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CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster

CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster

  • Johanna Sebald, 
  • Stefano Morettini, 
  • Valerie Podhraski, 
  • Cornelia Lass-Flörl, 
  • Alexandra Lusser


Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in viin vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in Chd1 mutants to be functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.


In contrast to most vertebrates, Drosophila melanogaster lacks an adaptive immune system, and host defense relies exclusively on various innate immunity mechanisms (reviewed e.g. in [1][5]). The activation of an immune response upon recognition of the invading microorganisms is controlled by a complex interplay of multiple signaling pathways that are widely conserved and serve similar roles in vertebrates. Two major signaling cascades regulate the production of antimicrobial peptide genes and other immune response reactions in the fly: the Toll and the immune deficiency (Imd) pathway. Both signal to transcription factors of the nuclear factor κB (NFκB)/Rel family, which are termed Dorsal, Dif and Relish [6].

The molecular mechanisms of Drosophila immune response have been studied in considerable detail in recent years, and various transcription factors beside the NFκB-like factors (e.g. dGATA, Caudal, Drifter, dAP1) are known to regulate the expression of immunity-related genes [3]. In contrast, relatively few studies have addressed the role of chromatin-based regulatory mechanisms for immunity-related genes in Drosophila. Modulation of chromatin structure and dynamics by posttranslational modification of histones, incorporation of variant histones or the action of ATP-dependent chromatin remodeling factors is a well-studied general mechanism in gene regulation (e.g. [7][10]). In vertebrates, histone modifications and the activity of SNF2-family chromatin remodeling factors have been demonstrated to play crucial roles in the regulation of immunity-related genes, such as NFκB target genes or other inflammatory response genes [11], [12].

The SNF2 family of proteins comprises a large group of ATP-utilizing motor proteins, the majority of which has chromatin-related functions (e.g. [13]). Of the 15 predicted subfamilies that were identified in Drosophila [14], only a handful has been studied in greater detail and even fewer with respect to their role in the immune response of the fly. Two prominent chromatin remodeling factors that have been found to be involved in the regulation of Drosophila defense mechanisms are the nucleosome remodeling factor (NURF) complex and Domino [15][17]. NURF, which contains the motor subunit ISWI, was demonstrated to act as a corepressor of STAT target genes, thereby modulating the JAK/STAT-mediated immune response [15], [18], [19]. Domino (Dom), a fly homolog of the yeast and mammalian Swr1 ATPases, has originally been described as a factor required for hemocyte formation [16], [20], [21] and was recently found to control the regulation of a large subset of immunity-related genes [17].

The chromatin remodeling factor chromo helicase domain protein 1 (CHD1) has been implicated in the regulation of transcription, in particular elongation. For example, CHD1 has been found to colocalize with the elongating form of RNA polymerase II, to interact with various elongation and mRNA processing factors and to affect the transcription of many genes in yeast and embryonic stem cells [22]. In Drosophila, we and others have found that CHD1 is required for full transcriptional induction of heat shock genes [23], [24]. In addition to its transcription-dependent functions, we have shown previously that CHD1 acts as a chromatin assembly and remodeling factor in vitro and that it is required for the transcription-independent incorporation of the histone H3 variant H3.3 during the reorganization of paternal pronuclear chromatin at fertilization in vivo [25], [26].

In an effort to further dissect the biological functions of CHD1 in Drosophila, we have performed gene expression profiling of Chd1 wild-type and mutant larvae. We found, that a considerable fraction of genes that are misregulated in the absence of CHD1 are genes involved in Drosophila immune response, stress response and detoxification processes. Since CHD1 has not been previously linked to function in immunity-related mechanisms in any organism, we decided to more directly investigate this potential new role of CHD1. We observed that loss of CHD1 rendered flies susceptible to infection by the gram-negative bacterium Pseudomonas aeruginosa upon ingestion of the bacteria but did not affect sensitivity upon septic injury. We found that in Chd1 mutants intestinal AMP levels and, at the same time, bacterial load of the gut were significantly elevated. Moreover, we show that guts of Chd1 mutant flies allowed the passage of large numbers of bacteria into the fly body upon challenge with P. aeruginosa, which ultimately may be the cause of the flies’ death. Thus, we propose that CHD1 should be considered as a novel player contributing to intestinal resistance against microbial assault.


Deletion of Chd1 Leads to Misregulation of Immunity-linked Genes in Drosophila Larvae

In a search for novel functions of CHD1 we performed microarray analysis with RNA from Chd1-deficient (Chd1−/−; [26]) third instar larvae. To minimize genetic background effects, we used a line bearing a wild-type Chd1 transgene in the Chd1-deficient background (hereafter termed Chd1WT/WT; [23]) to serve as the wild-type reference. We have previously shown that the expression levels of transgenic Chd1 in this line equal those of w1118 wild type flies [23]. Expression data were generated using Affymetrix GeneChip Drosophila Genome 2.0 arrays. Our analysis revealed that 602 genes were upregulated and 421 genes were downregulated at least 2-fold in Chd1−/− larvae (Tables S1 and S2). Subjecting the data to gene ontology analysis we found that a large portion of misregulated genes are linked to functions in metabolism, transport, detoxification and proteolysis. Interestingly, about 7% of upregulated and 9% of downregulated genes have assigned immunity-related functions (Figure 1A and Tables S1 and S2). Thus, in all at least 28% of the upregulated and 30% of the downregulated genes in Chd1 mutant flies belong to pathways that are involved in stress response in a wider sense.

Figure 1. Whole genome expression profiling of Chd1−/− larvae.

(A) Gene ontology classification of genes that display at least 2-fold up (left) or downregulation (right). Immunity-linked genes account for a considerable fraction of all misregulated genes. (B) Assignment of immunity-linked misregulated genes in Chd1−/− larvae to several functional subcategories. Color bars denote the magnitude of aberrant regulation.

Nothing has been known so far about a role of the CHD1 remodeler in immunity-linked processes. Therefore, we set out to more closely study this possibility. In a first step, we further analyzed the group of immunity-linked genes with regard to functional subcategories (Figure 1B, Tables S1 and S2). We found that various components of immunity-related signaling cascades were misregulated in the absence of CHD1. Examples are SPE (spätzle-processing enzyme), Spheroide and Easter, which are proteases involved in activating the Toll ligand Spätzle [1], [27] or the Toll pathway-specific transcription factor Dorsal, all of which showed higher expression levels in Chd−/− larvae. Several effector genes of the humoral response, which are targets of immunity-related signaling cascades, were downregulated with some of them showing exceptionally strong reduction in transcript levels (e.g. drosomycin 2, 50-fold; dro5, 11-fold; immune induced molecule 1, 10-fold; IM3, 7-fold). Strongly decreased transcription was also observed for the genes encoding the pattern recognition proteins PGRP-SB1/2 and PGRP-SC2, which are thought to negatively regulate host defense response [28][30]. Various up- and downregulated genes show gut specific expression patterns ( or have reported relevance for gut immunity. For example, two very strongly activated genes, CG16775 and CG11765, are predominantly expressed in the larval gut, and they have been found to undergo gene expression changes upon bacterial infection [31], [32]. Other genes in this group include CG31508, encoding a small protein with no characterized function, that had previously been identified to be strongly induced in the gut upon infection in a Rel-dependent manner [33], a fibrinogen-related protein gene (CG5550), a gene encoding a potential constituent of the gut peritrophic matrix (CG7248) and a peptidoglycan recognition protein gene (PGRP-SC1b), which acts as a negative regulator of immunity-regulating signal transduction pathways [29]. All of these genes have been related to infection-induced transcriptional misregulation [31], [34]. Other gut-specific genes that were downregulated were dro2 and PGRP-SC2. Furthermore, CG6104, a gene of the E(spl) region that has been implicated in gut stem cell maintenance [35] and CG4091, a gene encoding a putative caspase inhibitor that shows high expression levels in the larval midgut and was linked to autophagic cell death [36], were downregulated in Chd1−/− larvae.

CHD1 Mutants are Susceptible to Oral Infection by the Gram-negative Bacterium P. aeruginosa

In light of the fact that various immune response and stress response pathways were misregulated in Chd1-deficient larvae, we next sought to investigate, whether this aberrant transcription program affects the ability of the flies to combat microbial infections. In nature, infection of Drosophila typically occurs through the entry of microbes via ingestion or via the respiratory system eliciting a local immune response mediated by the epithelia. Thus, to study if CHD1 affects defense mechanisms in the intestine, flies were fed with sucrose solution containing either the gram-positive bacterium Staphylococcus aureus, the gram-negative Pseudomonas aeruginosa or the fungus Rhizopus oryzae. These experiments revealed that the absence of CHD1 severely hampered survival of the flies after infection with P. aeruginosa. About 50% of Chd1-mutant flies had succumbed to the infection after 5 days, and less than 5% were alive after 14 days compared to 80% of the Chd1WT/WT line (Figure 2A). Thus, Chd1-mutant flies show similar susceptibility to Pseudomonas infection as the Imd-pathway mutant DreddEP1412 (Fig. 2A). In contrast, only a small, albeit statistically significant, decrease in viability of Chd1−/− flies was observed after infection with S. aureus, and no differences to the wild type became apparent when R. oryzae spores had been ingested by the flies (Figure 2A). These results show that CHD1 is indeed involved in host defense mechanisms, in particular the local response to P. aeruginosa infection.

Figure 2. Loss of CHD1 renders flies more susceptible to oral but not to systemic infection by P. aeruginosa.

(A) Kaplan-Meier plots displaying survival rates of female Chd1−/−, Chd1WT/WT, Tlrv1/Tlr3 and DreddEP1412 flies after feeding with 5% sucrose solution containing either S. aureus, P. aeruginosa (both 109–1010 cfu/ml), R. oryzae (5×108 spores/ml) or no microbes for 15 h. Chd1-deficient flies are significantly more susceptible to oral infections with S. aureus and P. aeruginosa (*P<0.05; n = 80) than Chd1WT/WT flies. (B) Survival rates of female Chd1−/−, Chd1WT/WT, Tlrv1/Tlr3 and DreddEP1412 flies following septic injury with different microbe solutions or NaCl as above (106–107 cfu or spores/ml). No significant differences of survival of infected Chd1−/− (n = 200) and Chd1WT/WT (n = 320) flies were observed.

CHD1 is not Required for Fighting Systemic Infections in Drosophila

When microbes are able to escape the first line of Drosophila immune response that consists of local production of AMPs and reactive oxygen species (ROS) by the epithelia, or if they breach the epithelial barrier in case of wounding, a systemic immune response is elicited [1]. Since we have observed that the absence of CHD1 renders flies more susceptible to oral infection, we determined whether CHD1 is also required for the defense against systemic infection. To this end, we infected Chd1-deficient and control flies with S. aureus, P. aeruginosa and R. oryzae by septic injury. Interestingly, these experiments did not reveal evidence that Chd1−/− flies were more sensitive towards infection than the rescued line (Figure 2B). Conversely, the Toll-mutant Tlrv1/Tlr3 line [37] showed a clear decrease in viability upon infection with S. aureus or R. oryzae, and DreddEP1412 flies were highly susceptible to P. aeruginosa (Figure 2B). These findings are consistent with the notion that the Toll pathway predominantly acts in response to gram-positive bacteria and fungi and that the Imd pathway is necessary to combat infection by gram-negative bacteria [1].

Thus, in sharp contrast to oral infection, where loss of Chd1 renders flies susceptible to infection, CHD1 does not appear to impact on the systemic immune response.

Analysis of the Cellular Immune Response in Chd1-mutant Larvae

Due to the fact that our microarray analysis data indicated the misregulation of a number of genes linked to cellular host defense, such as Nimrod and Tetraspanin receptor genes (Fig. 1B, Tables S1, S2), we examined, whether increased susceptibility to infection was related to impaired hemocyte function. We found that hemocyte numbers as well as their ability for phagocytosis (as tested by injection of india ink into the larval hemocoel [38]) were similar in Chd1-mutant and –rescued larvae (Fig. S1A, B). Likewise, wound healing-coupled melanization proceeded without difference in wild-type and mutant larvae after injury of third instar larvae by pricking with a sterile needle (Fig. S1C). Thus, these results suggest that increased susceptibility of Chd1-mutant flies is likely not due to a defective cellular immune response.

AMP Genes are Derepressed in Chd1-mutant Flies

To investigate the molecular mechanisms of the contribution of CHD1 to intestinal immunity, we measured expression levels of different AMP genes by RT-qPCR in guts prepared from adult Chd1−/− and Chd1WT/WT flies. We analyzed mRNA levels of Attacin C (AttC) and Diptericin B (DipB), which are target genes of the Imd signaling pathway, and Metchnikowin (Mtk), which is known as a Toll-regulated gene in systemic immune response but is controlled by the Imd signaling pathway as well as by the transcription factor dGATAe in the gut [33], [39], [40]. Surprisingly, expression of AttC, DipB and Mtk was significantly higher in intestines from Chd1 mutant flies than in those from control flies (Figure 3A). We further tested the expression of the AMP genes Drosomycin 2 (dro2) and Drosomycin 3 (dro3), the latter of which had been shown to be controlled by the JAK/STAT pathway [33]. Similar to the other AMP genes, dro3 transcription was strongly overactivated in guts from Chd1−/− flies (Figure 3A). In contrast, dro2 mRNA levels were decreased in the absence of CHD1 (Figure 3A). Together these data indicate a latent activation of intestinal immune response in Chd1 mutant flies even in the absence of bacterial challenge.

Figure 3. CHD1 affects the expression of AMP genes in the gut.

(A) Expression of several AMP genes is significantly upregulated in Chd1−/− flies in the absence of infection. (B) The expression of several regulators of Imd pathway activity is not significantly altered in Chd1−/− flies. RT-qPCR analysis of isolated guts of unchallenged (−PA) and P. aeruginosa infected (PA; 15 h) Chd1−/− and Chd1WT/WT flies was performed. Transcript levels of indicated genes were normalized against Rpl32 and are expressed relative to those of the respective gene in Chd1WT/WT guts. Values represent mean +/− SD of at least 3 independent experiments with 50 guts each (*P<0.05).

Expression of AMP Genes upon Infection

Next we determined whether CHD1 is required for the regulation of AMP gene expression upon infection. We isolated RNA from guts of flies after 15 h of infection with P. aeruginosa and measured the expression of the selected AMP genes by RT-qPCR. We found that in the Chd1WT/WT flies, ingestion of P. aeruginosa elicited the activation of all tested AMP genes by various degrees ranging from ∼9-fold (AttC) to ∼2-fold induction (dro2; Figure 3A). In contrast, none of the tested AMPs showed statistically significant levels of induction in Chd1−/− flies upon bacterial ingestion (Figure 3A). Note, that variations in AMP expression levels upon infection were rather pronounced across different experiments. Although these experiments were performed up to 6 times with 50 guts each, this problem could not be solved. It is possible that different feeding behavior of the flies or subtle differences in the inoculum are responsible for this effect. However, because in some experiments AMP expression was clearly induced (Figure 3A), we conclude that, in principle, AMP activation is still possible in the mutant flies and therefore not dependent on CHD1.

Together these results indicate that CHD1 most likely does not impact on the induction of AMP expression in response to bacterial challenge, but that it contributes to the maintenance of proper AMP levels in the absence of infection. One possible way by which the chromatin remodeler CHD1 might affect this process is by directly or indirectly regulating transcriptional read-out of immune response signaling pathways, in particular of the Imd (e.g. AttC, DipB, Mtk) and the JAK/STAT (e.g. dro3) pathways.

AMP Overactivation in Chd1 Mutant Guts is not Due to a Misregulation of Immunosuppressive Genes

To examine, whether the elevated steady state AMP levels in Chd1 mutant guts are due to defects in the regulation of the intestinal Imd immune signaling pathway, we analyzed the expression levels of a number of genes that are known for their roles in this process. Beside the repressors of basal AMP transcription Caudal (Cad; [41]), Caspar [42] and dUSP36 [43], we tested transcript levels of Pirk/PIMS/Rudra, which mediates negative feedback regulation of Imd signaling [44][46], as well as of four members of the PGRP family (SB1, SC2, LB, LC; Fig. 3C). PGRP-LC is a membrane receptor involved in sensing gram-negative bacteria [47][49], PGRB-LB and –SC2 have been described as negative regulators of Imd pathway activity [30], [50], whereas no obvious functions in the immune response have been detected so far for PGRP-SB1 [28]. None of the negative regulator genes PGRP-LB, PGRP-SC2, Pirk, caspar, caudal and dUSP36 or the positively acting receptor PGRP-LC showed altered expression in Chd1-mutant compared to wild-type guts (Figure 3B). PGRP-SB1 transcript levels were slightly elevated in the mutant in the absence of bacterial challenge and strongly induced upon infection (Figure 3B). PGRP-SB1 has been shown to be induced in a Rel-dependent way by bacterial infection [33]. Therefore, its upregulation in the Chd1 mutant is consistent with the observed derepression of other Imd target genes, such as AttC, DipB and Mtk (Figure 3A). However, these data do not point to a role for CHD1 in interfering with the negative regulation of the Imd pathway.

Chd1−/− Flies have Normal Numbers of Intestinal Stem Cells

We next examined whether CHD1 plays a role not only in AMP production but also in gut cell homeostasis. Gut-specific immune response not only relies on Imd-mediated AMP expression but also on the production of ROS [1], [51]. A side effect of the release of ROS is the damage of epithelial cells. Gut cell renewal is orchestrated by a variety of signaling pathways, including the Notch, WNT, JAK/STAT, EGRF and p38 pathways [52][57]. As a consequence of their activity intestinal stem cell (ISC) division and differentiation of progenitor cells is induced [53], [58]. Misfunctioning of this replenishment cycle has been shown to gravely affect viability of the fly upon infection [54]. Using immunofluorescence microscopy we first examined CHD1 localization in guts from wild-type flies and found that CHD1 was present in all cell types, including ISCs and dividing cells, which were visualized by staining with antibodies against Delta (Dl) and phosphorylated histone H3 (PH3), respectively (Figure 4A–C). Because it was shown that mammalian CHD1 is required for the maintenance of pluripotency of mouse embryonic stem cells [59], we analyzed the number and distribution of Dl+ ISCs in intestines from Chd1−/− flies. These experiments revealed no obvious differences between Chd1−/− and Chd1WT/WT flies (Figure 4D). Similar results were obtained with guts from infected animals at 12 h, 2 and 4 days of infection (an exemplary image is shown in Figure 4D). Of note, the number of PH3-positive cells did not increase upon infection neither in wild-type nor in the mutant guts, indicating that no significant ISC proliferation was induced by the ingestion of P. aeruginosa in our system. Moreover, expression levels of the escargot (esg) gene, which is frequently used as a marker of stem cells and early differentiating cells, were not significantly altered in Chd1−/− versus Chd1WT/WT flies (data not shown).

Figure 4. Deletion of Chd1 does not cause aberrant numbers or distribution of ISCs in the fly intestine.

(A, A’, A”) Co-staining of isolated guts of Chd1WT/WT flies with antibodies against CHD1 and Delta revealed localization of CHD1 to ISCs. (B) CHD1 is present in the nuclei of large enterocytes. (C, C’) CHD1 colocalizes with mitotic, PH3-positive cells. CHD1, green; Dl, red; PH3, red; DNA was visualized by staining with DAPI (blue). (D) Guts of unchallenged and P. aeruginosa infected Chd1−/− and Chd1WT/WT flies were stained with anti-DI antibody. An area of the anterior midgut is shown. Images are presented with inverted colors to enhance clarity. Arrows indicate individual ISCs with cell membrane-associated Dl signal. No significant differences with respect to number or distribution of ISCs was observed in uninfected and infected wild-type and mutant flies.

Given that Chd1-mutant flies die with high frequency upon P. aeruginosa ingestion (Figure 2A), we would have expected enhanced ISC proliferation as a consequence of increased epithelial damage. Signaling from damaged cells has been shown to induce ISC proliferation [54]. To examine, if cell damage occurs upon infection in Chd1−/− guts, we performed immunostainings with antibodies against activated caspase 3 but were unable to detect increased cell death in the guts of Chd1−/− flies (data not shown). Note, that guts from control flies fed with SDS showed clear caspase 3 staining (Figure S2). Also, the overall appearance of intestines from non-infected or infected Chd1-mutant flies was similar (Figure S3). Thus, the strong susceptibility of Chd1−/− flies to oral infection by P. aeruginosa appears not to be due to major degeneration of gut epithelia. Together, these results indicate that cell renewal is not particularly stimulated under our infection conditions. Nevertheless, CHD1 appears to have no major role in the maintenance of the stem cell population in the gut.

Increased Microbial Colonization of Guts from Chd1−/− Flies

To further explore the causes for the increased mortality of Chd1−/− flies upon P. aeruginosa infection, we considered that elevated intestinal AMP levels, observed in the absence of CHD1, might affect the titers and/or community structure of commensal microbes. Such an effect has been observed before when the AMP-specific transcriptional repressor Cad was knocked down [41]. We examined the bacterial load in wild-type and mutant flies by qPCR. Interestingly, we found profoundly increased titers of bacteria in dissected guts from Chd1−/− flies in the absence of P. aeruginosa ingestion as well as after infection (Figure 5A and Figure S4A). To obtain evidence for potential alterations in the community structure of commensal bacteria in these densely colonized guts, we determined the relative abundance of two known gut-specific bacterial strains, Acetobacteriaceae strain EW911 and Gluconobacter sp. strain EW707 [41]. We found that in the absence of infection titers of Acetobacter EW911 were similar in wild-type and mutant flies (Figure 5C and Figure S4C). However, when taking into account the increase in overall bacterial titer in Chd1−/− intestines (Figure S4A), the proportion of Acetobacter EW911 within total microbiota actually is reduced (compare Figure S4A and C). The same results were obtained for Gluconobacter EW707 (Figure S5). Hence, the increased bacterial load in unchallenged guts in the absence of CHD1 appears not to be due to an even amplification of all bacteria but to be caused by an enrichment of (an) undefined species.

Figure 5. Bacterial load is elevated in Chd1-mutant flies.

(A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251659264P. aeruginosa titers are strongly increased in Chd1−/− flies after infection. qPCR as in (A) with primers specific for P. aeruginosa. (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911. Relative differences of bacterial genes and the fly Rpl32 gene are expressed as 2−ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.

Analysis of the bacterial load in fly bodies from which the gut had been removed (“rest fly”) revealed that it was similar in wild type and mutant in the unchallenged state. In contrast, we observed a dramatic increase of bacteria in mutant, but not in wild-type flies, at 3 and 4 days of infection, respectively, indicating invasion of microbes from the gut into the body cavity (Figure 5A and Figure S4A). We then measured P. aeruginosa titers in the absence of infection and found no differences between Chd1−/− and Chd1WT/WT guts (Figure 5B). However, in Chd1 mutant intestines P. aeruginosa showed 25 fold higher enrichment after 3 days of infection and ∼100 fold enrichment after 4 days of infection compared to wild-type guts (Figure 5B and Figure S4B). The accumulation of P. aeruginosa was even stronger in the body of mutant flies, where it increased to levels more than 300 fold higher than those of the wild-type flies (Figure 5B and Figure S4B). These data indicate that the intestinal environment in Chd1 mutant flies allows ingested P. aeruginosa to accumulate to considerably higher numbers. Moreover, the dramatically increased bacterial titers in the fly body outside the gut after oral infection suggest that the gut epithelia is much more permissible to the passage of P. aeruginosa and possibly other bacteria into the hemolymph, which may ultimately result in the death of the fly.


Misregulation of Humoral Response Genes in the Absence of CHD1

Our study shows that the chromatin remodeling factor CHD1 is involved in the regulation of immunity-related processes in Drosophila melanogaster. In particular, it appears to play an important part in gut-specific host defense against the gram-negative bacterium P. aeruginosa. We found that CHD1 affects the transcriptional regulation of AMP genes, which are under the control of the Imd as well as the JAK/STAT pathways. Intriguingly, gut-specific expression of AttC, DipB, Mtk and dro3 was significantly overactivated in adult Chd1−/− mutant flies in the absence of infection. There are several explanations that may account for this phenomenon. First, CHD1 might function as a negative transcriptional regulator of AMP genes. Although overexpression of these AMPs was not evident from our microarray analysis of third instar larvae, we did detect significantly higher levels of AttC, DipB and dro3 using RT-qPCR analysis of larval RNA (Figure S6). On the other hand, Mtk expression was downregulated in Chd1−/− larvae in contrast to adult guts, where it was overactivated (Figure S6 and Figure 3A). These findings may suggest that CHD1 is required for the proper regulation of immunity-associated genes in adults and in larvae. Our data from adult flies point to a corepressor function of CHD1. However, CHD1 has also been shown to act as a coactivator. For example, it is required in larvae to fully induce transcription of heat shock genes upon thermal stress [23]. Hence, CHD1 may act as both, a corepressor or coactivator of immune response genes depending on the developmental and/or tissue-specific context.

An alternative explanation for the observed upregulation of AMP levels in adult guts might be that CHD1 affects transcription of genes that are involved in the regulation of Imd pathway activity. Although we cannot formally rule out this possibility, our data showing wild-type-like levels of several such regulators (Cad, dUSP36, Casper, Pirk/PIMS/Rudra, PGRP-LB, LC, SC2, SB1) rather argue against it.

Overactivation of AMP Genes - the Cause for Increased Infection Sensitivity of Chd1−/− Flies?

Alternatively or in addition to the explanations proposed above for the effect of Chd1 deletion on AMP expression and fly survival upon infection, it is possible that CHD1 contributes to maintaining the fly intestine in a state that will allow for efficient combat of invading pathogens. It may be counterintuitive that guts, which express higher levels of AMPs, such as in Chd1−/− flies, should be more susceptible to infection by P. aeruginosa than those with normal AMP expression. Indeed, previous work has demonstrated that AMPs, in particular DipB, are critically involved in fighting oral infection by P. entomophila [60]. In our system, however, increased intestinal AMP levels caused by the absence of CHD1 did not confer resistance against P. aeruginosa. Although these results appear contradictory, they can be reconciled by considering the different degree of overexpression of DipB in both studies. While Liehl et al. [60] obtained strong overexpression of DipB using the UAS-Gal4 system (∼100 fold over uninfected state), Chd1 mutation caused only about 2–4 fold higher DipB levels (Figure 3A). Thus, in our system, elevated AMP levels in Chd1 mutants might not suffice to actively counteract P. aeruginosa infection. However, it is tempting to speculate that they may be adequate to exert continual selective pressure upon the commensal microbial community. As a result enrichment and overgrowth of less sensitive bacteria of the gut microbiota might occur, and these in turn may stimulate the expression of AMP genes, thus reinforcing the effects of Chd1-loss. This might weaken intestinal epithelia in a way to allow increased passage of P. aeruginosa into the body cavity. The facts that we find considerably elevated bacterial titers in the guts of Chd1−/− flies in the absence of infection (Figure 5A) as well as the strong enrichment of P. aeruginosa outside the gut upon infection (Figure 5B) are in good agreement with such a scenario. Of note, our experiments using septic injury did not show elevated susceptibility to P. aeruginosa infection of Chd1−/− compared to Chd1WT/WT flies. It may be possible that P. aeruginosa undergoes a switch in virulence when exposed to the altered bacterial colonization in the digestive tract of Chd1-mutant flies. For example, it has been shown before that coinfection of Drosophila with P. aeruginosa along with certain bacteria resulted in changed virulence factor gene expression and enhanced pathogenicity [61]. Alternatively and/or in addition, co-invasion of gut bacteria with P. aeruginosa into the hemolymph may cause increased lethality of the flies upon oral but not systemic infection.

Role of CHD1 in Gut Cell Homeostasis

Lee and colleagues have recently shown that increased AMP production can indeed have adverse effects on the animal [41]. Overactivation of AMPs by inactivation of the transcriptional repressor Cad resulted in a disturbance of the commensal microbial community structure and consequently in epithelial damage and increased fly mortality [41]. Since epithelial damage is counteracted by the activity of ISCs and because it has been reported previously that mouse CHD1 is required for the maintenance of embryonic stem cell pluripotency [59], we determined, if ISC number or proliferation/differentiation was altered in Chd1 mutants. In contrast to expectation, we observed normal numbers and distribution of ISCs in the absence of CHD1 arguing against an involvement of CHD1 in stem cell maintenance. In our experiments oral infection of either Chd1WT/WT or Chd1−/− flies with P. aeruginosa failed to cause gross morphological changes of the gut, induction of apoptosis in intestinal epithelia or ISC proliferation as was observed in other studies [55], [62]. Therefore, we cannot rule out that CHD1 plays a role in ISC proliferation or differentiation under different conditions.

The absence of gut tissue remodeling was also noted in a recent study, in which oral infection experiments were carried out with the P. aeruginosa P14 strain [63]. In this study it was concluded that the flies succumb to infection due to bacteremia rather than intestinal damage. Our data also suggest that oral infection of Chd1-mutant flies with P. aeruginosa leads to death by a mechanism that does not involve major gut degradation but instead correlates with increased bacterial load in the fly body.

Collectively, we have characterized a novel biological role for the chromatin remodeling and assembly factor CHD1 that is linked to immune response processes in Drosophila. Given the high degree of conservation of chromatin remodeling mechanisms across different species, it should be interesting to consider CHD1 also in studies of host defense in mammalian organisms.

Materials and Methods

Fly Strains

Flies were kept on standard cornmeal media at 25°C except for infection experiments, when flies were incubated at 29°C. Chd1-deficient (Chd1−/−) flies were obtained by crossing Df(2L)Chd11/CyO, GFP with Df(2L)Exel7014/CyO, GFP [26], and Chd1WT/WT flies were obtained from crosses of Df(2L)Chd11,P{Chd1WT}/CyO, GFP and Df(2L)Exel7014, P{Chd1WT}/CyO, GFP [23]. For Tl-deficient flies the temperature-sensitive Tlr3 and Tlrv1 alleles were combined [37]. DreddEP1412 flies [64] were obtained from the Bloomington Stock Center.

Microbial Strains

Rhizopus oryzae AS82 (clinical isolate) was cultivated on potato-dextrose agar and spore suspensions were generated in 0.9% NaCl/0.01% Tween 80. Clinical isolates of Pseudomonas aeruginosa (998) and Staphylococcus aureus (877) were grown in tryptic soy broth at 37°C.

Infection Experiments

Septic injury experiments were carried out by pricking the dorsal thorax of 2–4 day old female flies with a 27G needle that had been dipped into the respective microbe solution or 0.9% NaCl, respectively. The concentrations of the inocula were as follows: R. oryzae, 107 spores/ml; P. aeruginosa and S. aureus, 106 cfu/ml. Flies were incubated at 29°C and transferred to fresh food vials at every third day. For oral infection experiments bacterial pellets of overnight cultures were resuspended in 5% sucrose solution at an OD600 of 0.2–0.4 for P. aeruginosa and 1.8–2.0 for S. aureus and applied to Whatman filter discs covering the surface of a standard food vial. For fungal infection, food vials containing cornmeal glucose sucrose yeast extract agar for zygomycetes were inoculated with R. oryzae mycelia and fungal growth and sporulation was allowed for 3 days. 2–4 day old female flies were starved for 5 hours and then transferred to food vials containing contaminated filter discs/fungal mycelia or to filter discs soaked in sterile 5% sucrose solution for 15 h at 29°C. Subsequently, flies were transferred to fresh, uncontaminated vials and maintained at 29°C for 14 days with food changes at every third day. Dead flies were removed daily. Survival rates were calculated as percentage of living flies at each given time point. Data of 3–6 independent experiments with a minimum of 20 flies each were analyzed for statistical significance using the Kaplan-Meier log rank test (Prism 5.0 software).

Microarray Analysis and Statistics

Total RNA was extracted from 3 independent pools of 40 wandering Chd1−/− and Chd1WT/WT 3rd instar larvae. Probe generation, hybridization to Drosophila Genome 2.0 arrays (Affymetrix) and data normalization was performed by the in-house Expression Profiling Unit (Innsbruck Medical University). Data analysis was performed using CARMAweb (Comprehensive R based Microarray Analysis web service) software package [65]. Microarray data were preprocessed using the gcRMA method and differentially expressed genes were identified in each of the three biological replicates using a fold change cut–off of 2. Statistical significance of differential regulation was determined by unpaired t-test analysis. To correct for multiple hypothesis testing problems the Benjamini and Hochberg adjustment method [66] was used. Functional annotation of the resulting gene lists was performed manually using information available on FlyBase (


For gut-specific expression analysis, 50 guts (midgut plus hindgut) were dissected from female flies that were either uninfected or at 15 h after oral infection. Guts were frozen in liquid nitrogen immediately after dissection and RNA was prepared using the RNeasy Mini Kit (Qiagen). cDNA synthesis and RT-qPCR were performed as described [23]. qPCR primers are listed in Table S3. At least four independent experiments were performed for each condition and fly line. Statistical significance of differential regulation was determined using unpaired t-test analysis (Prism 5.0). The p-value for statistical significance was set at P<0.05.

Determination of Bacterial Load

Twenty 2–4 day old female flies were washed with 70% ethanol, the complete intestinal tract was removed and the remaining carcasses were frozen in liquid nitrogen and subsequently homogenized in TES buffer (1 mM EDTA, 10 mM Tris-HCl pH 7.5, 100 mM NaCl) with a sterilized pestle. For gut-specific analysis female flies were washed with 70% ethanol and subsequently 10 guts (midgut plus hindgut) were dissected into sterile TES buffer. Genomic DNA was isolated by consecutive incubation of the samples with lysozyme (50 U/ml; 15 min, 37°C) and 1% SDS/2 mg/ml proteinase K (2 h, 37°C) with gentle shaking followed by phenol-chloroform extraction and ethanol precipitation. DNA pellets were dissolved in water and digested with RNase A (0.2 mg/ml). qPCR reactions with primers specific for bacterial 16S rDNA, P. aeruginosa, Acetobacteriaceae strain EW911 or Gluconobacter sp. strain EW707 (Table S3) were conducted in triplicate using a StepONEPlus instrument (Life Technologies) and Power SYBR Green PCR master mix (Life Technologies). The Drosophila Rpl32 gene was used for normalization and 2−ΔCT values were plotted.

Immunocytochemistry and Microscopy

Guts of adult female flies were dissected into PBS and fixed for 15 min with methanol/heptane (1∶1). The guts were washed 3 times in 100% methanol, gradually transferred to PBST (PBS, 0.15% Triton X-100) and incubated with primary antibody diluted in 0.5%BSA/PBST overnight at 4°C. The following antibodies were used: PH3, Caspase 3 (both at 1∶250; Cell Signaling Technology), Delta (1∶50; Developmental Studies Hybridoma Bank) and CHD1 [67]. After secondary antibody (Alexa Fluor® 488 goat anti-rabbit IgG; Alexa Fluor® 594 goat anti-mouse IgG; Invitrogen) incubation, specimen were stained with DAPI and mounted in Vectashield (Vector Laboratories Inc.). Images were taken on a confocal laser scanning microscope (SP5, Leica). Images were processed using LSM Image Browser (version 4.2) software and Adobe Photoshop CS3.

Hemocyte Count, Phagocytosis and Melanization Tests

Hemocytes were collected from 14 wandering 3rd instar larvae and counted as described in [68]. Statistical differences were determined using unpaired t-test analysis (Prism 5.0). Phagocytosis was performed by injection of india ink into the body cavity of 3rd instar larvae essentially as described by [38], except that a 30G 1/2” needle was used for injections. Melanization capability was tested as follows: 3rd instar larvae were washed in PBS and immobilized on double-sided tape attached to a glass slide. Sterile wounding was performed by pricking the larvae with a 30G 1/2” needle at abdominal segment A4 or A5. After wounding, larvae were transferred to a fresh glass slide and melanization was monitored every 10 min for 1.5 hours.

Supporting Information

Figure S1.

Hemocyte function appears unaffected in Chd1-mutant larvae. (A) Hemocyte numbers are similar in Chd1WT/WT and Chd1−/− larvae. Hemocytes were collected from larvae and counted in a hemocytometer. (B) Phagocytosis activity of mutant hemocytes is indistinguishable from that of wild-type hemocytes. White arrows indicate phagocytized ink particles in hemocytes. (C) Wound healing and melanization occurs in a similar fashion in wild-type and Chd1-mutant larvae. Black arrows indicate the site of wounding after 1.5 h.


Figure S2.

Detection of apoptosis by caspase 3 staining. Guts from wild-type flies that were fed for 6 h with SDS or sucrose only were stained with antibodies against activated caspase 3 (green); DNA was visualized by DAPI staining (blue). A section of the anterior midgut is shown.


Figure S3.

Overall gut morphology is similar in Chd1-rescued and -mutant flies irrespective of the state of infection. Several light microscopic images were taken along the anterior posterior axis of dissected guts (midgut plus hindgut) and subsequently assembled into one picture using Photoshop.


Figure S4.

Bacterial load is elevated in Chd1-mutant flies. (A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251658240P. aeruginosa titers are strongly increased in Chd1/ flies after infection. qPCR as in (A) with primers specific for P. aeruginosa. (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911. The Drosophila Rpl32 gene was used for normalization, and enrichment relative to the non-infected (−PA) Chd1WT/WT line was calculated using the 2−ΔΔCT method. Values represent mean +/− SD of three independent experiments.


Figure S5.

Analysis of the gut-specific bacterium Gluconobacter EW707. qPCR as in Figure S3 with primers specific for Gluconobacter EW707. (A) The relative differences of GB EW707 and the fly Rpl32 gene are expressed as 2−ΔCT values. (B) Enrichment relative to the non-infected (−PA) Chd1WT/WT line was calculated using the 2−ΔΔCT method. Values represent mean +/− SD of three independent experiments.


Figure S6.

Several AMP genes are upregulated in Chd1/ larvae in the absence of infection. Indicated AMP genes that did not score in the microarray were analysed by RT-qPCR. Transcript levels were normalized against Rpl32 and are expressed relative to values obtained in Chd1WT/WT larvae. Values represent mean +/− SD of three independent experiments. Statistical significance was determined using unpaired t-test analysis (*P<0.05).


Table S1.

Upregulated genes in Chd1−/− flies.


Table S2.

Downregulated genes in Chd1/ flies.



We thank Drs G. Chamilos, D. Kontoyiannis, B. Lemaitre and the Bloomington stock center for providing fly strains. We thank H. Wörle, G. Scheran and C. Hörtnagl for technical assistance and C. Lamina for help with statistics. We are grateful to H. Haas for discussions and P. Loidl and J.T. Kadonaga for comments on the manuscript.

Author Contributions

Conceived and designed the experiments: JS SM AL. Performed the experiments: JS SM VP. Analyzed the data: JS SM AL. Contributed reagents/materials/analysis tools: CLF. Wrote the paper: JS AL.


  1. 1. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697–743.
  2. 2. Bae YS, Choi MK, Lee WJ (2010) Dual oxidase in mucosal immunity and host-microbe homeostasis. Trends Immunol 31: 278–287.
  3. 3. Uvell H, Engstrom Y (2007) A multilayered defense against infection: combinatorial control of insect immune genes. Trends Genet 23: 342–349.
  4. 4. Brennan CA, Anderson KV (2004) Drosophila: the genetics of innate immune recognition and response. Annu Rev Immunol 22: 457–483.
  5. 5. Dionne MS, Schneider DS (2008) Models of infectious diseases in the fruit fly Drosophila melanogaster. Dis Model Mech 1: 43–49.
  6. 6. Hetru C, Hoffmann JA (2009) NF-kappaB in the immune response of Drosophila. Cold Spring Harb Perspect Biol 1: a000232.
  7. 7. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21: 381–395.
  8. 8. Hargreaves DC, Crabtree GR (2011) ATP-dependent chromatin remodeling: genetics, genomics and mechanisms. Cell Res 21: 396–420.
  9. 9. Piatti P, Zeilner A, Lusser A (2011) ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition. Int J Mol Sci 12: 6544–6565.
  10. 10. Banaszynski LA, Allis CD, Lewis PW (2010) Histone variants in metazoan development. Dev Cell 19: 662–674.
  11. 11. Natoli G (2009) Control of NF-kappaB-dependent transcriptional responses by chromatin organization. Cold Spring Harb Perspect Biol 1: a000224.
  12. 12. Smale ST (2010) Selective transcription in response to an inflammatory stimulus. Cell 140: 833–844.
  13. 13. Clapier CR, Cairns BR (2009) The biology of chromatin remodeling complexes. Annu Rev Biochem 78: 273–304.
  14. 14. Flaus A, Martin DM, Barton GJ, Owen-Hughes T (2006) Identification of multiple distinct Snf2 subfamilies with conserved structural motifs. Nucleic Acids Res 34: 2887–2905.
  15. 15. Kwon SY, Xiao H, Glover BP, Tjian R, Wu C, et al. (2008) The nucleosome remodeling factor (NURF) regulates genes involved in Drosophila innate immunity. Dev Biol 316: 538–547.
  16. 16. Braun A, Lemaitre B, Lanot R, Zachary D, Meister M (1997) Drosophila immunity: analysis of larval hemocytes by P-element-mediated enhancer trap. Genetics 147: 623–634.
  17. 17. Walker J, Kwon SY, Badenhorst P, East P, McNeill H, et al. (2011) Role of elongator subunit Elp3 in Drosophila melanogaster larval development and immunity. Genetics 187: 1067–1075.
  18. 18. Kwon SY, Xiao H, Wu C, Badenhorst P (2009) Alternative splicing of NURF301 generates distinct NURF chromatin remodeling complexes with altered modified histone binding specificities. PLoS Genet 5: e1000574.
  19. 19. Kugler SJ, Gehring EM, Wallkamm V, Kruger V, Nagel AC (2011) The Putzig-NURF nucleosome remodeling complex is required for ecdysone receptor signaling and innate immunity in Drosophila melanogaster. Genetics 188: 127–139.
  20. 20. Braun A, Hoffmann JA, Meister M (1998) Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes. Proc Natl Acad Sci U S A 95: 14337–14342.
  21. 21. Ruhf ML, Braun A, Papoulas O, Tamkun JW, Randsholt N, et al. (2001) The domino gene of Drosophila encodes novel members of the SWI2/SNF2 family of DNA-dependent ATPases, which contribute to the silencing of homeotic genes. Development 128: 1429–1441.
  22. 22. Marfella CG, Imbalzano AN (2007) The Chd family of chromatin remodelers. Mutat Res 618: 30–40.
  23. 23. Morettini S, Tribus M, Zeilner A, Sebald J, Campo-Fernandez B, et al. (2011) The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization. Nucleic Acids Res 39: 3103–3115.
  24. 24. Petesch SJ, Lis JT (2008) Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134: 74–84.
  25. 25. Lusser A, Urwin DL, Kadonaga JT (2005) Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. Nat Struct Mol Biol 12: 160–166.
  26. 26. Konev AY, Tribus M, Park SY, Podhraski V, Lim CY, et al. (2007) CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 317: 1087–1090.
  27. 27. Valanne S, Wang JH, Ramet M (2011) The Drosophila Toll signaling pathway. J Immunol 186: 649–656.
  28. 28. Zaidman-Remy A, Poidevin M, Herve M, Welchman DP, Paredes JC, et al. (2011) Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function. PLoS One 6: e17231.
  29. 29. Bischoff V, Vignal C, Duvic B, Boneca IG, Hoffmann JA, et al. (2006) Downregulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2. PLoS Path 2: e14.
  30. 30. Paredes JC, Welchman DP, Poidevin M, Lemaitre B (2011) Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 35: 770–779.
  31. 31. Vodovar N, Vinals M, Liehl P, Basset A, Degrouard J, et al. (2005) Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. Proc Natl Acad Sci U S A 102: 11414–11419.
  32. 32. Xi Z, Gavotte L, Xie Y, Dobson SL (2008) Genome-wide analysis of the interaction between the endosymbiotic bacterium Wolbachia and its Drosophila host. BMC Genomics 9: 1.
  33. 33. Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B (2009) Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe 5: 200–211.
  34. 34. 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 U S A 98: 12590–12595.
  35. 35. Bardin AJ, Perdigoto CN, Southall TD, Brand AH, Schweisguth F (2010) Transcriptional control of stem cell maintenance in the Drosophila intestine. Development 137: 705–714.
  36. 36. Gorski SM, Chittaranjan S, Pleasance ED, Freeman JD, Anderson CL, et al. (2003) A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol 13: 358–363.
  37. 37. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86: 973–983.
  38. 38. Stofanko M, Kwon SY, Badenhorst P (2008) A misexpression screen to identify regulators of Drosophila larval hemocyte development. Genetics 180: 253–267.
  39. 39. Levashina EA, Ohresser S, Lemaitre B, Imler JL (1998) Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin. J Mol Biol 278: 515–527.
  40. 40. Senger K, Harris K, Levine M (2006) GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc Natl Acad Sci U S A 103: 15957–15962.
  41. 41. Ryu JH, Kim SH, Lee HY, Bai JY, Nam YD, et al. (2008) Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319: 777–782.
  42. 42. Kim M, Lee JH, Lee SY, Kim E, Chung J (2006) Caspar, a suppressor of antibacterial immunity in Drosophila. Proc Natl Acad Sci U S A 103: 16358–16363.
  43. 43. Thevenon D, Engel E, Avet-Rochex A, Gottar M, Bergeret E, et al. (2009) The Drosophila ubiquitin-specific protease dUSP36/Scny targets IMD to prevent constitutive immune signaling. Cell Host Microbe 6: 309–320.
  44. 44. Lhocine N, Ribeiro PS, Buchon N, Wepf A, Wilson R, et al. (2008) PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4: 147–158.
  45. 45. Aggarwal K, Rus F, Vriesema-Magnuson C, Erturk-Hasdemir D, Paquette N, et al. (2008) Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. PLoS Path 4: e1000120.
  46. 46. Kleino A, Myllymaki H, Kallio J, Vanha-aho LM, Oksanen K, et al. (2008) Pirk is a negative regulator of the Drosophila Imd pathway. J Immunol 180: 5413–5422.
  47. 47. Ramet M, Manfruelli P, Pearson A, Mathey-Prevot B, Ezekowitz RA (2002) Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644–648.
  48. 48. Gottar M, Gobert V, Michel T, Belvin M, Duyk G, et al. (2002) The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416: 640–644.
  49. 49. Choe KM, Werner T, Stoven S, Hultmark D, Anderson KV (2002) Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296: 359–362.
  50. 50. Zaidman-Remy A, Herve M, Poidevin M, Pili-Floury S, Kim MS, et al. (2006) The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24: 463–473.
  51. 51. Ryu JH, Ha EM, Lee WJ (2010) Innate immunity and gut-microbe mutualism in Drosophila. Dev Comp Immunol 34: 369–376.
  52. 52. Ohlstein B, Spradling A (2006) The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439: 470–474.
  53. 53. Ohlstein B, Spradling A (2007) Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling. Science 315: 988–992.
  54. 54. Buchon N, Broderick NA, Chakrabarti S, Lemaitre B (2009) Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev 23: 2333–2344.
  55. 55. Buchon N, Broderick NA, Kuraishi T, Lemaitre B (2010) Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection. BMC Biol 8: 152.
  56. 56. Lin G, Xu N, Xi R (2008) Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells. Nature 455: 1119–1123.
  57. 57. Jiang H, Patel PH, Kohlmaier A, Grenley MO, McEwen DG, et al. (2009) Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell 137: 1343–1355.
  58. 58. Micchelli CA, Perrimon N (2006) Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439: 475–479.
  59. 59. Gaspar-Maia A, Alajem A, Polesso F, Sridharan R, Mason MJ, et al. (2009) Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature 460: 863–868.
  60. 60. Liehl P, Blight M, Vodovar N, Boccard F, Lemaitre B (2006) Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model. PLoS Path 2: e56.
  61. 61. Sibley CD, Duan K, Fischer C, Parkins MD, Storey DG, et al. (2008) Discerning the complexity of community interactions using a Drosophila model of polymicrobial infections. PLoS Path 4: e1000184.
  62. 62. Apidianakis Y, Pitsouli C, Perrimon N, Rahme L (2009) Synergy between bacterial infection and genetic predisposition in intestinal dysplasia. Proc Natl Acad Sci U S A 106: 20883–20888.
  63. 63. Limmer S, Haller S, Drenkard E, Lee J, Yu S, et al. (2011) Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune response in a Drosophila melanogaster oral infection model. Proc Natl Acad Sci U S A 108: 17378–17383.
  64. 64. Elrod-Erickson M, Mishra S, Schneider D (2000) Interactions between the cellular and humoral immune responses in Drosophila. Curr Biol 10: 781–784.
  65. 65. Rainer J, Sanchez-Cabo F, Stocker G, Sturn A, Trajanoski Z (2006) CARMAweb: comprehensive R- and bioconductor-based web service for microarray data analysis. Nucleic Acids Res 34: W498–503.
  66. 66. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J Roy Statist Soc Ser: 289–300.
  67. 67. Podhraski V, Campo-Fernandez B, Worle H, Piatti P, Niederegger H, et al. (2010) CenH3/CID incorporation is not dependent on the chromatin assembly factor CHD1 in Drosophila. PLoS One 5: e10120.
  68. 68. Matova N, Anderson KV (2006) Rel/NF-kappaB double mutants reveal that cellular immunity is central to Drosophila host defense. Proc Natl Acad Sci U S A 103: 16424–16429.