• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of emborepLink to Publisher's site
EMBO Rep. Feb 2004; 5(2): 207–212.
Published online Jan 23, 2004. doi:  10.1038/sj.embor.7400073
PMCID: PMC1298984
Scientific Report

Parasite-specific immune response in adult Drosophila melanogaster: a genomic study

Abstract

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite-specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence.

Introduction

The majority of insect vectors for human parasites are found among dipterans. In an attempt to understand the immunological basis for Anopheles vector capacity, Schneider & Shahabuddin (2000) successfully used Drosophila melanogaster and the malaria parasite Plasmodium gallinaceum as a vector–parasite model system. Ookinetes injected into the fly haemocoel developed into sporozoites that were infective when injected into the chicken host. However, when feeding the flies with parasitized blood or ookinetes, parasite development was hampered, indicating that the important barrier for the parasite to develop resides in the gut of this insect. Either certain mosquito-specific invasion routes are not present in Drosophila, or the malaria parasites encountered immune response mechanisms, against which they have not evolved countermeasures as in Aedes or Anopheles hosts. None of the four immune genes tested were induced by the injected malaria parasites, and the question remains open as to whether antiparasitic response genes were induced.

In the search for genes encoding proteins involved in an antiparasitic response, we chose to infect Drosophila through a natural route with the protozoan parasite Octosporea muscaedomesticae and with fungi, bacteria and virus for comparison. To ensure the induction of genes that are needed to defeat the infection, our intention was to use previously known naturally infecting microorganisms that the fly would survive either completely or for a reasonably long time as compared to conditions in nature. In wildlife populations of D. melanogaster, the mean life expectancy for males is estimated as not more than 3 days (Rosewell & Shorrocks, 1987). Because the immune response in Drosophila has evolved under such premises, it seems logical to study the response during the first few days of its adult life. We assayed the responding genes at one time point that would cover the transcriptional activation of the most well-studied antimicrobial genes. Because in earlier studies, including oral feeding of Drosophila larvae with Erwinia carotovora (Basset et al, 2000), and oral feeding of adult Drosophila with Crithidia sp. (Boulanger et al, 2001) and Serratia marcescens (Kylsten et al, 1990), the assayed effector genes were expressed at 24 h, we chose to use this time point.

We selected a set of microorganisms that infect Drosophila in nature. The protozoan parasite O. muscaedomesticae (Microsporidia) was isolated from the proximal gut epithelium of infected houseflies, Musca domestica, and found to heavily infect, but not kill, D. melanogaster (Kramer, 1973). The fungus Beauveria bassiana infects by penetrating the exoskeleton and kills the host. Despite the fact that it kills within days, it was used as a standard in comparison with other genome-wide screens. The Gram-negative bacterium S. marcescens Db1140 is a derivative of the highly virulent strain Db11 isolated from Drosophila (Flyg & Xanthopoulos, 1983). The Db1140 strain was demonstrated by northern blots to induce expression of the Cecropin A gene in Drosophila when administered through the food (Kylsten et al, 1990). Drosophila C virus (DCV) is a picorna-like single-stranded RNA virus widely spread among Drosophila. When adult Drosophila were fed with DCV, 30–50% of the flies died within 6 days after infection (Gomariz-Zilber et al, 1995).

This is the first whole-genome study on antiparasitic response in D. melanogaster. We demonstrate that Drosophila responds by upregulating a new and specific set of genes on an oral infection with Octosporea. Many of the genes with unknown function have signal peptides and will be a subject for future analyses of antiparasitic activity.

Results and Discussion

Genome data analysis

The Drosophila gene expression in response to different microbes was examined after 24 h of natural infection of adult males. The RNA was hybridized to Affymetrix Drosophila GeneChips, and Affymetrix MAS 5.0 software was used for the calculation of expression and statistical analyses of the chips. (Contact the author at es.us.tmg@eyaf.dirgni for supplementary information table 1). Duplicates of each infection were compared to duplicates of normal flies in a 2 × 2 matrix (supplementary information text part A online). The genes that were significantly increased (P<0.0025, Wilcoxon's signed ranks test) in all four comparisons were defined as induced genes. In total, 427 genes were induced and selected for further analysis (supplementary information table 2 online). The fungal infection generated the strongest response, with 298 genes induced, and the parasitic infection induced 127 genes. In the viral and bacterial infections, a low number of genes were significantly induced: 11 and 10, respectively. The significantly induced genes are found in many different functional classes (Fig 1). A common feature in the four infections was that many of the genes encode enzymes, in particular serine proteases: Octosporea, 35% enzymes (13% serine proteases); Beauveria, 24% (8%); Serratia, 60% (50%); and DCV, 36% (27%) (supplementary information table 4 online).

Figure 1
Distribution of induced Drosophila genes according to their functional categories based on NetAffxTM GO.

Unique or common induction of a gene was determined by comparing the expression of each induced gene selected in one treatment with its expression in other treatments (supplementary information text part A online).

The numbers of uniquely induced genes were 214, 59 and 2 in response to Beauveria, Octosporea and DCV, respectively; this constitutes 65% of the 427 induced genes and thereby demonstrates specificity in the immune response (Fig 2). Many genes were induced in several infections; 16 genes are designated as common in response to all four infections. The genes in common encode the antimicrobial proteins Attacin A, Cecropin A1, Cecropin A2, Drosomycin and Metchnikowin, as well as acetyl-CoA homeostasis (CG8628), one serine protease (CG6483) and nine genes with unknown functions (supplementary information table 3 online).

Figure 2
Number of induced genes that are common or unique in the different infections. Significantly induced genes (cf. Fig 1) were compared with other infections for uniqueness.

Confirmation of genes responding to Beauveria infection

The antifungal peptide genes Drosomycin and Metchnikowin (Ekengren & Hultmark, 2001, and references therein) were heavily induced by Beauveria in our study: 14.3- and 19.9-fold, respectively (Table 1). In a similar experiment, where the D. melanogaster strain OregonR was naturally infected with the same strain of Beauveria, the response at 24 h was lower compared to our results: Drosomycin 6.4-fold and Metchnikowin 4.4-fold (De Gregorio et al, 2001). The Canton S flies used in our study died within 5 days (Fig 3), whereas 90% of the OregonR flies used by De Gregorio et al (2002) were still alive at that time point. This may indicate that our flies were more heavily infected, or that there is a certain genetic difference between these two wild-type isolates of D. melanogaster.

Figure 3
Survival of 20 flies from each experiment after the 24-h infection period using different microbes in sugar solution. The flies were placed on normal fly food at day 0. Data represent the mean value of duplicates. The results correspond well to earlier ...
Table 1
Fold changes of selected immunity-related genes

Turandot M (TotM) is a stress-induced humoral protein gene in Drosophila, earlier shown to be upregulated by the Gram-negative bacterium Enterobacter cloacae β12 when injected into adults (Ekengren & Hultmark, 2001). In our study, TotM is induced 13.7-fold by fungal infection (Table 1) and 2.4-fold by bacterial feeding. The strong fungal induction could reflect the stress response inferred by cuticular penetration. Notably, in De Gregorio's study TotM (CG14027) is, after 24 h, upregulated 3.6-fold by the fungal infection and 13.6-fold by septic injury. This is a recurring pattern of contrasting results on fungal versus bacterial infections, when we compare our results with theirs (see Speculation).

Serine proteases

Serine proteases and their inhibitors (serpins) are important early regulators in several invertebrate responses, including haemolymph coagulation, melanization and induction of antibacterial peptide genes (Jiang & Kanost, 2000). In the present study, 37 of all 240 annotated serine protease genes (http://flybase.bio.indiana.edu) were induced. Interestingly, serine proteases induced by DCV and Serratia and some of those induced by Octosporea infections, Ser4, Ser99Da, Ser99Db, CG2229, CG8869, CG8871 and CG16749, were downregulated in the Beauveria infection (supplementary information table 4 online). It is possible that one set of serine proteases is induced to encounter infections through the gut and another set is induced on infection through the cuticle. Moreover, Beauveria and Octosporea also induced different sets of serpins (supplementary information table 5 online).

Among the serine protease genes that were significantly changed in response to the microorganisms used in our study (supplementary information table 4 online), ten were designated as Drosophila immune response genes also by De Gregorio et al (2001): Ser4, Ser99Dc, CG5909, CG6639, CG8215, CG8869, CG8871, CG11841, CG11842 and CG16749. The serine protease genes that are up- or downregulated after fungal infection in our study are changed in the same direction in their study, although at a lower level. On the contrary, some of the serine protease genes upregulated by bacteria in our study are found to be downregulated after septic injury in the study of De Gregorio et al (2001) (see Speculation).

Parasite response genes among immunity-related genes

After parasitic infection, 127 genes were recorded as significantly induced. Since the flies infected with the parasite survived, we can conclude that this insect can produce all the factors needed to mount the parasite invasion (Fig 3). The Drosophila flies infected with Octosporea gave a fundamentally different response by known immunity-related genes as compared to the other microorganisms used in this study. For instance, several lysozyme genes, Lys B, C, D, E and CG16756, were induced by Octosporea, which is contrary to the response of the fungal infection, in which lysozyme genes were downregulated (Table 1). Although lysozymes are induced in response to bacterial injection of Lepidoptera pupae (Sun et al, 1991), a similar induction in Drosophila has not been reported before. Lys B, C, D and E, which were upregulated by Octosporea in the present study, are closely related genes that are normally constitutively expressed in the midgut of Drosophila adults (Daffre et al, 1994). An important characteristic of many lysozymes is their chitinase activity. Recently, it was experimentally demonstrated that Lys D from D. melanogaster displays chitinase activity (Regel et al, 1998). Chitin is known as a major component of arthropod cuticle, fungal cell walls and the exoskeleton of some algae. It is also a component of Octosporea spores and, as such, a substrate and target for this activity. An interesting observation in this context is that transgenic expression of two Plasmodium sporozoite surface antigens in Drosophila strongly affects the lysozyme gene expression profile (M. Shahabuddin, personal communication).

We found that out of the 127 genes upregulated in response to Octosporea, 59 were uniquely expressed and, among these, 48 are newly retrieved immune responsive genes (Table 2), not upregulated in other extensive genome studies (supplementary information text part B online), of responses to bacteria and fungi in Drosophila (De Gregorio et al, 2001; Irving et al, 2001; Boutros et al, 2002). In the following, we highlight some of the genes in Table 2, which have been earlier known to possess domains with putative immune functions.

Table 2
Parasite-specific genes

The parasite-induced gene CG2736 encodes a scavenger receptor with a CD36 family domain (http://flybase.bio.indiana.edu). A common feature of scavenger receptors is their ability to bind a broad range of ligands, including different microbes, indicating a potential role in innate host defence (Gough & Gordon, 2000). The scavenger receptor CD36 is involved in diverse cellular activities, like recognition and phagocytosis of apoptotic cells. Notably, CD36 is also a major receptor for the adherence of the Plasmodium falciparum-infected erythrocyte on endothelial cells (Tachado et al, 1997. and references therein). We speculate that the induction of the scavenger receptor gene could be due to the interaction between Octosporea and the endothelial cells.

The parasite-specific gene CG11315 is of particular interest, because it was earlier shown to contain two ML domains (MD-2-related lipid recognition), recognizing lipid moieties (Inohara & Nunez, 2002). In mammals, MD-2 serves as a cofactor for TLR4 (Toll-like receptor 4) and is essential for the response to lipopolysaccharides (LPS). Similar to bacterial LPS, several lipid-containing parasite antigens, like GPI links, are highly immunogenic in mammals (Tachado et al, 1997). Such molecules may be important targets for the recognition and signalling machinery also in insects. Apart from CG11315, seven other genes containing ML domains have been earlier identified in Drosophila (Inohara & Nunez, 2002).

Lectins are carbohydrate-binding proteins suggested to participate in agglutination of cells, glycoprotein clearance and innate immunity. One Drosophila gene (CG9978), which is parasite specific, contains a galactose-specific C-type lectin domain (CTLD) (Theopold et al, 1999). Similarly, a putative galactose-specific lectin was induced in the gut of Anopheles gambiae, on feeding with malaria-parasitized blood (Dimopoulos et al, 1997).

Most of the uniquely expressed genes encode gene products that lack known domains or other similarities to known proteins. To predict interesting candidates among these, to be tested for antiparasitic activity, we performed an in silico screen for the presence of signal peptide (http://www.cbs.dtu.dk/services/SignalP) and the absence of transmembrane domains (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html). From this screen, nine genes were found and, of these, eight have unknown functions and will be further characterized (Table 2).

The overall scope of this study was to identify parasite-specific response factors, and ultimately effector molecules that act on vector-borne protozoan parasites. Such molecules could serve as structural models for antiparasitic drug design. Another approach is the introduction of antiparasitic genes into bacterial symbionts residing in the gut of insect vectors. For this purpose, our search for strictly antiparasitic molecules is important.

Speculation

When we compared our results with the study of De Gregorio et al (2001), who also used Affymetrix Drosophila GeneChips, we observed striking differences. Out of 28 immune genes that are in common, 26 responded more strongly to bacteria than to fungi in their study, whereas our results were the opposite. Similarly, the three serine protease genes that were upregulated in our study after bacterial feeding were downregulated in their study. They challenged adult flies with nonpathogenic bacteria (Micrococcus luteus mixed with Escherichia coli) by septic injury or externally with pathogenic fungi, which means that both microorganisms entered through the cuticle. We used the same fungal infection, but oral feeding of bacteria (S. marcescens). The opposing results suggest that the method of infection is crucial for the specificity of the response. However, it is difficult at this point to draw firm conclusions, because a sterile injury control was not included in the study of De Gregorio et al (2001) and different bacterial species were used in the two studies, which could have given different responses.

Methods

Microbes. S. marcescens Db1140 (obtained from A. Boman, Karolinska Institutet, Sweden) was grown to OD600 0.5 in Luria–Bertani broth (LB) supplemented with streptomycin at 30 μg ml−1 (Flyg & Xanthopoulos, 1983).

DCV EB strain was obtained from P.D. Christian (NIBSC, UK) (Johnson & Christian, 1998) and the infectivity was verified in Drosophila Schneider S2 cells.

O. muscaedomesticae (parasitic protozoan) isolated from Sarcophaga bullata was obtained from L. Solter (Illinois Natural History Survey, Illinois, USA). B. bassiana (fungus) was obtained from B. Lemaitre (Centre de Genetique Moleculaire, Gif-sur-Yvette, France) and was sporulated on malt-agar plates.

Natural infections. Flies of the Canton S strain of D. melanogaster were obtained from M. Rasmusson (Umeå University, Sweden) and were maintained on cornmeal and yeast food at 25°C, at 70% humidity and a 12 h light/dark cycle. We collected 1–3-day-old male flies under ether anaesthesia and placed them in vials with a piece of wettex covering the bottom and soaked in 2 ml of 1% sugar solution or 1% sugar solution containing the microbe of interest. Flies were infected with B. bassiana by gently shaking them in spores according to Lemaitre et al (1997) and then transferring them to the 1% sugar solution diet. Control flies were fed on 1% sugar solution only. After 24 h, flies were ether anaesthetized and frozen in liquid N2 or kept in vials containing normal food for survival experiments.

Feeding and starvation. To determine the efficiency of feeding, approximately 100 flies were fed with 0.05% bromophenol blue (Schmid, Köngen, Germany) in 1% sugar solution for 30 or 150 min. As judged by their bluish abdomens, 75% of the flies had ingested sugar solution after 30 min and 95% after 150 min.

The survival of flies feeding on 1% sugar was compared with survival on normal food. Six vials with 20 flies in each were used for each treatment. No difference in survival was detected in the first 3 days, indicating that the flies were not heavily starved.

Sample preparation and analysis. For each infection as well as for the control, two independent experiments were performed, each of them used for one genome array analysis. For each experiment, total RNA was isolated from 100 male flies and hybridized to the Affymetrix Drosophila GeneChips in the Affymetrix core facility at NOVUM (Karolinska Institutet, Sweden), according to Affymetrix's protocols (supplementary information text part C online).

Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s1.xls, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s2.pdf, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s3.pdf, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s4.pdf, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s5.pdf, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s6.pdf, http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s7.pdf and http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400073s8.pdf).

Supplementary Material

Supplementary Table 1

Supplementary Information Text part A

Supplementary Information Table 2

Supplementary Information Table 4

Supplementary Information Table 3

Supplementary Information Table 5

Supplementary Information Text part B

Supplementary Information Text part C

Acknowledgments

We thank J. Lidén for helpful discussions. This project was supported by the Swedish Research Council to I.F., the Lars Hierta Foundation, the Hierta-Rietzius Foundation of the Royal Swedish Academy of Sciences, the Maud and Birger Gustavsson Foundation and the Nilsson-Ehle Foundation of the Royal Physiographic Society to O.T., and the Sven and Lilly Lawski Foundation to K.R.L. and O.T.

References

  • Basset A, Khush RS, Braun A, Gardan L, Boccard F, Hoffmann JA, Lemaitre B (2000) The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response. Proc Natl Acad Sci USA 97: 3376–3381 [PMC free article] [PubMed]
  • Boulanger N, Ehret-Sabatier L, Brun R, Zachary D, Bulet P, Imler JL (2001) Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp. Insect Biochem Mol Biol 31: 129–137 [PubMed]
  • Boutros M, Agaisse H, Perrimon N (2002) Sequential activation of signaling pathways during innate immune responses in Drosophila. Dev Cell 3: 711–722 [PubMed]
  • Daffre S, Kylsten P, Samakovlis C, Hultmark D (1994) The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract. Mol Gen Genet 242: 152–162 [PubMed]
  • De Gregorio E, Spellman PT, Rubin GM, Lemaitre B (2001) Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci USA 98: 12590–12595 [PMC free article] [PubMed]
  • De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B (2002) The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J 21: 2568–2579 [PMC free article] [PubMed]
  • Dimopoulos G, Richman A, Muller HM, Kafatos FC (1997) Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc Natl Acad Sci USA 94: 11508–11513 [PMC free article] [PubMed]
  • Ekengren S, Hultmark D (2001) A family of Turandot-related genes in the humoral stress response of Drosophila. Biochem Biophys Res Commun 284: 998–1003 [PubMed]
  • Flyg C, Xanthopoulos KG (1983) Insect pathogenic properties of Serratia marcescens. Passive and active resistance to insect immunity studied with protease-deficient and phage-resistant mutants. J Gen Microbiol 129: 453–464
  • Gomariz-Zilber E, Poras M, Thomas-Orillard M (1995) Drosophila C virus: experimental study of infectious yields and underlying pathology in Drosophila melanogaster laboratory populations. J Invertebr Pathol 65: 243–247 [PubMed]
  • Gough PJ, Gordon S (2000) The role of scavenger receptors in the innate immune system. Microbes Infect 2: 305–311 [PubMed]
  • Inohara N, Nunez G (2002) ML—a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem Sci 27: 219–221 [PubMed]
  • Irving P, Troxler L, Heuer TS, Belvin M, Kopczynski C, Reichhart JM, Hoffmann JA, Hetru C (2001) A genome-wide analysis of immune responses in Drosophila. Proc Natl Acad Sci USA 98: 15119–15124 [PMC free article] [PubMed]
  • Jiang H, Kanost MR (2000) The clip-domain family of serine proteinases in arthropods. Insect Biochem Mol Biol 30: 95–105 [PubMed]
  • Johnson KN, Christian PD (1998) The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family. J Gen Virol 79: 191–203 [PubMed]
  • Kramer JP (1973) Susceptibility of sixteen species of muscoid flies to the microsporidean parasite Octosporea muscaedomesticae. J NY Entomol Soc 81: 50–53
  • Kylsten P, Samakovlis C, Hultmark D (1990) The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. EMBO J 9: 217–224 [PMC free article] [PubMed]
  • Lemaitre B, Reichhart JM, Hoffmann JA (1997) Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci USA 94: 14614–14619 [PMC free article] [PubMed]
  • Regel R, Matioli SR, Terra WR (1998) Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function. Insect Biochem Mol Biol 28: 309–319 [PubMed]
  • Rosewell J, Shorrocks B (1987) The implications of survival rates in natural populations of Drosophila: capture–recapture experiments on domestic species. Biol J Linn Soc 32: 373–384
  • Schneider D, Shahabuddin M (2000) Malaria parasite development in a Drosophila model. Science 288: 2376–2379 [PubMed]
  • Sun S-C, Åsling B, Faye I (1991) Organization and expression of the immunoresponsive lysozyme gene in a giant silk moth, Hyalophora cecropia. J Biol Chem 266: 6644–6649 [PubMed]
  • Tachado SD, Gerold P, Schwarz R, Novakovic S, McConville M, Schofield L (1997) Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties. Proc Natl Acad Sci USA 94: 4022–4027 [PMC free article] [PubMed]
  • Theopold U, Rissler M, Fabbri M, Schmidt O, Natori S (1999) Insect glycobiology: a lectin multigene family in Drosophila melanogaster. Biochem Biophys Res Commun 261: 923–927 [PubMed]

Articles from EMBO Reports are provided here courtesy of The European Molecular Biology Organization
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...