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)