VVQQLRALETASQSAVASVVAFAREASEAKERAEKAVERAKSKKRGVDAA
TEAAARATAAAQRAETVVSDARKHAADLTAASKDAIETADESLRLLATC
EKA DEPIRTAAEKCTGAAAEVTSKSLESAFDALAELLPDGADEIREHGA
VFVVGLK SLEDDV RTAGEAKSEAEKAEGDANDAADGARAVLTGVCVLL
LLAALH
20
Binding to the trypanosome surface?
TsetseEP
Antibody
X
21
Does TsetseEP knockdown affect the prevalence of
T. congolense?
Infected at blood meal Mean percentage infected following dsTsetseEP injection (number of flies) Mean percentage infected following dsAmp control injection (number of flies)
3 33.33 (36) 9.50 (21)