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Differential expression of fat body genes in Glossina morsitans morsitans following infection with Trypanosoma brucei brucei
International Journal for Parasitology 38 (2008) 93–101 www.elsevier.com/locate/ijpara
Differential expression of fat body genes in Glossina morsitans morsitans following infection with Trypanosoma brucei brucei M.J. Lehane a
a,*
, W. Gibson b, S.M. Lehane
a
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK b School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK
Received 1 February 2007; received in revised form 16 May 2007; accepted 18 June 2007
Abstract To determine which fat body genes were differentially expressed following infection of Glossina morsitans morsitans with Trypanosoma brucei brucei we generated four suppression subtractive hybridisation (SSH) libraries. We obtained 52 unique gene fragments (SSH clones) of which 30 had a known orthologue at E 05 or less. Overall the characteristics of the orthologues suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly; (ii) that self-cured flies are mounting an oxidative stress response; and (iii) that self-cured flies are displaying increased energy usage. The three most consistently differentially expressed genes were further analysed by gene knockdown (RNAi). Knockdown of Glossina transferrin transcripts, which are upregulated in self-cured flies compared with flies infected with trypanosomes, results in a significant increase in the number of trypanosome infections establishing in the fly midgut, suggesting transferrin plays a role in the protection of tsetse flies from trypanosome infection. 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Glossina; Trypanosoma; SSH; Immunity; RNAi
1. Introduction The African trypanosomes responsible for sleeping sickness and nagana are cyclically transmitted by tsetse flies (Diptera: Glossinidae). The Word Health Organization (WHO) estimate that there are approximately 50,000 deaths annually and a loss of 1,598,000 disability adjusted life years (DALYs) caused by human African trypanosomiasis (HAT) with 60 million people at risk in 37 countries covering 40% of Africa (11 million km2) (WHO, 2002). After a devastating epidemic in the early 20th century when a million people died of HAT, the disease nearly disappeared in the 1960s only to re-emerge strongly in the 1990s (Ekwanzala et al., 1996; van Hove, 1996; Moore et al., 1999; TDR, 2003). In addition, animal African trypanosomiasis or nagana has restricted agricultural development and human nutrition in sub-Saharan Africa and *
Corresponding author. Tel.: +44 151 705 3316; fax: +44 151 705 3369. E-mail address: [email protected] (M.J. Lehane).
has a profound effect on the economy of much of the continent (Jordan, 1986) as recognised by the African Union (Kabayo, 2002). Despite the importance of these diseases, our understanding of tsetse/trypanosome interactions is still rudimentary (Aksoy et al., 2003). Not all tsetse flies which ingest trypanosomes of the subgenus Trypanozoon (brucei group) become infected. Even under ideal laboratory conditions, typically 40% or more of the flies challenged at the first bloodmeal will kill the trypanosomes they ingest and thus self-cure. From the third bloodmeal onwards, approximately 90% of trypanosomechallenged flies self-cure. We have no detailed knowledge of the differences between self-cured and infected flies and elucidating these differences remains a major challenge. One possible difference may lie in the insect immune system which is known to play a key role in determining the fate of the infection (Hao et al., 2001). Thus, stimulating an immune response in the fly by bacterial challenge to the haemolymph results in up to 80% decrease in infection levels in challenged flies. The fat body, which is a diffuse
0020-7519/$30.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.06.004
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organ found distributed throughout the haemocoel of the fly, is the insect’s major immune response organ as well as playing the central role in the intermediary metabolism of the insect. Consequently, in this study we have tried to identify genes which are differentially expressed in the fat body of trypanosome-infected tsetse flies and, separately, in flies which kill all the trypanosomes they ingest (selfcured flies). To identify such differentially expressed genes we used a suppression subtractive hybridisation (SSH) approach (Diatchenko et al., 1996; Gurskaya et al., 1996). We then used gene knockdown (RNAi) to determine if any of the most consistently differentially expressed genes directly influence the prevalence of trypanosome infection in the tsetse fly. 2. Materials and methods 2.1. Insects Glossina morsitans morsitans (originating from Zimbabwe) were maintained in a colony at the University of Bristol, UK and at the Liverpool School of Tropical Medicine. Flies were maintained at 26 ± 1 C and 60–70% relative humidity. All flies were fed through silicone membranes (Moloo, 1971) three times per week on sterile, defibrinated horse blood. The first bloodmeal received by experimental flies was infectious, containing Trypanosoma brucei brucei TSW 196 MSUS/CI/78/TSW196 [CLONE A], which is a fully fly-transmissible clone and able to undergo genetic exchange (Gibson, 1989). Typically, 200 lL of mouse blood (containing 4 · 106 bloodstream form parasites) were diluted in 5 mL of horse blood. Each fly typically takes 20 lL of blood and so receives approximately 1.6 · 104 parasites in the meal. Given the concentration of parasites in the meal, there is no realistic chance of fed flies avoiding ingestion of parasites. Unfed flies were removed, ensuring that all flies were initially infected. Midguts were dissected from cold anaesthetised (4 C) flies 8– 10 days after the infectious bloodmeal. Each midgut was lightly macerated in three drops of saline on a glass slide and the infection status was determined by searching for procyclic trypanosomes in 10 random fields by light microscopy (magnification 125·).
2.2. Suppression subtraction hybridisation (SSH) The Clontech PCR-select cDNA subtraction kit was used to obtain the differentially expressed transcripts by an SSH method according to manufacturer’s instructions. In brief, cDNA was prepared from fat bodies dissected from the abdomens of four batches of 50 G. m. morsitans of mixed sex. mRNA was isolated separately for each of the four batches using Dynabeads Oligo (dT)25 (Dynal). Batches 1 and 4 were self-cured flies which, despite being fed an infected meal, were not infected on dissection 8–10 days after the infectious bloodmeal. Batch 2 flies were non-infected controls. Batch 3 were flies which were infected on dissection 8–10 days after the infectious bloodmeal. Four subtracted libraries were produced by SSH using the TA Cloning kit (Invitrogen). In brief, these were: library A, self-cured flies (tester) minus non-infected flies (driver); library B, non-infected (tester) minus self-cured (driver); library C, infected (tester) minus self-cured (driver); library D, self-cured (tester) minus infected (driver) (Table 1, A–D) To select differentially expressed clones in each library (95% confidence level – Clontech) we used the PCRselect differential screening kit (Clontech) as recommended by the manufacturer. In brief, we randomly selected approximately 100–250 clones for each library (see Table 1) and arrayed PCR-generated cDNA from these, in duplicate, on nylon membranes. Only clones which hybridized to unsubtracted tester probes and/or forward subtracted probes, but not to reverse-subtracted or unsubtracted driver probes, were sequenced (see Table 1). We predicted that antimicrobial peptide genes would be common in the screens and, as these genes are already well known (Hao et al., 2001), we decided to eliminate them from the analysis. To do this we hybridised each array with a mixed probe containing Glossina defensin, diptericin and attacin cDNA. For each sequenced clone we used BlastX (http://www.ncbi.nlm.nih.gov) to try to identify the closest protein orthologue with a suggested function. All sequences of SSH-generated gene fragments have been deposited in dbEST (http://www.ncbi.nlm.nih. gov/dbEST/).
Table 1 Total number of clones processed for the selection of differentially expressed clones Library
Tester
A B C D
Self-cured Non-infected Infected Self-cured
Minus Minus Minus Minus
Driver
No. clones arrayed
Positives
Positives minus def/att/dipt
Non-infected Self-cured Self-cured Infected
237 108 226 175
25 28 27 18
25 28 24 18
Positives, clones which hybridized to unsubtracted tester probes and/or forward subtracted probes but not to reverse-subtracted or unsubtracted driver probes. If these clones did not hybridise to a mixture probe of defensin/attacin/diptericin then they were sequenced. Def, defensin; att, attacin; dipt, diptericin.
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2.3. RNAi Double-stranded RNA (dsRNA) was transcribed using a MEGAscript High Yield T7 Transcription kit (Ambion, Huntingdon, UK). Gene templates were available as clones either directly from the SSH cloning or from the tsetse expressed sequence tag (EST) program (Lehane et al., 2003). Double-stranded AMP (dsAMP) was generated using pBluescript II SK+ as a template. Template DNA was removed from the transcription reaction by DNase treatment, and dsRNA was purified using MEGAclear columns (Ambion) and eluted in nuclease-free water. Eluates were concentrated in a Christ (Osterode, Germany) 2–18 rotational vacuum concentrator to approximately 5 lg/lL. Borosilicate glass capillaries (2.00 mm outside diameter) were formed into a fine point using a needle puller (PC10; Narishige, Japan). Flies were anaesthetised by chilling and approximately 10 lg of dsRNA were dissolved in 2 lL of nuclease-free water and injected using a glass needle into the dorsolateral surface of the thorax of male flies. (The optimal quantity of dsRNA for RNAi of a series of genes in G. m. morsitans has been determined in the Lehane laboratory to be 6–10 lg – Lehane, unpublished data). Flies were given a bloodmeal on the day after emergence (day 1). They were injected with 8 lg dsRNA on day 2. Control groups were injected with dsAMP or nuclease-free water. Flies were given a second bloodmeal on day 3. The degree of knockdown achieved was measured using semi-quantitative RT-PCR and Northern analysis on day 5. Midguts and the remainder of the carcass were dissected and snap frozen in liquid nitrogen in groups of five. The intimate association of the fat body with the midgut makes it impossible to remove all traces of fat body from dissected midguts. Experimental flies were fed a third, T. b. brucei-infected bloodmeal (as described above) on day 5. Following this, they were fed uninfected bloodmeals every 48 h until they were killed and dissected on day 12 and scored for midgut infection with trypanosomes. The fate of midgut infections is usually clear by day 6 and is unequivocal by 12 days p.i. (Gibson and Bailey, 2003). Fat bodies were dissected from these flies and snap frozen in liquid nitrogen in groups of five infected or five uninfected samples. 2.4. RT-PCR For RT-PCR, total RNA was extracted from individual tissues using Trizol (Invitrogen, Paisley, UK) and treated with RQ1 RNase-Free DNase. RNA was quantified using a Nanodrop ND-1000 (Wilmington, DE) spectrophotometer. A Promega Access RT-PCR System (Promega, Southampton, UK) was used for amplification of transcripts. G. m. morsitans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Accession No. DQ016434) was used to normalise samples. PCR cycling conditions were: 48 C for 45 min, 94 C for 2 min, followed by 30 cycles of
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94 C for 30 s, 57 C for 1 min, 68 C for 2 min and a final extension of 68 C for 7 min. Primers used in the analysis of the major genes studied are shown in Table 2.
Table 2 Primers used in RT-PCR analysis of the major genes studied SSH identifier
EST contig
Primers
A13
Gmm-3242
GCTGGTCAGAACTTAG ACAACCCACATGCGTG GTACTTATGTTGCCAAC CAATTGTCAACAGCTTG GCCGAGGTACTGATTAC GTTAGCAAGGCAATTG GGAATGCGTTATGAAG CACACCAAGTTGAGCTC CAGGTATTGGCTTGGATG AGGTACGTGGAACTATAG CAATTGTCTAGCTACCTC GGACGAAAGCTGATGTAG CATAAACAACGGGCTTG GAAGATGGTGTCTACCAC GCTGTATGCTTGTCCTG CTGGATGGTTCCAACATG CAGGTACAAATCGTTAC TAATCGGTGAGCCAATC GGACATTTCTGTGAGAC GCTTTCGAAGAATGTTG GTGAGTGCGGCAAACTTG TGACCTTGGCCACTGCAG GGTACATTTCAAGGAAG GTACTAAATGCTTCGTG GAGGTACTTCAGCTTTG CAGGTACAAGCTGGATC GCCGAGGTACATAATAC ACCTGAAGGCACCAAAG ATAACGAAACCGGCAAC CAACTTGCAATGGAGAC CAACTTGCAATGGAGAC CTTGGATCCACTAGTTAC TCGTTAAGAAGGCGGTG CTTCAGCTTTGGCCAAG CGAGGTACCACGATTAG GTTCTGGTTCAGTGCGT CTCTATAATTCCGGTTTC TTCGTGGTAGCTATGTG GATCCCTCAACTCCTGAAG ATTGCGACCCTTGATGTTG GGTACGACAAATGGTAG CACGGTGTTCGAAACAC GATACCGTTAAGGGATTGT CTTTACTGTAATGAGCCAC GACTCACTATAGGGCTC CTACACCAGAGCCATCAAG GAGGAGCTTTCGAATTG GCACTTTCACCAAACAG CTTCTCGTCCATTCTTC CCACTGAATCAACCGAC CAGGTATTGGCTGAAGAG CAGCATTCAACGGCAATAG GTCATTCTAGGAACTC GAGAGTGAATACGAATG
A91-R 2A101 2A119 2A132
Gmm-2710
2A188
Gmm-3135
2A192
Gmsg-9400
2A36 2A4
Gmm-3263
2A92
Gmm-3323
B151
Gmm-3330
B161
Gmm-2697
B164 B176 B189-1
Gmm-3228
B193 B198 B204 C380 2C46 D451 D473 D531 D582 2D113 2D25 2D79
Gmm-10350
The relevant expressed sequence tag (EST) contig is given where this information was used in the study – see: http://www.genedb.org/genedb/ glossina/index.jsp. Primers used to generate a single-stranded probe for Northern analysis are shown in bold.
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2.5. Northern analysis
using Gene Tools software on a Gene Genius Bio Imaging System (Syngene).
The NorthernMax Formaldehyde-Based System for Northern Blots (Ambion) was used to perform Northern analysis. For analysis of differential expression of genes 20 lg of total RNA per lane were loaded on a 1% formaldehyde agarose gel; for monitoring knockdown levels with RNAi only 10 lg per lane were loaded. The Strip-EZ PCR probe synthesis and removal kit (Ambion) was used to synthesise single-stranded DNA probes which were labelled with [a-32P]dATP (MP Biomedicals). The primers used are listed in Table 2. Membranes were hybridised overnight at 42 C, given 2 · 5 min low stringency washes and 2 · 15 min high stringency washes before exposure to Kodak BioMax MR film. Band intensities were measured
2.6. Statistics Logistic regression analysis was carried out using the STATA statistical package v.8. 3. Results Four SSH libraries were produced. We reasoned that these would contain the following: library A, genes which are upregulated in self-cured flies, which are potentially the most interesting gene set if self-cure is a result of specific, novel gene action. Library B, genes which are
Table 3 Genes selected by suppression subtractive hybridisation from libraries A, B, C and D are identified by their clone number Clone
Accession Nos.
Homologue
Library A A13 A91R 2A101 2A119 2A132 2A188 2A192 2A36 2A4 2A 92
CV507139 CV507140 CV507141 CV507142 CV507143 CV507144 CV507145 CV507146 CV507147 CV507148
Salivary antigen 5 related Ribosomal protein L14 Lipocalin Dihydrodiol reductase Prolidase Lambda crystallin Transferrin Translocase like-protein Beta subunit ATP synthase Glycerol phosphate dehydrogenase
Library B B151 B161 B164 B176 B189-1 B193 B198 B204 B293a
CV507149 CV507150 CV507151 CV507152 CV507153 CV507154 CV507155 CV507156 CV507157
Library C 2C46 C380 C397a Library D D451 D473 D543 D582 2D113 2D25 2D79 2D106
Duplicates
Best BlastX match
Species
E value
1 1 3 1 1 1 1 1 1 1
AF259957 Y09766 NM_078728 AE003583 AY061085 BC004074 AAM46784 AC011066 X71013 X59076
Gm Dm Dm Dm Dm Mm Gm Dm Dm Dv
1e-53 1e-32 4e-09 1e-13 7e-48 5e-38 4e-86 4e-25 2e-59 3e-71
Chymotrypsin Trypsin Serine protease Carboxypeptidase Serine protease Serine protease Serine protease Ring finger protein 5 Poly-adenylate binding protein
9 10 2 1 1 1 1 1 1
AF252868 AF252869 AF302472 AE003618 AE003569 AE003523 AF302474 NP_062276.1 P21187
Gm Gm Cb Dm Dm Dm Cb Mm Dm
7e-99 6e-64 2e-12 1e-55 5e-32 2e-25 1e-05 9e-07 7e-21
CV507158 CV507159 CV507160
16s mitochondrial RNA C1-THF synthase 60s ribosomal protein L18a
1 17 1 1
AF072373 AE003688 AAL48844
Gm Dm Dm
3e-81 6e-24 8e-95
CV507161 CV507162 CV507163 CV507164 CV507165 CV507166 CV507167 CV507168
Sphingomyelin phosphodiesterase 1 Ubiquitin specific protease G-protein coupled receptor Alpha esterase Nucleoside diphosphate kinase ATPase 3-Hydroxyisobutyrate dehydrogenase Catalase
1 1 1 1 1 2 1 1 1
AC009460 NM 003470 CG9643 AY058345 AAF57188 AF368918 AF368918 CAA59444
Dm Hs Dm Dm Dm Dm Gm Cj
3e-40 3e-21 3e-17 1e-58 2e-56 3e-85 3e-85 9e-11
Blastx searches were performed and the closest protein orthologue with a suggested function are reported along with the accession number of the orthologue and the species from which it originates. An E value is given for each. In addition to those shown above, 12 genes in A, two in B, six in C and eight in D showed no informative orthogues in the BlastX search and are not listed in the table. In genes selected from library A the lipocalin-like gene was selected three times. In genes selected from library B Gm chymotrypsin occurred nine times, Gm trypsin 10 times and the Cb serine protease represented by B164 twice. In library C the 16s mitochondrial RNA gene occurred 17 times. In genes selected from library D the nucleoside diphosphate kinase gene occurred twice. Cb, Chrysomya bezziana; Mm, Mus musculus; Dm, Drosophila melanogaster; Dv, Drosophila virilis; Sp, Sarcophaga peregrina; Gm, Glossina morsitans morsitans; Hs, Homo sapiens; Pc, Pinus caribaea; Cj, Campylobacter jejuni. a These genes only produced orthogues when the corresponding expressed sequence tag was used for BlastX searching.
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downregulated from the normal non-infected state during the self-curing process. Library C, genes which are transcribed in infected flies but not in self-cured flies. These genes may be beneficial to the trypanosome infection process. Library D, genes which are transcribed in self-cured flies but not in infected flies. As with library A, this library may be a guide to key genes upregulated during the selfcuring process. Adding results from all four libraries, 98 clones were selected as being differentially expressed both by the SSH and the initial differential screening process. Surprisingly, only three were discarded because they hybridised to the defensin/attacin/diptericin probe. The remaining 95 clones were sequenced. Forty-three clones proved to be duplicates (see below). Of the 52 unique gene fragments, BlastX searches revealed that 28 had an orthologue at a value of E 05 or less. These are listed in Table 3. This process was relatively successful, first because significant genomic resources are now available for G. m. morsitans with over 70,000 eESTs sequenced (http://www.genedb.org/genedb/ glossina/index.jsp), and second because there is so much similarity between many Drosophila and Glossina transcripts (Lehane et al., 2003). Those sequences that did not produce an orthologue in the Blast search consisted of 12 genes in library A, two in B, three in C and seven in D. For these we used BlastN to search the Glossina EST database (http://www.sanger.ac.uk/Projects/G_morsitans/) (Lehane et al., 2003). We obtained perfect matches for five of these SSH clones. The EST contigs were always longer sequences than the SSH clones reported here. Consequently, we used this longer contig to search the NCBI databases using BlastX and in this way managed to produce an orthologue for a further two SSH clones, giving a total of 30 SSH clones with an identified orthologue at E 05 or less (Table 3). We took these 30 SSH clones forward for further analysis but did not pursue the remaining 22 gene fragments. When the full Glossina genome project is complete (Aksoy et al., 2005) (http://iggi.sanbi.ac.za/ wiki/index.php/Main_Page) it will be valuable to return to these fragments for further analysis. The duplicates were as follows: in library A the lipocalin-like gene was selected three times. In library B the G. m. morsitans chymotrypsin-like gene occurred nine times, the G. m. morsitans trypsin-like gene 10 times and the Chrysomya bezziana serine protease-like gene represented by B164 occurred twice. In library C the 16s mitochondrial RNA gene was represented 17 times. In library D the nucleoside diphosphate kinase gene occurred twice. False positives can be a problem with SSH (Byers et al., 2000). Consequently, the 30 SSH clones selected by the PCR-select differential screening kit were screened further, first using semi-quantitative RT-PCR analysis and then quantitatively using Northern analysis. For five of these 30 genes we failed to generate primers giving satisfactory results in RT-PCR analysis. Analysis of the other 25 genes resulted in eight genes consistently showing differential expression measured by semi-quantitative RT-PCR. For
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these eight genes we then performed two biological replicates of a Northern analysis to further test their differential expression. Digital analysis of the negative images from the Northerns showed that three genes consistently showed differential expression (Fig. 1). These were: 2a192, an orthologue of transferrin; B151, an orthologue of chymotrypsin; and B161, an orthologue of trypsin (Table 3). These results suggest that if selection of a consistently differentially regulated gene is the aim of such an SSH study then this needs to be confirmed by suitable quantitative methods such as Northern analysis. We used RNAi to determine if any of the three genes selected in the Northern analysis screen were influencing the success of trypanosome establishment in the fly midgut. RNAi protocols had previously been optimised in the laboratory (data not shown). We monitored the degree of knockdown achieved separately in the midgut and remaining carcass of the fly using semi-quantitative RT-PCR and Northern analysis. Significant knockdown was achieved for all three genes (Fig. 2). 2A192 was not detected in the midgut, in agreement with other studies. (Guz et al., 2007). B151 was not detected in the carcass using Northern analysis but could be detected by RT-PCR, suggesting this gene produces a low abundance transcript in the fat body. Only knockdown of transcripts from the gene represented by SSH clone 2A192 (transferrin) has a statistically significant impact on trypanosome prevalence causing almost a doubling in the number of trypanosome infections which successfully established in the fly midgut (Table 4).
A13
2A4
2A92
2A132
2A188
GAPDH I S 2A192
I S B151
I S
I S
I S
B161
I = infected S = self-cured GAPDH I S
I S
I S
Fig. 1. Northern analysis of eight selected genes. For each gene the blot in the left lane is from the fat body of infected flies (I), the blot in the right lane is from the fat body of self-cured flies (S). Gene A13 did not give a product despite four attempts. All images were digitally analysed directly from the negative. The three genes most clearly differentially expressed were 2A192 (149.6% – self-cured as a percentage of infected); B151 (67.1%); B161 (40.4%).
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1
2
3
4
2A192 GAPDH Lane 1 = midgut, specific dsRNA. Lane 2 = midgut, dsAMP control. Lane 3 = rest of carcass, specific dsRNA. Lane 4 = rest of carcass, dsAMP control.
B151 GAPDH B161 GAPDH
% Knockdown Midgut
% Knockdown Carcass
2A192
ND
100
B151
81.6
ND
B161
55.8
100
Fig. 2. Results of Northern analysis to determine the level of knockdown achieved in the RNA interference experiments. Lane 1, midgut, specific doublestranded RNA (dsRNA). Lane 2, midgut, double-stranded ampicillin (dsAMP) control. Lane 3, remains of the carcass, specific dsRNA. Lane 4, remains of the carcass, dsAMP control. Band intensities were normalised separately for each lane using the corresponding GAPDH band intensities. Results are expressed as the percentage knockdown achieved using the named, gene-specific dsRNA compared with control dsAMP. ND, transcripts not detected by Northern analysis. Two biological replicates were performed with equivalent results in both replicates.
Table 4 Logistic regression analysis of infected versus self-cured fly numbers following knockdown of the specified gene transcripts using RNA interference Injected dsRNA
2A192 B161 Amp B151 Amp
Biological replicates
3 3 3 3 3
Infection rate
8/67 = 11.9% 7/67 = 10.4% 4/67 = 6.0% 5/70 = 7.1% 3/69 = 4.3%
Adjusted for biological replicate effects Odds ratio (95% CI)
P
2.136 1.838 1 1.692 1
0.002 0.428 — 0.613 —
(1.318–3.461) (0.408–8.275) — (0.220–13.020) —
Experimental flies are compared with the ampicillin (Amp) controls. N.B. The 95% confidence interval (CI) unadjusted for replicate effects is (0.386–7.413) (P = 0.485).
4. Discussion The differential fat body immune responses reported here are in reaction to a parasite which completes the insect stage of its life cycle entirely within the lumen of the alimentary canal and salivary glands. Consequently, differential regulation of gene activity in the fat body at the time of dissection may be brought about by signalling molecules (e.g. cytokines) acting between the alimentary canal epithelium and the fat body (Boulanger et al., 2002). Alternatively, it is known that some trypanosomes do invade the haemolymph (Mshelbwala, 1972; Otieno, 1973; Otieno
et al., 1976) although they are killed there (Croft et al., 1982). These haemolymph-entering trypanosomes may act directly on fat body-associated receptor systems although our previous work suggests trypanosomes alone are unlikely to stimulate a marked fat body immune response (Hao et al., 2001). Another alternative is that such trypanosomes penetrating the gut wall might permit microbial contamination of the haemolymph from the midgut lumen and it may be this which induces the fat body immune response. Further experimental evidence is needed to determine the exact nature of the trypanosome-related stimulus to the fat body. For logistical reasons we limited the number of RNAi experiments to those genes which were the most consistently differentially regulated. However, we consider there is also useful information available from genes selected in the other screens and below we discuss genes selected as differentially expressed through at least the first of the three screening steps. Library A contains the gene fragment 2A192 which is part of the transcript of a Glossina transferrin gene (GenBank Accession No. AF368908) (Strickler-Dinglasan et al., 2006). The best BlastP alignments of the translated protein are with the transferrin precursors of Sarcophaga peregrina (Q26643) (76.6% identity) and Drosophila sylvestris (AAC77913) (62.5% identity) and so it fits into the Tsf1 family of transferrins (Dunkov and Georgieva, 2006; Strickler-Dinglasan et al., 2006). However, the Glossina
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transferrin lacks two of the five iron binding motifs found in the N-terminal region of these two species (EYRYT in S. peregrina; EYRYS in D. silvestris) and thus the protein may have other functions (Dunkov and Georgieva, 2006; Strickler-Dinglasan et al., 2006). Trypanosomes, at least when in their vertebrate hosts, use transferrin receptors to capture host transferrin and associated iron (Gerrits et al., 2002). A modified form of transferrin has been implicated in insect immune responses where it is believed to sequester iron, thus denying invading organisms access to iron (Yoshiga et al., 1997). Vertebrate transferrins have two iron binding sites but insect transferrins involved in the immune response have lost the C-terminal binding site which prevents pathogen receptors binding the transferrin (Yoshiga et al., 1997) and this Glossina transferrin is typical in having this modified C-terminal region (Strickler-Dinglasan et al., 2006). Transferrin is consistently downregulated in infected tsetse flies. This is consistent with other studies in insects which commonly show changes in the transcriptional level of transferrin under immune challenge. Thus transferrin is upregulated in mosquitoes infected with filarial worms (Beerntsen et al., 1994) or with a fungus (Aguilar et al., 2005). The protein is modified in Drosophila in response to fungal infection (Levy et al., 2004) and there is evidence for its differential expression during infection in other insects (Seitz et al., 2003; Thompson et al., 2003; Ursic-Bedoya and Lowenberger, 2007). The RNAi experiments provide direct evidence for the involvement of transferrin in tsetse trypanosome interactions (Table 4) although the mechanism remains unknown. Recent results from vertebrates suggest a possible immune signalling function for transferrin resulting in upregulation of the free radical nitrous oxide (Stafford and Belosevic, 2003) so it is interesting to note that it has been proposed that reactive oxygen intermediates (ROI) may be involved in tsetse trypanosome interactions (Hao et al., 2003; Lehane et al., 2003; Munks et al., 2005; Macleod et al., 2007). The putative signalling function of transferrin in vertebrates requires the proteolytic cleavage of the transferrin molecule (Stafford and Belosevic, 2003) and it is also interesting to note that the cleavage site is conserved in insect transferrins (Harizanova et al., 2005; Guz et al., 2007). Reactive oxygen intermediates (ROI) are thought to be important in insect defences (Nappi and Vass, 1998; Ha et al., 2005) including that of tsetse flies (Hao et al., 2003; Lehane et al., 2003; Munks et al., 2005; Macleod et al., 2007). Thus, addition of glucosamine (a known anti-oxidant (Xin et al., 2006)) or other anti-oxidants to the blood meal of tsetse flies results in increased prevalence of trypanosome infections (Maudlin and Welburn, 1987; Macleod et al., 2007). A fly utilising ROI in its defences would need protection from these molecules. Consequently, it is interesting to note the upregulation in library A of lambda crystallin and in library D of catalase, both of which are involved in protection from ROI (Missirlis et al., 2003; Ma et al., 2004). Differential expression of these
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genes in self-cured flies compared with non-infected and infected flies together with other published data (Hao et al., 2003; Macleod et al., 2007) suggest immune protection using superoxides deserves more study in tsetse trypanosome interactions. A lipocalin family member is also present in library A. Members of the lipocalin family are mainly extracellular carriers of lipophilic molecules. However, a subset of the family are cytokines and others have protease inhibiting properties (Logdberg and Wester, 2000) both of which could be important in the insect’s immune response. Clone 2A132 in library A is a homologue of a Drosophila member of the dipeptidase family involved in the hydrolysis of Xaa-Pro dipeptides (EC 3.4.13.9). It is known that proline is central to the metabolism of the fly (Bursell, 1977). It is also interesting to note that the EP isoform of the procyclin external coat of the procyclic, insect form of T. b. brucei is predicted to have 22–30 Glu-Pro (EP) tandem repeats in its C-terminal domain and that the other major procylin coat isoform, GPEET, also has Xaa-Pro in its five or six pentapeptide repeats (Roditi et al., 1987; Richardson et al., 1988; Clayton and Mowatt, 1989). Library B contains a series of proteolytic enzymes which have been downregulated in self-cured flies. This is consistent with studies in other immune-challenged insects where differential regulation of proteolytic enzymes have been noted in microarray experiments. However, the patterns of response reported to date are inconsistent and highly variable, being dependent on the immunogen and route of delivery (De Gregorio et al., 2001; Roxstrom-Lindquist et al., 2004). In addition to downregulation of these proteolytic enzymes, an orthologue of a gene encoding polyA binding protein is downregulated in library B, and this protein is involved in the positive regulation of translation. Given that the fat body is the major organ involved in intermediary metabolism in insects these results taken together may suggest a down regulation of general protein metabolism in self-cured flies. In addition, the upregulation of the genes represented by SSH clones D582, 2D113, 2D79, 2A4 in library D may indicate an upregulation of energy usage in self-cleared flies. This all suggests that trypanosome infection has a considerable effect on metabolism in the tsetse fly, a view which is consistent with recent findings in Drosophila that infection causes a fundamental alteration in metabolism (Dionne et al., 2006). Library C contains a homologue of C1-THF synthase which produces a co-factor used in purine biosynthesis. Trypanosomes, as with other protozoan parasites, are incapable of synthesising purines which they must acquire from their hosts (Carter et al., 2001). The simplest explanation for this observation is the infected fly is replenishing the pool of available purines that the trypanosomes have depleted. This would imply that the trypanosomes have access to the purine pool despite being ‘outside’ the body of the fly (i.e. in the midgut lumen) but how that might be achieved is completely unknown.
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Clone D451, which is differentially regulated in library D, is a homologue of sphingomyelin phosphodiesterase 1. Trypanosome membranes contain sphingosine derivatives and in particular the GPI-anchored proteins of the procyclic coat are associated with sphingolipid/sterol-rich rafts in the trypanosome plasma membrane (Denny et al., 2001). In summary, the SSH approach taken here suggests that a large number of genes are differentially regulated during the trypanosome infection process in tsetse flies. The emerging patterns suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly, (ii) that self-cured flies may be mounting an oxidative stress response and displaying an increase in energy usage. This study provides direct evidence for the involvement of transferrin in protecting flies from trypanosome infection and identifies this as a target for future research. It is probable that many genes are involved in the tsetse/trypanosome interaction. When the G. m. morsitans genome is published, it will open the way for microarray or proteomic-based studies which can efficiently target such genes for further study.The differentially expressed gene lists provided here will form an excellent comparator for such further studies. Acknowledgement This work was supported by funding from the Wellcome Trust. References Aguilar, R., Jedlicka, A.E., Mintz, M., Mahairaki, V., Scott, A.L., Dimopoulos, G., 2005. Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochemistry and Molecular Biology 35, 709–719. Aksoy, S., Gibson, W.C., Lehane, M.J., 2003. Interactions between tsetse and trypanosomes with implications for the control of trypanosomiasis. Advances in Parasitology 53, 1–83. Aksoy, S., Berriman, M., Hall, N., Hattori, M., Hide, W., Lehane, M.J., 2005. A case for a Glossina genome project. Trends in Parasitology 21, 107–111. Beerntsen, B.T., Severson, D.W., Christensen, B.M., 1994. Aedes aegypti – characterization of a hemolymph polypeptide expressed during melanotic encapsulation of filarial worms. Experimental Parasitology 79, 312–321. Boulanger, N., Brun, R., Ehret-Sabatier, L., Kunz, C., Bulet, P., 2002. Immunopeptides in the defense reactions of Glossina morsitans to bacterial and Trypanosoma brucei brucei infections. Insect Biochemistry and Molecular Biology 32, 369–375. Bursell, E., 1977. Synthesis of proline by fat body of the tsetse fly (Glossina morsitans). Metabolic pathways. Insect Biochemistry 7, 427–434. Byers, R.J., Hoyland, J.A., Dixon, J., Freemont, A.J., 2000. Subtractive hybridization – genetic takeaways and the search for meaning. International Journal of Experimental Pathology 81, 391–404. Carter, N.S., Landfear, S.M., Ullman, B., 2001. Nucleoside transporters of parasitic protozoa. Trends in Parasitology 17, 142–145. Clayton, C.E., Mowatt, M.R., 1989. The procyclic acidic repetitive proteins of Trypanosoma brucei – purification and post-translational modification. Journal of Biological Chemistry 264, 15088–15093. Croft, S.L., East, J.S., Molyneux, D.H., 1982. Antitrypanosomal factor in the haemolymph of Glossina. Acta Tropica 39, 293–302. De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide analysis of the Drosophila immune response by using
oligonucleotide microarrays. Proceedings of the National Academy of Sciences of the United States of America 98, 12590–12595. Denny, P.W., Field, M.C., Smith, D.F., 2001. GPI-anchored proteins and glycoconjugates segregate into lipid rafts in kinetoplastida. FEBS Letters 491, 148–153. Diatchenko, L., Lau, Y.F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the United States of America 93, 6025–6030. Dionne, M.S., Pham, L.N., Shirasu-Hiza, M., Schneider, D.S., 2006. Akt and foxo dysregulation contribute to infection-induced wasting in Drosophila. Current Biology 16, 1977–1985. Dunkov, B., Georgieva, T., 2006. Insect iron binding proteins: insights from the genomes. Insect Biochemistry and Molecular Biology 36, 300–309. Ekwanzala, M., Pepin, J., Khonde, N., Moliho, S., Bruneel, H., De Wals, P., 1996. In the heart of darkness:sleeping sickness in Zaire. Lancet 348, 1427–1430. Gerrits, H., Mussmann, R., Bitter, W., Kieft, R., Borst, P., 2002. The physiological significance of transferrin receptor variations in Trypanosoma brucei. Molecular and Biochemical Parasitology 119, 237–247. Gibson, W.C., 1989. Analysis of a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei. Parasitology 99, 391–402. Gibson, W., Bailey, M., 2003. The development of Trypanosoma brucei within the tsetse fly midgut observed using green fluorescent trypanosomes. Kinetoplastid Biology and Disease 2, 1. Gurskaya, N.G., Diatchenko, L., Chenchik, A., Siebert, P.D., Khaspekov, G.L., Lukyanov, K.A., Vagner, L.L., Ermolaeva, O.D., Lukyanov, S.A., Sverdlov, E.D., 1996. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Analytical Biochemistry 240, 90–97. Guz, N., Attardo, G.M., Wu, Y., Aksoy, S., 2007. Molecular aspects of transferrin expression in the tsetse fly (Glossina morsitans morsitans). Journal of Insect Physiology 53 (7), 715–723. Ha, E.M., Oh, C.T., Ryu, J.H., Bae, Y.S., Kang, S.W., Jang, I.H., Brey, P.T., Lee, W.J., 2005. An antioxidant system required for host protection against gut infection in Drosophila. Developmental Cell 8, 125–132. Hao, Z., Kasumba, I., Lehane, M.J., Gibson, W.C., Kwon, J., Aksoy, S., 2001. Tsetse immune responses and trypanosome transmission: implications for the development of tsetse-based strategies to reduce trypanosomiasis. Proceedings of the National Academy of Sciences of the United States of America 98, 12648–12653. Hao, Z.G., Kasumba, I., Aksoy, S., 2003. Proventriculus (cardia) plays a crucial role in immunity in tsetse fly (Diptera: Glossinidiae). Insect Biochemistry and Molecular Biology 33, 1155–1164. Harizanova, N., Georgieva, T., Dunkov, B.C., Yoshiga, T., Law, J.H., 2005. Aedes aegypti transferrin. Gene structure, expression pattern, and regulation. Insect Molecular Biology 14, 79–88. Jordan, A.M., 1986. Trypanosomiasis Control and African Rural Development. Longman, London. Kabayo, J.P., 2002. Aiming to eliminate tsetse from Africa. Trends in Parasitology 18, 473–475. Lehane, M.J., Aksoy, S., Gibson, W., Kerhornou, A., Berriman, M., Hamilton, J., Soares, M.B., Bonaldo, M.F., Lehane, S., Hall, N., 2003. Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes. Genome Biology 4, R63 (art. no). Levy, F., Bulet, P., Ehret-Sabatier, L., 2004. Proteomic analysis of the systemic immune response of Drosophila. Molecular and Cellular Proteomics 3, 156–166. Logdberg, L., Wester, L., 2000. Immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses. Biochimica et Biophysica Acta – Protein Structure and Molecular Enzymology 1482, 284–297.
M.J. Lehane et al. / International Journal for Parasitology 38 (2008) 93–101 Macleod, E.T., Maudlin, I., Darby, A.C., Welburn, S.C., 2007. Antioxidants promote establishment of trypanosome infections in tsetse. Parasitology, 1–5. Ma, W., Li, D., Sun, F., Kleiman, N.J., Spector, A., 2004. The effect of stress withdrawal on gene expression and certain biochemical and cell biological properties of peroxide-conditioned cell lines. FASEB Journal 18, 480–488. Maudlin, I., Welburn, S.C., 1987. Lectin mediated establishment of midgut infections of Trypanosoma congolense and Trypanosoma brucei in Glossina morsitans. Tropical Medicine and Parasitology 38, 167–170. Missirlis, F., Rahlfs, S., Dimopoulos, N., Bauer, H., Becker, K., Hilliker, A., Phillips, J.P., Jackle, H., 2003. A putative glutathione peroxidase of Drosophila encodes a thioredoxin peroxidase that provides resistance against oxidative stress but fails to complement a lack of catalase activity. Biological Chemistry 384, 463–472. Moloo, S.K., 1971. Artificial feeding technique for Glossina. Parasitology 63, 507–512. Moore, A., Richer, M., Enrile, M., Losio, E., Roberts, J., Levy, D., 1999. Resurgence of sleeping sickness in Tambura County, Sudan. American Journal of Tropical Medicine and Hygiene 61, 315–318. Mshelbwala, A.S., 1972. Trypanosoma brucei infections in the haemocoel of tsetse flies. Transactions of the Royal Society of Tropical Medicine and Hygiene 66, 637–643. Munks, R.J., Sant’Anna, M.R., Grail, W., Igglesden, T., Yoshiyama, M., Lehane, S.M., Lehane, M.J., 2005. Antioxidant gene expression in the blood-feeding fly Glossina morsitans morsitans. Insect Molecular Biology 14 (5), 483–491. Nappi, A.J., Vass, E., 1998. Hydrogen peroxide production in immunereactive Drosophila melanogaster. Journal of Parasitology 84, 1150– 1157. Otieno, L.H., 1973. Trypanosoma brucei in the haemolymph of experimentally infected young Glossina morsitans. Transactions of the Royal Society of Tropical Medicine and Hygiene 67, 886–887. Otieno, L.H., Darji, N., Onyango, P., 1976. development of Trypanosoma (Trypanozoon) brucei in Glossina morsitans inoculated into the tsetse haemocoel. Acta Tropica 33, 143–150. Richardson, J.P., Beecroft, R.P., Tolson, D.L., Liu, M.K., Pearson, T.W., 1988. Procyclin – an unusual immunodominant glycoprotein surface-
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antigen from the procyclic stage of African trypanosomes. Molecular and Biochemical Parasitology 31, 203–216. Roditi, I., Carrington, M., Turner, M., 1987. Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature 325, 272–274. Roxstrom-Lindquist, K., Terenius, O., Faye, I., 2004. Parasite-specific immune response in adult Drosophila melanogaster: a genomic study. EMBO Reports 5, 207–212. Seitz, V., Clermont, A., Wedde, M., Hummel, M., Vilcinskas, A., Schlatterer, K., Podsiadlowski, L., 2003. Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach. Developmental and Comparative Immunology 27, 207–215. Stafford, J.L., Belosevic, M., 2003. Transferrin and the innate immune response of fish: identification of a novel mechanism of macrophage activation. Developmental and Comparative Immunology 27, 539– 554. Strickler-Dinglasan, P.M., Guz, N., Attardo, G., Aksoy, S., 2006. Molecular characterization of iron binding proteins from Glossina morsitans morsitans (Diptera: Glossinidae). Insect Biochemistry and Molecular Biology 36, 921–933. T.D.R., 2003. WHO/Tropical Disease Research Unit, Geneva Switzerland. Meeting report. 4–8 June 2001 Geneva, Switzerland, 1–182. Thompson, G.J., Crozier, Y.C., Crozier, R.H., 2003. Isolation and characterization of a termite transferrin gene up-regulated on infection. Insect Molecular Biology 12, 1–7. Ursic-Bedoya, R.J., Lowenberger, C.A., 2007. Rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization. Developmental and Comparative Immunology 31, 109–120. van Hove, D., 1996. Sleeping sickness in Zaire. Lancet 349, 438. W.H.O., 2002. World Health Report. Xin, R.G., Liu, S., Guo, Z.Y., Yu, H.H., Li, C.P., Ji, X., Feng, J.H., Li, P.C., 2006. The antioxidant activity of glucosamine hydrochloride in vitro. Bioorganic and Medicinal Chemistry 14, 1706–1709. Yoshiga, T., Hernandez, V.P., Fallon, A.M., Law, J.H., 1997. Mosquito transferrin, an acute-phase protein that is up-regulated upon infection. Proceedings of the National Academy of Sciences of the United States of America 94, 12337–12342.
Differential expression of fat body genes in Glossina morsitans morsitans following infection with Trypanosoma brucei brucei M.J. Lehane a
a,*
, W. Gibson b, S.M. Lehane
a
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK b School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK
Received 1 February 2007; received in revised form 16 May 2007; accepted 18 June 2007
Abstract To determine which fat body genes were differentially expressed following infection of Glossina morsitans morsitans with Trypanosoma brucei brucei we generated four suppression subtractive hybridisation (SSH) libraries. We obtained 52 unique gene fragments (SSH clones) of which 30 had a known orthologue at E 05 or less. Overall the characteristics of the orthologues suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly; (ii) that self-cured flies are mounting an oxidative stress response; and (iii) that self-cured flies are displaying increased energy usage. The three most consistently differentially expressed genes were further analysed by gene knockdown (RNAi). Knockdown of Glossina transferrin transcripts, which are upregulated in self-cured flies compared with flies infected with trypanosomes, results in a significant increase in the number of trypanosome infections establishing in the fly midgut, suggesting transferrin plays a role in the protection of tsetse flies from trypanosome infection. 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Glossina; Trypanosoma; SSH; Immunity; RNAi
1. Introduction The African trypanosomes responsible for sleeping sickness and nagana are cyclically transmitted by tsetse flies (Diptera: Glossinidae). The Word Health Organization (WHO) estimate that there are approximately 50,000 deaths annually and a loss of 1,598,000 disability adjusted life years (DALYs) caused by human African trypanosomiasis (HAT) with 60 million people at risk in 37 countries covering 40% of Africa (11 million km2) (WHO, 2002). After a devastating epidemic in the early 20th century when a million people died of HAT, the disease nearly disappeared in the 1960s only to re-emerge strongly in the 1990s (Ekwanzala et al., 1996; van Hove, 1996; Moore et al., 1999; TDR, 2003). In addition, animal African trypanosomiasis or nagana has restricted agricultural development and human nutrition in sub-Saharan Africa and *
Corresponding author. Tel.: +44 151 705 3316; fax: +44 151 705 3369. E-mail address: [email protected] (M.J. Lehane).
has a profound effect on the economy of much of the continent (Jordan, 1986) as recognised by the African Union (Kabayo, 2002). Despite the importance of these diseases, our understanding of tsetse/trypanosome interactions is still rudimentary (Aksoy et al., 2003). Not all tsetse flies which ingest trypanosomes of the subgenus Trypanozoon (brucei group) become infected. Even under ideal laboratory conditions, typically 40% or more of the flies challenged at the first bloodmeal will kill the trypanosomes they ingest and thus self-cure. From the third bloodmeal onwards, approximately 90% of trypanosomechallenged flies self-cure. We have no detailed knowledge of the differences between self-cured and infected flies and elucidating these differences remains a major challenge. One possible difference may lie in the insect immune system which is known to play a key role in determining the fate of the infection (Hao et al., 2001). Thus, stimulating an immune response in the fly by bacterial challenge to the haemolymph results in up to 80% decrease in infection levels in challenged flies. The fat body, which is a diffuse
0020-7519/$30.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.06.004
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organ found distributed throughout the haemocoel of the fly, is the insect’s major immune response organ as well as playing the central role in the intermediary metabolism of the insect. Consequently, in this study we have tried to identify genes which are differentially expressed in the fat body of trypanosome-infected tsetse flies and, separately, in flies which kill all the trypanosomes they ingest (selfcured flies). To identify such differentially expressed genes we used a suppression subtractive hybridisation (SSH) approach (Diatchenko et al., 1996; Gurskaya et al., 1996). We then used gene knockdown (RNAi) to determine if any of the most consistently differentially expressed genes directly influence the prevalence of trypanosome infection in the tsetse fly. 2. Materials and methods 2.1. Insects Glossina morsitans morsitans (originating from Zimbabwe) were maintained in a colony at the University of Bristol, UK and at the Liverpool School of Tropical Medicine. Flies were maintained at 26 ± 1 C and 60–70% relative humidity. All flies were fed through silicone membranes (Moloo, 1971) three times per week on sterile, defibrinated horse blood. The first bloodmeal received by experimental flies was infectious, containing Trypanosoma brucei brucei TSW 196 MSUS/CI/78/TSW196 [CLONE A], which is a fully fly-transmissible clone and able to undergo genetic exchange (Gibson, 1989). Typically, 200 lL of mouse blood (containing 4 · 106 bloodstream form parasites) were diluted in 5 mL of horse blood. Each fly typically takes 20 lL of blood and so receives approximately 1.6 · 104 parasites in the meal. Given the concentration of parasites in the meal, there is no realistic chance of fed flies avoiding ingestion of parasites. Unfed flies were removed, ensuring that all flies were initially infected. Midguts were dissected from cold anaesthetised (4 C) flies 8– 10 days after the infectious bloodmeal. Each midgut was lightly macerated in three drops of saline on a glass slide and the infection status was determined by searching for procyclic trypanosomes in 10 random fields by light microscopy (magnification 125·).
2.2. Suppression subtraction hybridisation (SSH) The Clontech PCR-select cDNA subtraction kit was used to obtain the differentially expressed transcripts by an SSH method according to manufacturer’s instructions. In brief, cDNA was prepared from fat bodies dissected from the abdomens of four batches of 50 G. m. morsitans of mixed sex. mRNA was isolated separately for each of the four batches using Dynabeads Oligo (dT)25 (Dynal). Batches 1 and 4 were self-cured flies which, despite being fed an infected meal, were not infected on dissection 8–10 days after the infectious bloodmeal. Batch 2 flies were non-infected controls. Batch 3 were flies which were infected on dissection 8–10 days after the infectious bloodmeal. Four subtracted libraries were produced by SSH using the TA Cloning kit (Invitrogen). In brief, these were: library A, self-cured flies (tester) minus non-infected flies (driver); library B, non-infected (tester) minus self-cured (driver); library C, infected (tester) minus self-cured (driver); library D, self-cured (tester) minus infected (driver) (Table 1, A–D) To select differentially expressed clones in each library (95% confidence level – Clontech) we used the PCRselect differential screening kit (Clontech) as recommended by the manufacturer. In brief, we randomly selected approximately 100–250 clones for each library (see Table 1) and arrayed PCR-generated cDNA from these, in duplicate, on nylon membranes. Only clones which hybridized to unsubtracted tester probes and/or forward subtracted probes, but not to reverse-subtracted or unsubtracted driver probes, were sequenced (see Table 1). We predicted that antimicrobial peptide genes would be common in the screens and, as these genes are already well known (Hao et al., 2001), we decided to eliminate them from the analysis. To do this we hybridised each array with a mixed probe containing Glossina defensin, diptericin and attacin cDNA. For each sequenced clone we used BlastX (http://www.ncbi.nlm.nih.gov) to try to identify the closest protein orthologue with a suggested function. All sequences of SSH-generated gene fragments have been deposited in dbEST (http://www.ncbi.nlm.nih. gov/dbEST/).
Table 1 Total number of clones processed for the selection of differentially expressed clones Library
Tester
A B C D
Self-cured Non-infected Infected Self-cured
Minus Minus Minus Minus
Driver
No. clones arrayed
Positives
Positives minus def/att/dipt
Non-infected Self-cured Self-cured Infected
237 108 226 175
25 28 27 18
25 28 24 18
Positives, clones which hybridized to unsubtracted tester probes and/or forward subtracted probes but not to reverse-subtracted or unsubtracted driver probes. If these clones did not hybridise to a mixture probe of defensin/attacin/diptericin then they were sequenced. Def, defensin; att, attacin; dipt, diptericin.
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2.3. RNAi Double-stranded RNA (dsRNA) was transcribed using a MEGAscript High Yield T7 Transcription kit (Ambion, Huntingdon, UK). Gene templates were available as clones either directly from the SSH cloning or from the tsetse expressed sequence tag (EST) program (Lehane et al., 2003). Double-stranded AMP (dsAMP) was generated using pBluescript II SK+ as a template. Template DNA was removed from the transcription reaction by DNase treatment, and dsRNA was purified using MEGAclear columns (Ambion) and eluted in nuclease-free water. Eluates were concentrated in a Christ (Osterode, Germany) 2–18 rotational vacuum concentrator to approximately 5 lg/lL. Borosilicate glass capillaries (2.00 mm outside diameter) were formed into a fine point using a needle puller (PC10; Narishige, Japan). Flies were anaesthetised by chilling and approximately 10 lg of dsRNA were dissolved in 2 lL of nuclease-free water and injected using a glass needle into the dorsolateral surface of the thorax of male flies. (The optimal quantity of dsRNA for RNAi of a series of genes in G. m. morsitans has been determined in the Lehane laboratory to be 6–10 lg – Lehane, unpublished data). Flies were given a bloodmeal on the day after emergence (day 1). They were injected with 8 lg dsRNA on day 2. Control groups were injected with dsAMP or nuclease-free water. Flies were given a second bloodmeal on day 3. The degree of knockdown achieved was measured using semi-quantitative RT-PCR and Northern analysis on day 5. Midguts and the remainder of the carcass were dissected and snap frozen in liquid nitrogen in groups of five. The intimate association of the fat body with the midgut makes it impossible to remove all traces of fat body from dissected midguts. Experimental flies were fed a third, T. b. brucei-infected bloodmeal (as described above) on day 5. Following this, they were fed uninfected bloodmeals every 48 h until they were killed and dissected on day 12 and scored for midgut infection with trypanosomes. The fate of midgut infections is usually clear by day 6 and is unequivocal by 12 days p.i. (Gibson and Bailey, 2003). Fat bodies were dissected from these flies and snap frozen in liquid nitrogen in groups of five infected or five uninfected samples. 2.4. RT-PCR For RT-PCR, total RNA was extracted from individual tissues using Trizol (Invitrogen, Paisley, UK) and treated with RQ1 RNase-Free DNase. RNA was quantified using a Nanodrop ND-1000 (Wilmington, DE) spectrophotometer. A Promega Access RT-PCR System (Promega, Southampton, UK) was used for amplification of transcripts. G. m. morsitans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Accession No. DQ016434) was used to normalise samples. PCR cycling conditions were: 48 C for 45 min, 94 C for 2 min, followed by 30 cycles of
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94 C for 30 s, 57 C for 1 min, 68 C for 2 min and a final extension of 68 C for 7 min. Primers used in the analysis of the major genes studied are shown in Table 2.
Table 2 Primers used in RT-PCR analysis of the major genes studied SSH identifier
EST contig
Primers
A13
Gmm-3242
GCTGGTCAGAACTTAG ACAACCCACATGCGTG GTACTTATGTTGCCAAC CAATTGTCAACAGCTTG GCCGAGGTACTGATTAC GTTAGCAAGGCAATTG GGAATGCGTTATGAAG CACACCAAGTTGAGCTC CAGGTATTGGCTTGGATG AGGTACGTGGAACTATAG CAATTGTCTAGCTACCTC GGACGAAAGCTGATGTAG CATAAACAACGGGCTTG GAAGATGGTGTCTACCAC GCTGTATGCTTGTCCTG CTGGATGGTTCCAACATG CAGGTACAAATCGTTAC TAATCGGTGAGCCAATC GGACATTTCTGTGAGAC GCTTTCGAAGAATGTTG GTGAGTGCGGCAAACTTG TGACCTTGGCCACTGCAG GGTACATTTCAAGGAAG GTACTAAATGCTTCGTG GAGGTACTTCAGCTTTG CAGGTACAAGCTGGATC GCCGAGGTACATAATAC ACCTGAAGGCACCAAAG ATAACGAAACCGGCAAC CAACTTGCAATGGAGAC CAACTTGCAATGGAGAC CTTGGATCCACTAGTTAC TCGTTAAGAAGGCGGTG CTTCAGCTTTGGCCAAG CGAGGTACCACGATTAG GTTCTGGTTCAGTGCGT CTCTATAATTCCGGTTTC TTCGTGGTAGCTATGTG GATCCCTCAACTCCTGAAG ATTGCGACCCTTGATGTTG GGTACGACAAATGGTAG CACGGTGTTCGAAACAC GATACCGTTAAGGGATTGT CTTTACTGTAATGAGCCAC GACTCACTATAGGGCTC CTACACCAGAGCCATCAAG GAGGAGCTTTCGAATTG GCACTTTCACCAAACAG CTTCTCGTCCATTCTTC CCACTGAATCAACCGAC CAGGTATTGGCTGAAGAG CAGCATTCAACGGCAATAG GTCATTCTAGGAACTC GAGAGTGAATACGAATG
A91-R 2A101 2A119 2A132
Gmm-2710
2A188
Gmm-3135
2A192
Gmsg-9400
2A36 2A4
Gmm-3263
2A92
Gmm-3323
B151
Gmm-3330
B161
Gmm-2697
B164 B176 B189-1
Gmm-3228
B193 B198 B204 C380 2C46 D451 D473 D531 D582 2D113 2D25 2D79
Gmm-10350
The relevant expressed sequence tag (EST) contig is given where this information was used in the study – see: http://www.genedb.org/genedb/ glossina/index.jsp. Primers used to generate a single-stranded probe for Northern analysis are shown in bold.
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2.5. Northern analysis
using Gene Tools software on a Gene Genius Bio Imaging System (Syngene).
The NorthernMax Formaldehyde-Based System for Northern Blots (Ambion) was used to perform Northern analysis. For analysis of differential expression of genes 20 lg of total RNA per lane were loaded on a 1% formaldehyde agarose gel; for monitoring knockdown levels with RNAi only 10 lg per lane were loaded. The Strip-EZ PCR probe synthesis and removal kit (Ambion) was used to synthesise single-stranded DNA probes which were labelled with [a-32P]dATP (MP Biomedicals). The primers used are listed in Table 2. Membranes were hybridised overnight at 42 C, given 2 · 5 min low stringency washes and 2 · 15 min high stringency washes before exposure to Kodak BioMax MR film. Band intensities were measured
2.6. Statistics Logistic regression analysis was carried out using the STATA statistical package v.8. 3. Results Four SSH libraries were produced. We reasoned that these would contain the following: library A, genes which are upregulated in self-cured flies, which are potentially the most interesting gene set if self-cure is a result of specific, novel gene action. Library B, genes which are
Table 3 Genes selected by suppression subtractive hybridisation from libraries A, B, C and D are identified by their clone number Clone
Accession Nos.
Homologue
Library A A13 A91R 2A101 2A119 2A132 2A188 2A192 2A36 2A4 2A 92
CV507139 CV507140 CV507141 CV507142 CV507143 CV507144 CV507145 CV507146 CV507147 CV507148
Salivary antigen 5 related Ribosomal protein L14 Lipocalin Dihydrodiol reductase Prolidase Lambda crystallin Transferrin Translocase like-protein Beta subunit ATP synthase Glycerol phosphate dehydrogenase
Library B B151 B161 B164 B176 B189-1 B193 B198 B204 B293a
CV507149 CV507150 CV507151 CV507152 CV507153 CV507154 CV507155 CV507156 CV507157
Library C 2C46 C380 C397a Library D D451 D473 D543 D582 2D113 2D25 2D79 2D106
Duplicates
Best BlastX match
Species
E value
1 1 3 1 1 1 1 1 1 1
AF259957 Y09766 NM_078728 AE003583 AY061085 BC004074 AAM46784 AC011066 X71013 X59076
Gm Dm Dm Dm Dm Mm Gm Dm Dm Dv
1e-53 1e-32 4e-09 1e-13 7e-48 5e-38 4e-86 4e-25 2e-59 3e-71
Chymotrypsin Trypsin Serine protease Carboxypeptidase Serine protease Serine protease Serine protease Ring finger protein 5 Poly-adenylate binding protein
9 10 2 1 1 1 1 1 1
AF252868 AF252869 AF302472 AE003618 AE003569 AE003523 AF302474 NP_062276.1 P21187
Gm Gm Cb Dm Dm Dm Cb Mm Dm
7e-99 6e-64 2e-12 1e-55 5e-32 2e-25 1e-05 9e-07 7e-21
CV507158 CV507159 CV507160
16s mitochondrial RNA C1-THF synthase 60s ribosomal protein L18a
1 17 1 1
AF072373 AE003688 AAL48844
Gm Dm Dm
3e-81 6e-24 8e-95
CV507161 CV507162 CV507163 CV507164 CV507165 CV507166 CV507167 CV507168
Sphingomyelin phosphodiesterase 1 Ubiquitin specific protease G-protein coupled receptor Alpha esterase Nucleoside diphosphate kinase ATPase 3-Hydroxyisobutyrate dehydrogenase Catalase
1 1 1 1 1 2 1 1 1
AC009460 NM 003470 CG9643 AY058345 AAF57188 AF368918 AF368918 CAA59444
Dm Hs Dm Dm Dm Dm Gm Cj
3e-40 3e-21 3e-17 1e-58 2e-56 3e-85 3e-85 9e-11
Blastx searches were performed and the closest protein orthologue with a suggested function are reported along with the accession number of the orthologue and the species from which it originates. An E value is given for each. In addition to those shown above, 12 genes in A, two in B, six in C and eight in D showed no informative orthogues in the BlastX search and are not listed in the table. In genes selected from library A the lipocalin-like gene was selected three times. In genes selected from library B Gm chymotrypsin occurred nine times, Gm trypsin 10 times and the Cb serine protease represented by B164 twice. In library C the 16s mitochondrial RNA gene occurred 17 times. In genes selected from library D the nucleoside diphosphate kinase gene occurred twice. Cb, Chrysomya bezziana; Mm, Mus musculus; Dm, Drosophila melanogaster; Dv, Drosophila virilis; Sp, Sarcophaga peregrina; Gm, Glossina morsitans morsitans; Hs, Homo sapiens; Pc, Pinus caribaea; Cj, Campylobacter jejuni. a These genes only produced orthogues when the corresponding expressed sequence tag was used for BlastX searching.
M.J. Lehane et al. / International Journal for Parasitology 38 (2008) 93–101
downregulated from the normal non-infected state during the self-curing process. Library C, genes which are transcribed in infected flies but not in self-cured flies. These genes may be beneficial to the trypanosome infection process. Library D, genes which are transcribed in self-cured flies but not in infected flies. As with library A, this library may be a guide to key genes upregulated during the selfcuring process. Adding results from all four libraries, 98 clones were selected as being differentially expressed both by the SSH and the initial differential screening process. Surprisingly, only three were discarded because they hybridised to the defensin/attacin/diptericin probe. The remaining 95 clones were sequenced. Forty-three clones proved to be duplicates (see below). Of the 52 unique gene fragments, BlastX searches revealed that 28 had an orthologue at a value of E 05 or less. These are listed in Table 3. This process was relatively successful, first because significant genomic resources are now available for G. m. morsitans with over 70,000 eESTs sequenced (http://www.genedb.org/genedb/ glossina/index.jsp), and second because there is so much similarity between many Drosophila and Glossina transcripts (Lehane et al., 2003). Those sequences that did not produce an orthologue in the Blast search consisted of 12 genes in library A, two in B, three in C and seven in D. For these we used BlastN to search the Glossina EST database (http://www.sanger.ac.uk/Projects/G_morsitans/) (Lehane et al., 2003). We obtained perfect matches for five of these SSH clones. The EST contigs were always longer sequences than the SSH clones reported here. Consequently, we used this longer contig to search the NCBI databases using BlastX and in this way managed to produce an orthologue for a further two SSH clones, giving a total of 30 SSH clones with an identified orthologue at E 05 or less (Table 3). We took these 30 SSH clones forward for further analysis but did not pursue the remaining 22 gene fragments. When the full Glossina genome project is complete (Aksoy et al., 2005) (http://iggi.sanbi.ac.za/ wiki/index.php/Main_Page) it will be valuable to return to these fragments for further analysis. The duplicates were as follows: in library A the lipocalin-like gene was selected three times. In library B the G. m. morsitans chymotrypsin-like gene occurred nine times, the G. m. morsitans trypsin-like gene 10 times and the Chrysomya bezziana serine protease-like gene represented by B164 occurred twice. In library C the 16s mitochondrial RNA gene was represented 17 times. In library D the nucleoside diphosphate kinase gene occurred twice. False positives can be a problem with SSH (Byers et al., 2000). Consequently, the 30 SSH clones selected by the PCR-select differential screening kit were screened further, first using semi-quantitative RT-PCR analysis and then quantitatively using Northern analysis. For five of these 30 genes we failed to generate primers giving satisfactory results in RT-PCR analysis. Analysis of the other 25 genes resulted in eight genes consistently showing differential expression measured by semi-quantitative RT-PCR. For
97
these eight genes we then performed two biological replicates of a Northern analysis to further test their differential expression. Digital analysis of the negative images from the Northerns showed that three genes consistently showed differential expression (Fig. 1). These were: 2a192, an orthologue of transferrin; B151, an orthologue of chymotrypsin; and B161, an orthologue of trypsin (Table 3). These results suggest that if selection of a consistently differentially regulated gene is the aim of such an SSH study then this needs to be confirmed by suitable quantitative methods such as Northern analysis. We used RNAi to determine if any of the three genes selected in the Northern analysis screen were influencing the success of trypanosome establishment in the fly midgut. RNAi protocols had previously been optimised in the laboratory (data not shown). We monitored the degree of knockdown achieved separately in the midgut and remaining carcass of the fly using semi-quantitative RT-PCR and Northern analysis. Significant knockdown was achieved for all three genes (Fig. 2). 2A192 was not detected in the midgut, in agreement with other studies. (Guz et al., 2007). B151 was not detected in the carcass using Northern analysis but could be detected by RT-PCR, suggesting this gene produces a low abundance transcript in the fat body. Only knockdown of transcripts from the gene represented by SSH clone 2A192 (transferrin) has a statistically significant impact on trypanosome prevalence causing almost a doubling in the number of trypanosome infections which successfully established in the fly midgut (Table 4).
A13
2A4
2A92
2A132
2A188
GAPDH I S 2A192
I S B151
I S
I S
I S
B161
I = infected S = self-cured GAPDH I S
I S
I S
Fig. 1. Northern analysis of eight selected genes. For each gene the blot in the left lane is from the fat body of infected flies (I), the blot in the right lane is from the fat body of self-cured flies (S). Gene A13 did not give a product despite four attempts. All images were digitally analysed directly from the negative. The three genes most clearly differentially expressed were 2A192 (149.6% – self-cured as a percentage of infected); B151 (67.1%); B161 (40.4%).
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1
2
3
4
2A192 GAPDH Lane 1 = midgut, specific dsRNA. Lane 2 = midgut, dsAMP control. Lane 3 = rest of carcass, specific dsRNA. Lane 4 = rest of carcass, dsAMP control.
B151 GAPDH B161 GAPDH
% Knockdown Midgut
% Knockdown Carcass
2A192
ND
100
B151
81.6
ND
B161
55.8
100
Fig. 2. Results of Northern analysis to determine the level of knockdown achieved in the RNA interference experiments. Lane 1, midgut, specific doublestranded RNA (dsRNA). Lane 2, midgut, double-stranded ampicillin (dsAMP) control. Lane 3, remains of the carcass, specific dsRNA. Lane 4, remains of the carcass, dsAMP control. Band intensities were normalised separately for each lane using the corresponding GAPDH band intensities. Results are expressed as the percentage knockdown achieved using the named, gene-specific dsRNA compared with control dsAMP. ND, transcripts not detected by Northern analysis. Two biological replicates were performed with equivalent results in both replicates.
Table 4 Logistic regression analysis of infected versus self-cured fly numbers following knockdown of the specified gene transcripts using RNA interference Injected dsRNA
2A192 B161 Amp B151 Amp
Biological replicates
3 3 3 3 3
Infection rate
8/67 = 11.9% 7/67 = 10.4% 4/67 = 6.0% 5/70 = 7.1% 3/69 = 4.3%
Adjusted for biological replicate effects Odds ratio (95% CI)
P
2.136 1.838 1 1.692 1
0.002 0.428 — 0.613 —
(1.318–3.461) (0.408–8.275) — (0.220–13.020) —
Experimental flies are compared with the ampicillin (Amp) controls. N.B. The 95% confidence interval (CI) unadjusted for replicate effects is (0.386–7.413) (P = 0.485).
4. Discussion The differential fat body immune responses reported here are in reaction to a parasite which completes the insect stage of its life cycle entirely within the lumen of the alimentary canal and salivary glands. Consequently, differential regulation of gene activity in the fat body at the time of dissection may be brought about by signalling molecules (e.g. cytokines) acting between the alimentary canal epithelium and the fat body (Boulanger et al., 2002). Alternatively, it is known that some trypanosomes do invade the haemolymph (Mshelbwala, 1972; Otieno, 1973; Otieno
et al., 1976) although they are killed there (Croft et al., 1982). These haemolymph-entering trypanosomes may act directly on fat body-associated receptor systems although our previous work suggests trypanosomes alone are unlikely to stimulate a marked fat body immune response (Hao et al., 2001). Another alternative is that such trypanosomes penetrating the gut wall might permit microbial contamination of the haemolymph from the midgut lumen and it may be this which induces the fat body immune response. Further experimental evidence is needed to determine the exact nature of the trypanosome-related stimulus to the fat body. For logistical reasons we limited the number of RNAi experiments to those genes which were the most consistently differentially regulated. However, we consider there is also useful information available from genes selected in the other screens and below we discuss genes selected as differentially expressed through at least the first of the three screening steps. Library A contains the gene fragment 2A192 which is part of the transcript of a Glossina transferrin gene (GenBank Accession No. AF368908) (Strickler-Dinglasan et al., 2006). The best BlastP alignments of the translated protein are with the transferrin precursors of Sarcophaga peregrina (Q26643) (76.6% identity) and Drosophila sylvestris (AAC77913) (62.5% identity) and so it fits into the Tsf1 family of transferrins (Dunkov and Georgieva, 2006; Strickler-Dinglasan et al., 2006). However, the Glossina
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transferrin lacks two of the five iron binding motifs found in the N-terminal region of these two species (EYRYT in S. peregrina; EYRYS in D. silvestris) and thus the protein may have other functions (Dunkov and Georgieva, 2006; Strickler-Dinglasan et al., 2006). Trypanosomes, at least when in their vertebrate hosts, use transferrin receptors to capture host transferrin and associated iron (Gerrits et al., 2002). A modified form of transferrin has been implicated in insect immune responses where it is believed to sequester iron, thus denying invading organisms access to iron (Yoshiga et al., 1997). Vertebrate transferrins have two iron binding sites but insect transferrins involved in the immune response have lost the C-terminal binding site which prevents pathogen receptors binding the transferrin (Yoshiga et al., 1997) and this Glossina transferrin is typical in having this modified C-terminal region (Strickler-Dinglasan et al., 2006). Transferrin is consistently downregulated in infected tsetse flies. This is consistent with other studies in insects which commonly show changes in the transcriptional level of transferrin under immune challenge. Thus transferrin is upregulated in mosquitoes infected with filarial worms (Beerntsen et al., 1994) or with a fungus (Aguilar et al., 2005). The protein is modified in Drosophila in response to fungal infection (Levy et al., 2004) and there is evidence for its differential expression during infection in other insects (Seitz et al., 2003; Thompson et al., 2003; Ursic-Bedoya and Lowenberger, 2007). The RNAi experiments provide direct evidence for the involvement of transferrin in tsetse trypanosome interactions (Table 4) although the mechanism remains unknown. Recent results from vertebrates suggest a possible immune signalling function for transferrin resulting in upregulation of the free radical nitrous oxide (Stafford and Belosevic, 2003) so it is interesting to note that it has been proposed that reactive oxygen intermediates (ROI) may be involved in tsetse trypanosome interactions (Hao et al., 2003; Lehane et al., 2003; Munks et al., 2005; Macleod et al., 2007). The putative signalling function of transferrin in vertebrates requires the proteolytic cleavage of the transferrin molecule (Stafford and Belosevic, 2003) and it is also interesting to note that the cleavage site is conserved in insect transferrins (Harizanova et al., 2005; Guz et al., 2007). Reactive oxygen intermediates (ROI) are thought to be important in insect defences (Nappi and Vass, 1998; Ha et al., 2005) including that of tsetse flies (Hao et al., 2003; Lehane et al., 2003; Munks et al., 2005; Macleod et al., 2007). Thus, addition of glucosamine (a known anti-oxidant (Xin et al., 2006)) or other anti-oxidants to the blood meal of tsetse flies results in increased prevalence of trypanosome infections (Maudlin and Welburn, 1987; Macleod et al., 2007). A fly utilising ROI in its defences would need protection from these molecules. Consequently, it is interesting to note the upregulation in library A of lambda crystallin and in library D of catalase, both of which are involved in protection from ROI (Missirlis et al., 2003; Ma et al., 2004). Differential expression of these
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genes in self-cured flies compared with non-infected and infected flies together with other published data (Hao et al., 2003; Macleod et al., 2007) suggest immune protection using superoxides deserves more study in tsetse trypanosome interactions. A lipocalin family member is also present in library A. Members of the lipocalin family are mainly extracellular carriers of lipophilic molecules. However, a subset of the family are cytokines and others have protease inhibiting properties (Logdberg and Wester, 2000) both of which could be important in the insect’s immune response. Clone 2A132 in library A is a homologue of a Drosophila member of the dipeptidase family involved in the hydrolysis of Xaa-Pro dipeptides (EC 3.4.13.9). It is known that proline is central to the metabolism of the fly (Bursell, 1977). It is also interesting to note that the EP isoform of the procyclin external coat of the procyclic, insect form of T. b. brucei is predicted to have 22–30 Glu-Pro (EP) tandem repeats in its C-terminal domain and that the other major procylin coat isoform, GPEET, also has Xaa-Pro in its five or six pentapeptide repeats (Roditi et al., 1987; Richardson et al., 1988; Clayton and Mowatt, 1989). Library B contains a series of proteolytic enzymes which have been downregulated in self-cured flies. This is consistent with studies in other immune-challenged insects where differential regulation of proteolytic enzymes have been noted in microarray experiments. However, the patterns of response reported to date are inconsistent and highly variable, being dependent on the immunogen and route of delivery (De Gregorio et al., 2001; Roxstrom-Lindquist et al., 2004). In addition to downregulation of these proteolytic enzymes, an orthologue of a gene encoding polyA binding protein is downregulated in library B, and this protein is involved in the positive regulation of translation. Given that the fat body is the major organ involved in intermediary metabolism in insects these results taken together may suggest a down regulation of general protein metabolism in self-cured flies. In addition, the upregulation of the genes represented by SSH clones D582, 2D113, 2D79, 2A4 in library D may indicate an upregulation of energy usage in self-cleared flies. This all suggests that trypanosome infection has a considerable effect on metabolism in the tsetse fly, a view which is consistent with recent findings in Drosophila that infection causes a fundamental alteration in metabolism (Dionne et al., 2006). Library C contains a homologue of C1-THF synthase which produces a co-factor used in purine biosynthesis. Trypanosomes, as with other protozoan parasites, are incapable of synthesising purines which they must acquire from their hosts (Carter et al., 2001). The simplest explanation for this observation is the infected fly is replenishing the pool of available purines that the trypanosomes have depleted. This would imply that the trypanosomes have access to the purine pool despite being ‘outside’ the body of the fly (i.e. in the midgut lumen) but how that might be achieved is completely unknown.
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Clone D451, which is differentially regulated in library D, is a homologue of sphingomyelin phosphodiesterase 1. Trypanosome membranes contain sphingosine derivatives and in particular the GPI-anchored proteins of the procyclic coat are associated with sphingolipid/sterol-rich rafts in the trypanosome plasma membrane (Denny et al., 2001). In summary, the SSH approach taken here suggests that a large number of genes are differentially regulated during the trypanosome infection process in tsetse flies. The emerging patterns suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly, (ii) that self-cured flies may be mounting an oxidative stress response and displaying an increase in energy usage. This study provides direct evidence for the involvement of transferrin in protecting flies from trypanosome infection and identifies this as a target for future research. It is probable that many genes are involved in the tsetse/trypanosome interaction. When the G. m. morsitans genome is published, it will open the way for microarray or proteomic-based studies which can efficiently target such genes for further study.The differentially expressed gene lists provided here will form an excellent comparator for such further studies. Acknowledgement This work was supported by funding from the Wellcome Trust. References Aguilar, R., Jedlicka, A.E., Mintz, M., Mahairaki, V., Scott, A.L., Dimopoulos, G., 2005. Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochemistry and Molecular Biology 35, 709–719. Aksoy, S., Gibson, W.C., Lehane, M.J., 2003. Interactions between tsetse and trypanosomes with implications for the control of trypanosomiasis. Advances in Parasitology 53, 1–83. Aksoy, S., Berriman, M., Hall, N., Hattori, M., Hide, W., Lehane, M.J., 2005. A case for a Glossina genome project. Trends in Parasitology 21, 107–111. Beerntsen, B.T., Severson, D.W., Christensen, B.M., 1994. Aedes aegypti – characterization of a hemolymph polypeptide expressed during melanotic encapsulation of filarial worms. Experimental Parasitology 79, 312–321. Boulanger, N., Brun, R., Ehret-Sabatier, L., Kunz, C., Bulet, P., 2002. Immunopeptides in the defense reactions of Glossina morsitans to bacterial and Trypanosoma brucei brucei infections. Insect Biochemistry and Molecular Biology 32, 369–375. Bursell, E., 1977. Synthesis of proline by fat body of the tsetse fly (Glossina morsitans). Metabolic pathways. Insect Biochemistry 7, 427–434. Byers, R.J., Hoyland, J.A., Dixon, J., Freemont, A.J., 2000. Subtractive hybridization – genetic takeaways and the search for meaning. International Journal of Experimental Pathology 81, 391–404. Carter, N.S., Landfear, S.M., Ullman, B., 2001. Nucleoside transporters of parasitic protozoa. Trends in Parasitology 17, 142–145. Clayton, C.E., Mowatt, M.R., 1989. The procyclic acidic repetitive proteins of Trypanosoma brucei – purification and post-translational modification. Journal of Biological Chemistry 264, 15088–15093. Croft, S.L., East, J.S., Molyneux, D.H., 1982. Antitrypanosomal factor in the haemolymph of Glossina. Acta Tropica 39, 293–302. De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide analysis of the Drosophila immune response by using
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