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1 CEA, DSV, iRTSV, 17 rue des Martyrs, Grenoble F-38054, France
2 INSERM U873, Grenoble F-38054, France
3 Université Joseph Fourier, Grenoble F-38000, France
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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During evolution, pathogens have developed numerous strategies to inhibit host defense mechanisms such as toxins directly targeting RhoGTPases functions (Aktories & Barbieri 2005). The Exoenzyme S (ExoS) of the Gram-negative pathogen Pseudomonas aeruginosa contains a GAP domain at its N-terminus that targets RhoGTPases and consequently inhibits cytoskeletal reorganization and phagocytosis in cultured macrophages (Goehring et al. 1999; Rocha et al. 2003). ExoS is directly injected into the host cell cytoplasm by the type III secretion system (TTSS), a set of virulence factors found in many pathogenic Gram-negative bacteria that can be crucial for the initiation of infection and survival of the pathogen in the host (Galan & Collmer 1999; Cornelis & Van Gijsegem 2000). Pseudomonas aeruginosa is a major opportunistic pathogen for humans associated with infections in patients with diverse pathologies such as AIDS, cancer, severe burns and wounds, and cystic fibrosis. We and others demonstrated that this bacteria use common mechanisms of virulence to infect insects and mammals (Rahme et al. 2000; D’Argenio et al. 2001; Fauvarque et al. 2002). In particular, we previously demonstrated that the TTSS contributed to fast fly killing induced by P. aeruginosa (Fauvarque et al. 2002) and that the GAP domain of ExoS (ExoSGAP) inhibited fly immunity by preventing hemocytes-dependent pathogen phagocytosis (Avet-Rochex et al. 2005).
Since RhoGTPases are the main putative targets of ExoSGAP domain both in cultivated cells (Goehring et al. 1999; Krall et al. 2002) and in Drosophila (Avet-Rochex et al. 2005), we assessed in this study the potential contribution of each RhoGTPase, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42, to fly resistance to P. aeruginosa infections. We found that Rac2 is required for fly resistance to P. aeruginosa and to a large set of Gram-negative and Gram-positive bacteria. We further showed that Rac2 is dispensable for the induction of antimicrobial peptide synthesis by either Toll or the Immune deficiency (Imd) pathway, as well as for JNK-dependent transcriptional activation of its nuclear target puckered (puc) in response to bacterial infections. The Rac2 immune-deficient phenotype is mainly attributable to defective engulfment of pathogens since Rac2-mutant plasmatocytes exhibited strong reduction in the level of phagocytosis of both Gram-negative and Gram-positive bacterial particles. This immune phenotype is reminiscent to that observed in transgenic strains expressing ExoSGAP, suggesting that Rac2 function is inhibited by ExoSGAP in these cells. Indeed, co-expression of Rac2 and ExoSGAP led to normal survival kinetic of flies following P. aeruginosa infection. All together, our data show that Drosophila Rac2 is a key player in the cellular immune response to bacterial infection in vivo.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We previously demonstrated that the GAP domain of the P. aeruginosa toxin ExoS (ExoSGAP) inhibited fly immunity by preventing hemocyte-dependent pathogen phagocytosis (Avet-Rochex et al. 2005). Since RhoGTPases are the main actors of cytoskeleton reorganization required for phagocytosis and since they all are putative targets of ExoSGAP domain, we assessed the contribution of each RhoGTPase, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42, to fly resistance to infections by P. aeruginosa. We tested several mutant combinations affecting either Rho1, Cdc42 or each of the three Drosophila RacGTPases Rac1, Rac2 and Mtl for their resistance to P. aeruginosa infections by septic injury. In the case of Rho1 and cdc42 whose loss-of-function mutations are embryonic lethal (Fehon et al. 1997; Magie et al. 1999; Genova et al. 2000), heterozygous flies Rho172R/+ and Cdc424/+ or heteroallelic Cdc424/Cdc422 flies were infected by P. aeruginosa PAO1 strain. In all cases, flies displayed normal resistance compared to control flies (not shown). Loss-of-function mutants of each of the three Rac-encoding genes Rac1 (Rac1j11), Rac2 (Rac2
) and Mtl (Mtl) were described to be viable (Hakeda-Suzuki et al. 2002; Ng et al. 2002). However, only Rac2 and the strong hypomorphic mutation Rac1j10 displayed normal viability and no developmental delay, and could be then further tested for their resistance to infections. In contrast, Rac1J11 and Mtl were either poorly viable or presented strong developmental delay; they were thus tested in heterozygous, Rac1J11/+ and Mtl/+, or heteroallelic Rac1j10/Rac1J11, or homozygous hypomorphic Rac1j10/Rac1J10 conditions. All genetic combinations carrying either Rac1 or Mtl mutations displayed normal resistance following infection with P. aeruginosa (shown for Rac1J10, Fig. 1A). In contrast, Rac2 homozygous flies displayed a strong sensitivity to P. aeruginosa, exhibiting mortality kinetic almost similar to that displayed by the immune-deficient TAK12 flies (Fig. 1B). In addition, Rac2/Df(3L)pbl-X1 flies, where Df(3L)pbl-X1 includes the Rac2 genetic unit, behaved as Rac2 homozygous flies following Pseudomonas infections (Fig. 1B), indicating that Rac2 sensitivity is not due to a cryptic mutation and that the loss-of-function Rac2 behaved, as expected, as a null allele. These results suggest that Rac2 plays a major role among RhoGTPases in fly resistance to P. aeruginosa.
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flies were infected by feeding with medium contaminated by P. aeruginosa. Once again, Rac2 mutant flies died more rapidly than Rac2/+ control flies (Fig. 1C). The contribution of Rac2 in fly resistance to other kind of bacterial infections was further evaluated by infecting Rac2
mutants with several Gram-negative and Gram-positive pathogenic bacterial strains. Compared to Rac2/+ control flies, Rac2 mutant displayed a general sensitivity to pathogenic bacteria such as the Gram-negative bacteria Enterobacter cloacae and Agrobacterium tumefaciens (Fig. 1D,E), and the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis (Fig. 1F,G). In contrast, Rac2 flies were not sensitive to the strain Escherichia coli (Fig. 1H). By comparison, 60% of TAK12 mutant flies, which are impaired in antimicrobial peptide synthesis, died in 3–5 days in the same experimental conditions (Fig. 1H). All these results indicate that Rac2 plays a major non-redundant role in Drosophila innate immunity.
Rac2 does not affect antimicrobial peptides synthesis
Antimicrobial peptides production by fat body cells is a major immune mechanism contributing to bacterial clearance following fly infection. The Imd pathway is strongly stimulated by Gram-negative pathogens resulting in the activation of the NF-
B transcription factor Relish which in turn activates the transcription of numerous genes, in particular of the antimicrobial peptide diptericin (Lemaitre et al. 1995; Georgel et al. 2001). We observed the same diptericin expression profile in Rac2 mutant flies as in Rac2/+ or w1118 control flies following infection by either P. aeruginosa (Fig. 2A) or Enterobacter cloacae (Fig. 2B), indicating that Rac2 is not required in vivo for the activation of the Imd pathway following Gram-negative infections. In both cases, a normal induction kinetic of antimicrobial peptides was observed at earlier time points (see additional quantifications below the Northern pictures in Fig. 2A,B). The Toll pathway is strongly activated by fungi and Gram-positive bacterial infection resulting in the activation of the NF-B-like molecule Dif which in turn activates another set of antimicrobial peptides encoding genes, including drosomycin (Lemaitre et al. 1996). The expression of drosomycin was normally activated in Rac2 mutant compared to Rac2/+ control flies following infection by the Gram-positive bacteria Enterococcus faecalis (Fig. 2C). The Toll pathway is also activated and required in the case of P. aeruginosa infections (Apidianakis et al. 2005). Indeed, a strong drosomycin expression was observed at 14 and 20 h following P. aeruginosa infection both in Rac2/+ or w1118 control flies and in Rac2 mutant flies (Fig. 2D). In an independent experiment, we could observe the early activation of drosomycin, from 6 h post-infection, which might partly depend on co-activation by the Imd pathway since P. aeruginosa is a Gram-negative bacteria. Once again, this activation was not affected in Rac2 flies (Fig. 2D, additional quantification on the right). Our results indicate that Rac2 is not required for the activation of the two Drosophila NF-B-like pathways, Toll and Imd, following bacterial infections by septic injury.
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The JNK pathway induces the transcriptional activation of several host defense-response genes during stress and immune response (Rincon et al. 2000; Boutros et al. 2002; Zhuang et al. 2006). The JNK pathway was reported to be activated early, about 30–60 min, after fly septic injury with Enterobacter cloacae (Park et al. 2004) or with a mixture of Gram-negative and Gram-positive pathogens (De Gregorio et al. 2001). In these studies, JNK signaling towards the nucleus was observed through the induction of its nuclear target puckered (puc). Since RacGTPases are capable of controlling the activation of the JNK pathway during Drosophila development (Noselli & Agnes 1999; Woolner et al. 2005) as well as the JNK-dependent actin stabilization in activated hemocytes (Williams et al. 2006), we further evaluated whether JNK-dependent transcriptional activation would be affected in Rac2-mutant flies during the course of a bacterial infection. We observed a slight induction of the JNK-responsive gene puc at 30 min and 1 h following infection by Enterobacter cloacae which was not significantly affected in Rac2 mutant flies (not shown). In addition, we detected a strong induction of puc at 14–20 h post-infection by P. aeruginosa (Fig. 2A) and a modest induction in the case of Enterobacter cloacae (Fig. 2B). This late activation of puc was previously observed in the case of an oral infection by P. entomophila (Vodovar et al. 2005) but, to our knowledge, was not reported following septic injury. This late activation of puc was not affected in Rac2-mutant compared to Rac2/+ or w1118
control flies. In addition, we observed that the puc transcription level remained constant and was unaffected in Rac2-mutant flies following infection by the Gram-positive bacteria Enterococcus faecalis (not shown). Our experiments indicate that Rac2 is not essential for the JNK-dependent activation of puc during bacterial infections in vivo at the systemic level.
Rac2 may not be involved in JNK signaling towards the nucleus or, alternatively, it might play redundant function with other RhoGTPases in this process. If this would be the case, ectopic expression of Rac2 should induce JNK-dependent activation of puc. To assess this hypothesis, UASRac2 transgenic lines were constructed and over-expression of Rac2 was obtained by crossing UASRac2 flies with HSGal4 driver lines (see Experimental procedures). In these conditions, we observed a strong induction of Rac2 expression itself, but no significant induction of puc (Fig. 3). These results indicate that Rac2 does not contribute to JNK-dependent transcriptional activation of its nuclear target puc.
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In genomic transcriptional studies, Rac2 was found to be expressed in the hemocyte lineage (Irving et al. 2005). As already reported (Williams et al. 2005), we observed that plasmatocytes and crystal cells differentiated normally in Rac2-mutant larvae even if, repeatedly, a slight decreased number and mis-shape of these cells could be seen in Rac2-mutant compared to Rac2/+ control larvae (not shown). Lamellocytes also differentiated normally after wasp egg infection although they were not able to form a proper capsule around the invading pathogen (Williams et al. 2005). Our observations and other studies thus indicate that the Rac2 mutation does not significantly affect larval hematopoiesis but rather cell morphogenetic changes. Moreover, previous studies indicate that Rac2 is expressed in Drosophila SL2 cells upon LPS stimulation (Boutros et al. 2002) and that it would be required for bacterial engulfment in S2 cells (Stuart et al. 2005; Stroschein-Stevenson et al. 2006). However, these in vitro studies on S2 cells provided conflicting results (see Discussion), and Rac2 requirement in larval primary macrophages has not been assessed yet. We thus evaluated whether Rac2 would be required for bacterial phagocytosis by circulating plasmatocytes. For that purpose, the phagocytic index of wild-type and Rac2-mutant plasmatocytes was quantified on circulating blood cells isolated from late wandering third-stage larvae. Rac2 mutation led to a reduction of 66% of the phagocytic index (P < 0.01) in the case of E. coli bioparticles and of 35% (P < 0.01) in the case of S. aureus (Fig. 4). Our study thus demonstrates that Rac2 contributes to bacterial phagocytosis by larval circulating plasmatocytes which constitutes the primary macrophages in Drosophila.
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Since Rac2 is specifically required for bacterial phagocytosis whereas it is dispensable for the fat-body-dependent expression of antimicrobial peptides, its function might be required mainly in the hemocyte lineage during Drosophila immune response. To assess this hypothesis, we expressed the UASRac2 transgene in the hemocyte lineage through the srpGal driver line in either a wild-type or a Rac2 mutant context. In fact, we first observed that hemocyte-directed expression of Rac2 resulted in fly lethality: most of srpGal4, UASRac2 did not hatch at 20 or 25 °C. Therefore, flies were raised at 17 °C and put at 25 °C only 24 h before the infection experiments to circumvent developmental lethality of Rac2 over-expressing flies. We further observed that these surviving flies presented an increased sensitivity to P. aeruginosa following septic or oral infections (shown in the case of an oral infection, Fig. 5A). The loss of viability and increased sensitivity to Pseudomonas infection phenotypes, of flies over-expressing Rac2, were stronger with the srpGal4 driver line than with the daGal4 driver line (Fig. 5B), which might induce ubiquitous expression of Rac2, but at lower level, especially into the hemocyte lineage. The increased sensitivity of Rac2 over-expressing flies to P. aeruginosa suggests that Rac2 activity dominantly causes a misregulation of cytoskeleton changes in the hemocyte lineage, thus perturbing their function during the cellular immune response.
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phenotype in the case of P. aeruginosa infections by septic injury. Nevertheless, hemocyte-directed expression of the UASRac2 transgene was able to partially rescue the increased sensitivity of Rac2 mutant flies to P. aeruginosa in the case of an oral infection (Fig. 5C). These results support both the hypothesis that Rac2 is mainly required in phagocytic cells during the Drosophila immune response and that the level of Rac2 activity needs to be tightly regulated in these cells. ExoSGAP counteracts ectopic Rac2 activity during eye and wing morphogenesis
The immune phenotype of Rac2 mutant, which is associated with defective phagocytosis, is very similar to the immune-deficient phenotype observed when expressing the GAP domain of the P. aeruginosa ExoS toxin in transgenic flies (Avet-Rochex et al. 2005). We previously demonstrated that ExoSGAP acted as a negative regulator of the ectopic activity of the RhoGTPases Rac1, Cdc42 and Rho1 in the Drosophila eye (Avet-Rochex et al. 2005). Given the contribution of Rac2 in Drosophila resistance to P. aeruginosa, we tested whether the expression of ExoSGAP would similarly inhibit Rac2 activity in the adult fly. Accordingly, Rac2 was over-expressed in the developing eyes and wings using GMRGal4/UASRac2 and enGal4/UASRac2 flies, respectively. Tissue-directed expression of Rac2 induced strongly roughen and reduced eyes, and strong morphological defects in the wing (Fig. 6C,G compared to control flies, Fig. 6A,E). Co-expression of ExoSGAP with Rac2 restored eye and wing morphologies towards a normal shape and structure (Fig. 6D,H), with limited defects that appear similar to those observed when expressing the ExoSGAP toxin alone (Fig. 6B,F). These results demonstrate that ExoSGAP acts as a negative regulator of ectopic Rac2 activity in the adult fly.
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As previously reported, directed expression of the ExoS toxin GAP domain either ubiquitously, through the driver line daGal4, or mainly in the hemocyte lineage, through the driver line srpGal4, induced an increased fly susceptibility to P. aeruginosa infections (Avet-Rochex et al. 2005). Since Rac2 is specifically required for normal fly resistance to P. aeruginosa, we postulated that Rac2 could be one of the main endogenous host factor inhibited by ExoSGAP in the hemocyte lineage. In this case, over-expression of Rac2 might rescue the effect of expressing ExoSGAP on fly immunity. Unfortunately, although flies co-expressing Rac2 with ExoSGAP with the srpGal4 driver line at 17 °C displayed a mortality kinetic similar to control flies (not shown); this result was not informative, since, in these conditions, the ExoSGAP expression level was not sufficient to significantly increase fly sensitivity to P. aeruginosa (not shown). Then, we used the daGal4 driver line to co-express ExoSGAP and UASRac2 at 20 °C, since, as mentioned above, daGal4-driven expression of Rac2 was less deleterious compared to srpGal4 (Fig. 5A,B). In these flies, the co-expression of Rac2 rescued the increased sensitivity induced by ExoSGAP expression, following oral infection with P. aeruginosa (Fig. 5D). These results indicate that Rac2 over-expression can rescue the inhibitory effect of ExoSGAP on fly cellular immunity, indicating that Rac2 might be the main host factors whose activity is inhibited by the GAP domain of ExoS in these cells during the course of an infection.
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Discussion |
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Due to the poor viability or early lethality of null mutants affecting the other RhoGTPases-encoding genes (Rac1, Mtl, Rho1 and Cdc42), only partial loss-of-function mutants have been tested. Although these mutants displayed no increase in susceptibility to P. aeruginosa, we cannot exclude a contribution of these GTPases to fly immunity. Interestingly, Rac2 plays a key role in the melanization process during clot or capsule formation (Bidla et al. 2007; Williams et al. 2007), and Rac2 expression, but not Rac1, Mtl, Rho1 and Cdc42, is activated in microarray experiments in SL2 cells upon LPS stimulation (Boutros et al. 2002). Specific activation of Rac2 in phagocytic cells following immune challenge is in accordance with our observation that Rac2 plays a non-redundant function in fly resistance to bacterial infections.
The sensitivity of Rac2 mutant to bacterial infections is not due to a reduced level of antimicrobial peptides production, since we observed a normal induction of both Toll and Imd pathways following septic injury by either Gram-positive and Gram-negative bacteria, respectively. If minor changes occurred, they were either not detectable or not significant, and they could not account for the observed immune-deficient phenotype. The JNK pathway is another signaling pathway which is induced by bacterial infection, both at the systemic level and in the hemocyte lineage where its activation control cell shape changes. We show here that in Rac2 mutant, JNK signaling to the nucleus is not affected at the systemic level. However, this does not exclude a function of Rac2 in the control of JNK-dependent cytoskeleton reorganization in larval plasmatocytes and during lamellocytes differentiation as previously reported (Williams et al. 2005). Since Rac2 plasmatocytes displayed strong defects in the internalization of both E. coli and S. aureus bioparticles, the sensitivity of Rac2 mutants to bacterial infection could be attributable to defective phagocytosis by these cells which correspond to the Drosophila primary macrophages. Accordingly, Rac2 mutant displayed no sensitivity to the strain E. coli. Indeed, previous studies demonstrated that inhibiting phagocytosis by latex beads did not result in fly sensitivity to E. coli (Elrod-Erickson et al. 2000). In final support of the hypothesis that Rac2 is essentially required in the hemocyte lineage during Drosophila immune response, the hemocyte-directed expression of Rac2, although being very deleterious for fly survival and resistance to infection, was capable of partially rescuing the Rac2 loss-of-function phenotype, at least in the case of an oral infection by P. aeruginosa.
In previous studies, RNAi experiments provided conflicting results about the differential contribution of Rac2 to bacterial internalization by S2 cells. Rac2 as well as Rac1 and Cdc42 were found to contribute to the phagocytosis of both Gram-negative E. coli or of the yeast Candida albicans in S2 cells in an extensive RNAi genetic screen (Stroschein-Stevenson et al. 2006). In contrast, Rac2 was described to be required for the internalization of the Gram-positive S. aureus but to a lesser extent for that of E. coli in S2 cells in an independent study (Stuart et al. 2005). Finally, Rac2 was also described to be required for the internalization and the killing of the intracellular pathogen Mycobacterium fortuitum by S2 cells (Philips et al. 2005). Our study provides physiological evidence that Rac2 is in fact required for the internalization of both Gram-negative and Gram-positive bacterial particles by living plasmatocytes, a defect which is correlated with an increased sensitivity of mutant flies towards both types of pathogens.
Among RhoGTPases, Rac1 and Rac2 proteins are often considered to have redundant function; however, they display differential activities in several organisms. In mammals, even if Rac1 and Rac2 share 92% identical peptide sequence, few differences exist within the C-terminus which are important for the specificity of each of them in mouse leukocytes (reviewed in Bokoch 2005); in particular, Rac2-null neutrophils exhibit more severe defects than Rac1-null neutrophils (Koh et al. 2005). The two Drosophila Rac1 and Rac2 show 92% identity but display two different amino acids in position 150 and the C-terminus tail differs slightly: Rac1 has four basic residues, three of them being contiguous, whereas Rac2 contains three basic residues with only two of them being sequential. These basic residues may participate in differential cell localization and differential effectors in the cells and consequently for the different phenotype of Rac1 and Rac2 mutants during development (Hakeda-Suzuki et al. 2002; Ng et al. 2002) and during the immune response (Williams et al. 2005, 2006; this study). In mammalian cells, differential localization affects the RhoGAP specificity of the ExoS toxin (Zhang et al. 2007). Since the activity of both Rac1 and Rac2 are down-regulated when they were co-expressed with ExoSGAP in non-vital organ, both proteins might be targeted by the toxin during fly immune response (Avet-Rochex et al. 2005; this study). However, our present in vivo study indicates that Rac2 might be one of the main host factors whose activity would be prevented by the toxin in the phagocytic cells. First, Rac2 seems to be the major actor of cellular-dependent fly resistance to P. aeruginosa infections, and second, its over-expression can rescue the inhibitory effect of the ExoS toxin GAP domain on fly immunity.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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A cDNA fragment encompassing Rac2 coding sequence was ordered from DGRC (Drosophila Genomics Resource Center <http://dgrc.cgb.indiana.edu>) and subcloned (EcoRI/XhoI) into the pUAST plasmid. Transgenic lines were obtained following standard procedures (Robertson et al. 1988).
Fly stocks and crosses
Flies were grown on standard medium at 25 °C. Directed expression of Rac2 or ExoSGAP in fly tissues was induced by crossing UAS transgenic lines with various Gal4 driver lines (Brand & Perrimon 1993). The Glass Multimer Reporter Gal4 (GMRGal4) driver line was used to express transgenes in the fly eye, where Gal4 that drives the UAS target transgenes in all cells posterior to the morphogenetic furrow in the developing imaginal eye disc (Ellis et al. 1993). The enGal4 (engrailed) driver line was used for the UAS-targeted gene expression in the posterior part of the wing imaginal disc (Fietz et al. 1995). Ubiquitous expression of Rac2 was achieved by crossing UASRac2 transgenic flies with the driver lines daughterless Gal4 (daGal4) or heat-shock Gal4 (HSGal4). In the case of HSGal4, the adult progeny was submitted to heat shock as follows: 30 min at 37 °C, followed by 30 min at 17 °C, and followed by 30 min at 37 °C. Hemocyte-directed expression of Rac2 was achieved with the serpent Gal4 (srpGal4) driver line (Crozatier et al. 2004). The Rac2, Rac1J11, mtl null mutations and Rac1J10 hypomorphic mutation were a gift from Dr Luo (Hakeda-Suzuki et al. 2002; Ng et al. 2002). The amorph mutant cdc424 (Fehon et al. 1997), Rho72R (Strutt et al. 1997), the hypomorphic cdc422 (Fehon et al. 1997) and the deficiency covering Rac2 genetical unit Df(3L)pbl-X1 were provided by the Bloomington stock center.
Bacterial strains and infection conditions
Pseudomonas aeruginosa strains correspond to the wild-type reference and sequenced isolate PAO1 (Stover et al. 2000) or to the clinical isolate CHA inducing rapid fly mortality (Fauvarque et al. 2002). Enterobacter cloacae, Agrobacterium tumefaciens and Escherichia coli were used as Gram-negative strains, and Staphylococcus aureus and Enterococcus faecalis were used as Gram-positive strains. Pseudomonas aeruginosa were grown on Pseudomonas isolation agar (PIA, Difco) plates, whereas the other bacterial species were grown on Luria-Broth (LB) at 37 °C, except for A. tumefaciens which was grown at 30 °C. Infections were performed at 25 °C as described in Avet-Rochex et al. 2005.
Northern blot analysis
Northern blots were performed with total RNA isolated from 20 flies using RNA-plus kit (MP Biomedicals). Northern blots (15 µg RNA/lane) were probed with 32P-labeled diptericin (1 kb), drosomycin (376 bp), puc (1 kb) and actin, as internal loading control (1239 bp), cDNAs. The signals from Northern blots were quantified with PHOSPHOIMAGER (Bio-Rad, Personal Molecular Imager® FX) with the internal software (QUANTITY ONE). The levels of diptericin, puckered or drosomycin expression, respectively, were normalized with corresponding value of actin signal.
Phagocytosis tests
Ex vivo phagocytosis tests on isolated hemocytes were performed as described in Pearson et al. (2003) and Avet-Rochex et al. (2005).
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Acknowledgements |
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Footnotes |
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aThese authors contributed equally to the work.
bPresent address: CNRS, UMR 5547, Université Paul Sabatier, F-31062 Toulouse, France
* Correspondence: E-mail: marie-odile.fauvarque{at}cea.fr |
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Received: 3 April 2007
Accepted: 16 July 2007
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