The Interactive Fly
Zygotically transcribed genes
Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms
Sequential activation of signaling pathways during innate immune responses
Immunodeficient Drosophila mutants: Constitutive expression of a single antimicrobial peptide can restore wild-type resistence to infection
Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through either the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).
Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).
In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).
In adult Drosophila, gram-negative bacteria elicit an antimicrobial response mediated by a signaling pathway that involves the intracellular factors Imd, Tak1, IkappaB kinase Kenny (Key), and Rel. On the basis the expression profiling results, it was reasoned that the temporal waves of transcriptional activity in SL2 cells might reflect different signaling pathway contributions. It was therefore asked whether selectively removing signaling components by RNA interference (RNAi) would block induction of all, or only parts, of the transcriptional response to LPS (Boutros, 2002).
The effect of removing key or rel by RNAi was investigated. The expression profiles demonstrate that removing key or rel diminishes the induction of antimicrobial peptides. However, the induction of cytoskeletal and proapoptotic factors was not affected. In contrast, removing tak1 reduces the level of induction or repression for all identified genes, indicating that LPS-induced signaling is transmitted through Tak1 and that specific pathways branch downstream of Tak1 (Boutros, 2002).
In the Rel-independent group, several transcripts were identified that are indicative of other signaling events. For example, puc is transcriptionally regulated by JNK signaling during embryonic development. Therefore, the effect of removing SAPK/JNK activity was tested on LPS-induced transcripts. mkk4/hep dsRNA-treated cells lose the ability to induce the Rel-independent cluster, indicating that LPS signaling branches downstream of Tak1 into separate Rel- and JNK-dependent branches. To validate the results obtained from the microarray experiments, quantitative PCR (qPCR) was performed using puc and cec mRNA levels as indicators for Imd/Rel- or Mkk4/Hep-dependent pathways. Additionally, the effect of removing imd, which, in vivo, acts upstream of Tak1, was tested to clarify whether, in addition to Tak1, other known upstream components of a gram-negative signaling pathway are required for both Rel- and Mkk4/Hep-dependent pathways. These qPCR experiments confirm that cec is dependent for its expression on Imd, Tak1, Rel, and Key, whereas LPS-induced puc expression is dependent on Imd, Tak1, and Mkk4/Hep. Hence, the immunity signaling pathway in response to LPS bifurcates downstream of Imd and Tak1 into Rel- and SAPK/JNK-dependent branches. Both the Rel and SAPK/JNK pathways regulate different functional groups of downstream target genes (Boutros, 2002).
While both Rel and Mkk4/Hep pathways are downstream of Imd and Tak1 in response to LPS, the two downstream branches elicit different temporal expression patterns. It was then asked whether the first transcriptional response is controlled by downstream targets that might negatively feed back into the signaling circuit. puc was a candidate for such a transcriptionally induced negative regulator. Expression profiles of cells depleted for puc were tested before and after a 60 min LPS treatment. These experiments showed that transcripts dependent on the Mkk4/Hep branch of LPS signaling are upregulated, even without further LPS stimulus. In contrast, Rel branch targets are not influenced. puc dsRNA-treated cells show loss of the typical round cell shape. These cells appear flat and have a delocalized Actin staining, consistent with a deregulation of cytoskeletal modulators in puc-deficient cells (Boutros, 2002).
The analysis of expression profiles shows that, while SAPK/JNK and Rel signaling are controlled by the same Imd/Tak1 cascade, they appear to have different feedback loops. Whereas Rel signaling induces Rel expression and thereby generates a self-sustaining loop, possibly leading to the maintenance of target gene expression, the SAPK/JNK branch induces an inhibitor and thereby establishes a self-correcting feedback loop. These results may explain how a single upstream cascade can lead to different dynamic patterns (Boutros, 2002).
Septic injury of adult Drosophila is a widely used model system to study innate immune responses in vivo. To explore the signaling pathways that control induced genes in vivo, genome-wide expression profiles were generated of adult Drosophila infected by septic injury. Equal numbers of male and female adult Oregon R flies were infected with a mixture of E. coli (gram negative) and M. luteus (gram positive). Subsequently, flies were collected at 1, 3, 6, 24, 48, and 72 hr time points post-septic injury to measure temporal changes in gene expression levels. Computational analysis identified a list of 223 genes that were differentially regulated and matched the filtering criteria for at least two time points after microbial infection. This set includes 197 genes that are transiently upregulated and 26 that are transiently downregulated upon immune challenge. Different temporal profiles of gene expression can be detected in this analysis; clusters of genes differed significantly in the timing and persistence of induction. For example, whereas many genes are expressed transiently shortly after infection, others are induced late and are still upregulated at a 72 hr time point. A significant number of genes of both early and late clusters are differentially expressed at a 6 hr time point after infection, which was chosen for further analysis (Boutros, 2002).
The signaling requirements for these differentially expressed transcripts were examined in mutant alleles of known Toll and Imd/Rel pathway components, reasoning that additional pathways might be uncovered by analyzing patterns that cannot be reconciled with expected signaling patterns. Flies homozygous for loss-of-function mutations in tube, key, or rel were infected with gram-negative and gram-positive bacteria, and expression profiles were generated for a 6 hr time point after infection. In addition, noninfected Tl10b, a gain-of-function allele of the receptor, and cact, a homolog of the inhibitory factor IkappaB, were used to monitor transcripts that are constitutively expressed in gain-of-function signaling mutants. The antimicrobial peptides dipt and drosomycin (drs) are representative targets for the Toll and Imd/Rel pathways, respectively. dipt induction is not detectable in the expression profiles in either a rel or key mutant background, whereas its expression is not affected in tube mutants. In contrast, drs relies on Tube to convey a Toll-dependent signal. Consistently, the expression profiles show that, in a tube mutant background, drs expression is diminished. These experiments showed that the analysis of mutant expression profiles can be used to deduce signaling requirements for distinct target groups (Boutros, 2002).
Toward a computational annotation of signaling pathways, a pattern-matching strategy was employed to rank transcripts by similarity to bona fide Toll or Imd/Rel pathway targets, such as dipt and drs. A set of 91 transcripts that matched the filtering criteria was analyzed for differential expression at a 6 hr time point after septic injury. To determine their dependence on known immunity signaling pathways, the correlation coefficients were calculated of the individual gene expression level in mutant backgrounds to binary Toll or Imd/Rel patterns. Genes were subsequently ordered according to their correlation coefficients for each pathway signature. Using this strategy, transcripts were separated that primarily belong to either the Toll or Imd/Rel pathway groups. For example, genes that show a high correlation coefficient for a Toll pathway pattern include drs, transferrin, a secreted iron binding protein, IM2, and a cluster of homologous secreted peptides at 55C9. These genes have a low correlation coefficient for an Imd/Rel pattern, indicating that they are primarily dependent on Toll pathway signaling in response to microbial infection. In contrast, a group of genes score low for a Toll pathway pattern but have high correlation coefficients for an Imd/Rel pattern. This group includes known gram-negative antimicrobial peptides, such as cec and dipt, peptidoglycan receptor-like genes (PGRP-SD, PGRP-SB1), other small transcripts (CG10332), and genes coding for putative transmembrane proteins, such as CG3615 (Boutros, 2002).
Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).
It was reasoned that the patterns observed in the mutant analysis might reflect the contributions of additional signaling pathways. Also, these noncanonical clusters show distinct temporal expression patterns, suggesting that they are separately controlled. One group of genes consists primarily of cytoskeletal regulators and structural proteins that are expressed early on, with peak expression at 3 hr. These include several muscle-specific proteins, thus possibly reflecting the organ that is injured during injection. For example, flightin (fln) encodes a cytoskeletal structural protein expressed in the indirect flight muscle (Boutros, 2002).
Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep1 (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).
The clustering revealed a second noncanonical group with small proteins that are expressed late and transiently with peak expression at 6 hr after septic injury. One of the clustered transcripts, CG11501, encodes a small Cys-rich protein that is 115 amino acids long and is strongly induced after septic injury. By RT-PCR, it was confirmed that CG11501 is upregulated after septic injury. In order to characterize how CG11501 is controlled after microbial challenge, a candidate pathway approach was undertaken. In an independent study, it was found that totM gene induction, which is part of the same cluster, is dependent on a JAK/STAT signaling pathway. Whether CG11501 induction requires JAK/STAT signaling was examined. Mutations in JAK/STAT pathways in Drosophila have been implicated in various processes during embryonic and larval development. In Anopheles, STAT is activated in response to bacterial infection. Similarly, gain-of-function STAT has been implicated in the transcriptional control of thiolester proteins. Mutant alleles of hopscotch (hop), the Drosophila homolog of JAK were examined. Quantitative PCR shows that CG11501 induction after septic injury is diminished in hop loss-of-function mutants, whereas the expression of Toll and Imd targets drs, and cec is not affected (Boutros, 2002).
This study shows that in addition to known innate immune cascades, JNK and JAK/STAT are required for the transcriptional response during microbial challenge. One transcriptional signature of small secreted peptides can be traced to JAK/STAT signaling. Additionally, JNK signaling controls cytoskeletal genes after an LPS stimulus and after septic injury in vivo. Both in cells and in vivo, JNK pathways are connected to the same upstream signaling cassette that induces NFkappaB targets. Altogether, these results suggest that innate immune signaling pathways closely link cytoskeletal remodeling, as required for tissue repair, and direct antimicrobial actions. The data also provide insights into the connection of temporal patterns and the activation of distinct signaling pathways (Boutros, 2002).
NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).
Strikingly, cytoskeletal gene expression during innate immune responses is controlled by JNK through the same upstream signaling cascade that activates NFkappaB pathways. JNK pathways act downstream of microbial stimuli, both in vivo and in cells, to induce cytoskeletal regulators. In SL2 cells, JNK signaling is required for the induction of a cluster of cytoskeletal, cell adhesion regulators and proapoptotic factors. Interestingly, both NFkappaB and JNK branches share the same upstream components, Tak1 and Imd, indicating that the activation of both processes are tightly linked. MMP-1, a matrix metalloproteinase that is one of the most markedly upregulated genes after LPS stimulation, has been implicated in wound-healing responses in mammals. Compared with experiments in cells, the situation in vivo after septic injury is likely more complex. Gene expression profiling in whole organisms likely has a lower sensitivity for transcriptional changes that occur in rather small numbers of cells. Also, tissue-specific differences in signaling pathway activity may not reflect the transcriptional changes observed in the cell culture model. Muscle-specific cytoskeletal factors, possibly because they were injected into the thoracic muscle, are not inducible in a JNK-deficient genetic background. However, since it was necessary to remove both Mkk4 and Hep (Mkk7) in cells to deplete JNK pathway activity, an experiment that cannot be performed in vivo because of the lack of an Mkk4 mutant, these experiments might not have uncovered all JNK-dependent transcripts. SAPK/JNK modules can also be linked to different upstream activating cascades. For example, a recent study reported the activation of p38a through a cascade involving Toll, TRAF6, and TAB. Similarly, during innate immune responses JNK pathways can be activated by both Toll and Imd pathways in vivo (Boutros, 2002).
The activation of JNK signaling is reminiscent of signaling during dorsal and thorax closure. In dorsal closure, SAPK/JNK signaling controls cytoskeletal rearrangements that lead to the epithelial sheet movements of the embryonic epidermis. SAGE analysis of embryos with activated SAPK/JNK signaling has shown an induction of cytoskeletal factors. Also, dorsal closure movements are proposed to be similar to the reepithelization that occurs during wound healing. In other developmental contexts, SAPK/JNK signaling has been implicated in cytoskeletal rearrangements and cell motility, such as the generation of planar polarity in Drosophila and convergent-extension movements in vertebrates. A common theme of SAPK/JNK pathways might be their control of cytoskeletal regulators for diverse biological processes. The finding that, in response to LPS, SAPK/JNK and NFkappaB targets are coregulated through the same intracellular pathway suggests a close linkage of directed antimicrobial activities and tissue repair processes (Boutros, 2002).
In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).
One of the characteristics of the host defense of insects is the rapid synthesis of a variety of potent antibacterial and antifungal peptides. To date, seven types of inducible antimicrobial peptides (AMPs) have been characterized in Drosophila. The importance of these peptides in host defense is supported by the observation that flies deficient for the Toll or Immune deficiency (Imd) pathway, which affects AMP gene expression, are extremely susceptible to microbial infection. A genetic approach has been developed to address the functional relevance of a defined antifungal or antibacterial peptide in the host defense of Drosophila adults. AMP genes have been expressed via the control of the UAS/GAL4 system in imd;spätzle double mutants that do not express any known endogenous AMP gene. These results clearly show that constitutive expression of a single peptide in some cases is sufficient to rescue imd;spätzle susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).
Antimicrobial peptides (AMPs) are a key component of innate immunity. Their distribution throughout the animal and plant kingdom is ubiquitous, reflecting the importance of these molecules in host defense. In insects, systemic infection induces the synthesis of combinations of AMPs that are secreted from the immune organs, mainly the fat body, an analog of the mammalian liver, into the hemolymph, where the AMPs reach high concentrations. In Drosophila, at least seven types of AMPs (plus isoforms) have been described. Their activities have been either determined in vitro by using peptides directly purified from flies or produced in heterologous systems, or deduced by comparison with homologous peptides isolated in other insect species: (1) Drosomycin and Metchnikowin show antifungal activity; (2) Cecropins have both antibacterial and antifungal activities; (3) Drosocin and Defensin are predominantly active against Gram-negative and -positive bacteria, respectively, and (4) Attacins and Diptericins are similar to peptides from other insects that show antibacterial activity (Tzou, 2002 and references therein).
Analysis of the in vivo roles of each AMP on microbial infection is complicated by the numerous AMP genes present in the fly, as well as the redundant defense mechanisms within the innate immune system. The importance of AMPs, however, is supported by the sensitive phenotype of mutants that do not express AMP-encoding genes. A clear correlation is observed between the lack of expression of antibacterial peptide genes in mutants of the Immune deficiency (Imd) pathway and their susceptibility to Gram-negative bacteria. Conversely, mutations in the Toll pathway block Drosomycin expression and result in susceptibility to fungal infection. Finally, mutants deficient in both the Imd and Toll pathways failed to express any known AMP genes after infection and are extremely susceptible to both fungal and bacterial infections. These evidences of the importance of AMPs in fighting infection, however, are still indirect, because it cannot be exclude that these mutations affect other defense reactions. The Toll pathway, for example, has also been reported to regulate hemocyte proliferation. To study unambiguously the in vivo role of each AMP in Drosophila host defense, imd;spätzle (spz) double mutant flies have been created that are deficient for both the Imd and Toll pathways but that constitutively express different AMPs under the control of a noninducible promoter. These flies express only one AMP on infection and, consequently, a simple survival experiment can be used to monitor the contribution of this peptide in resistance to infection by various microorganisms. This powerful assay allowed the analysis, in vivo, of the spectrum of activity of each peptide and, by combining two different transgenes, any potential synergy among them. These results clearly show that expression of a single peptide, in some cases, is sufficient to rescue the imd;spz susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).
In this assay, the AMP genes are expressed via the UAS/GAL4 system at a level similar to that observed in wild-type induction of the endogenous AMP genes (except Defensin and Diptericin). However, there are still some differences between this assay and the wild-type physiological condition. In the UAS-Pep flies, AMP genes are expressed ubiquitously and constitutively, contrasting to the wild-type flies in which peptides are made mainly by the fat body in an acute phase profile. The accumulation of AMP, therefore, through constitutive gene expression before infection may be critical to confer an effective protection (Tzou, 2002).
This study provides an alternative method for monitoring and comparing the antimicrobial activity of the various Drosophila AMPs. Defensin is the most potent peptide against Gram-positive bacteria, whereas Attacin A and Drosomycin are active against Gram-negative bacteria and fungi, respectively. One copy of UAS-Def is sufficient to protect flies to wild-type level against M. luteus, B. subtilis, and S. aureus. The efficiency of Defensin may explain why the endogenous Defensin gene is transcribed to lower levels than the other AMP genes after infection. One copy of UAS-Drs is sufficient to protect against N. crassa, whereas two copies are required to induce a complete and partial protection against F. oxysporum and A. fumigatus, respectively. These results are consistent with the Minimum Inhibitory Concentration assay of Drosomycin required in vitro to kill these three fungi: 0.3-0.6 µM for N. crassa, 1.2-2.5 µM for F. oxysporum, and 20-40 µM for A. fumigatus. In addition, Diptericin in Drosophila contributes to resistance against some Gram-negative bacteria, although its activity is probably underestimated because of the low levels of Diptericin expression generated by the constructs used in this study. Surprisingly, no clear protective effect of Cecropin A could be detected in this assay, whereas Cecropin A peptide shows strong in vitro activity. The possibility cannot be excluded that in the lines used, Cecropin A is not effectively produced or well processed to the active form. Alternatively, a higher level of Cecropin A expression may be required to generate a protective effect, considering that the Drosophila genome contains three other inducible Cecropin genes (Tzou, 2002).
These results also underline the differential activities of Drosophila AMPs: such is the case of Attacin A and Drosocin in resistance to some Gram-negative bacterial species. Thus the existence of numerous AMPs may help widen the protection against a large number of microorganisms. In the case of Gram-negative bacterial infection, none of the peptides are able to restore a wild-type resistance in imd;spz double mutants. These results and the observation that the Drosophila genome encodes a high number of AMP genes with activity directed against Gram-negative bacteria suggest that the elimination of this class of bacteria may require the global toxicity generated by multiple, rather than one or two, AMPs (Tzou, 2002).
This study does not reveal a striking synergistic activity among any pair of AMPs tested. In some cases, a rather cooperative effect is observed between two AMPs such as Attacin A when coexpressed with either Diptericin or Drosocin in resistance to some Gram-negative bacteria. These observations suggest that the multiple Drosophila AMPs may function in an additive way, rather than synergistically (Tzou, 2002).
Host-pathogen interactions are antagonistic relationships in
which the success of each organism depends on its ability to overcome
the other. The production of AMPs is a common strategy to eliminate the
invading microbes and, consequently, pathogens have evolved strategies
to prevail over these defenses. The assay used provides a powerful tool
to compare the resistance of various bacteria to different AMPs,
because in these experiments, microbes were injected in an environment
previously enriched in peptides. The time race between pathogen and the
host defense is clearly illustrated by the observation that a
preexisting level of Defensin is sufficient to ensure a complete
resistance against B. subtilis, a Gram-positive bacterium
highly pathogenic for flies. This observation indicates that B. subtilis is sensitive to Drosophila AMP but nevertheless can overtake the Drosophila immune response by its rapid growth. The
observation that 'immunizing' flies with nonpathogenic bacteria
fully protects Drosophila from a subsequent infection by
B. subtilis is consistent with this
hypothesis. These results also show that the kinetics of infection by
P. aeruginosa or B. bassiana, two highly
entomopathogenic microbes, are not delayed in flies expressing AMP
genes, suggesting that these microbes have developed some mechanisms to
escape the AMP activity. The observation that Drosomycin
expression does not confer any protection against B. bassiana is unexpected, because Toll-mediated defense against this pathogen has been reported. This observation suggests that other antifungal peptides (e.g., Metchnikowin) or a yet uncharacterized defense reaction may be required to resist this fungus. Finally, the
human pathogen, S. aureus, is also highly pathogenic to
Drosophila and shows a better resistance to a high level of Defensin compared with other Gram-positive bacteria. These results underline the correlation between pathogenicity and increased resistance to AMPs (Tzou, 2002).
From a forward genetic screen for phagocytosis mutants in Drosophila, a mutation was identified that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, the data suggest that the Drosophila gene PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram+ bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-L-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, it was found that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis (Garver, 2006).
Host receptors must recognize a microbe before any immune response can begin. After recognition, signaling pathways are activated and result in effector responses such as induction of antimicrobial peptides (AMPs), melanization, and phagocytosis, which are important for controlling the infection. Much is known about the signaling pathways important for the AMP response in Drosophila, but less is known about the regulation of the other two responses. In Drosophila, if phagocytosis is blocked in a mutant that is already impaired for the AMP response, a normally innocuous Escherichia coli infection becomes lethal. Phagocytosis is also likely to be a first line of defense, because the response involves specific interaction between microbe and host receptors and occurs within half an hour, whereas the induction of AMPs occurs over several hours and AMPs can act on diverse groups of microbes (Garver, 2006).
The peptidoglycan recognition proteins (PGRPs) are critical receptors in the Drosophila immune response that are required for the recognition of peptidoglycan, a component of bacterial cell walls (Dziarski, 2004; Steiner, 2004), and for subsequent activation of AMP gene expression (Choe, 2002; Werner, 2003). PGRPs were first characterized in the moths Bombyx mori and Trichoplusia ni and proposed to be receptors that can trigger immune responses. PGRPs have also been identified in mammals, and mutant PGRP mice have been generated, but the most comprehensive characterization of PGRPs has been performed in Drosophila (Garver, 2006).
Drosophila has 13 PGRP genes, six long (L) forms with four that are predicted to reside in the plasma membrane, and seven short forms (S) that are all predicted to be secreted. PGRPs share homology with N-acetylmuramoyl-L-alanine amidases, which cleave peptidoglycan at the lactylamide bond between the glycan backbone and the stem peptides. Some PGRPs, such as PGRP-LC, -LE -SA, and -SD, lack a critical cysteine in the catalytic pocket and are not able to cleave peptidoglycan. PGRP-LC, -LE, and -SA have been demonstrated to bind peptidoglycan and are necessary for expression of AMP genes, supporting the hypothesis that PGRPs directly recognize bacteria and activate immune responses. The identification of mutations in PGRP-SA (seml) and PGRP-LC (ird7 or totem) indicated that these genes are necessary for activation of the two signaling pathways regulating AMP gene expression, the Toll pathway that responds to Gram+ bacteria and fungi and the Imd pathway that responds to Gram– bacteria. Drosophila uses PGRP-SA and PGRP-LC to distinguish between Gram+ and Gram– PGN for activation of the Toll and Imd signaling pathways (Leulier, 2003). Gram+ PGN and Gram– PGN differ in the stem peptide portion; typical Gram+ bacteria have a lysine as the third amino acid, whereas Gram– bacteria and the Gram+ Bacillus have a diaminopimelic acid (DAP) in that position. Two other noncatalytic PGRPs, PGRP-LE and PGRP-SD, also play a role in activation of the Imd and Toll pathways, respectively. PGRP-LC, PGRP-LE double mutants show a more dramatic phenotype to Bacillus and Gram– bacterial infection than either mutation alone, suggesting that PGRP-LE acts with PGRP-LC in the recognition of Gram– DAP-type peptidoglycan for the activation of the Imd pathway. PGRP-SD may be playing a similar role with PGRP-SA for activation of the Gram+/Toll pathway (Garver, 2006 and references therein).
Catalytic PGRPs, such as PGRP-SC1a and -SC1b, include this cysteine residue in the active site, and are potent enzymes that cleave peptidoglycan between the N-acetylmuramic acid of the backbone and the L-alanine in the stem peptide. After digestion with PGRP-SC1b, staphylococcal peptidoglycan exhibits less activation of the AMP genes in a Drosophila blood cell line, so it was hypothesized that catalytic PGRPs may act as scavengers to limit an inflammatory response to free peptidoglycan (Mellroth, 2003). This may not be absolute, since PGRP-SC1b-digested Gram– peptidoglycan is still able to activate the AMP response in S2 cells, albeit at a higher dose. However, it was of interest to examine the role of these genes in vivo (Garver, 2006).
In a genetic screen for phagocytosis mutants, a novel mutant, picky was identified. picky flies fail to phagocytose S. aureus particles but can phagocytose E. coli, Bacillus subtilis, and Saccharomyces cerevisiae zymosan particles. picky mutants have defects in the activation of the Toll pathway. picky maps to the location of the PGRP-SC1a, -SC1b, and -SC2 genes, and picky flies express significantly less PGRP-SC1a. The picky defects can be rescued by expression of PGRP-SC1a, indicating that PGRP-SC1a is important for phagocytosis and activation of AMP responses. These results differ from the prevailing model of catalytic PGRPs as scavengers and suggest that in vivo, PGRP-SC1a is required for initiating immune pathways. By comparing a wild-type PGRP-SC1a with a cysteine-serine mutant PGRP-SC1a for rescue of picky phenotypes, it was found that the catalytic activity is essential for phagocytosis of live S. aureus, but not for activation of the Toll pathway (Garver, 2006).
To identify genes important for phagocytosis, a collection of ethylmethane sulfonate-induced adult viable Drosophila mutants was screened using an in vivo phagocytosis assay. To determine what a mutant might look like, the PGRP-LC (ird7) and PGRP-SA (seml) mutants were examined. PGRP-LC has been reported to be a recognition receptor for phagocytosis of E. coli in S2 cells. However, this study contradicted another report citing that ird7 blood cells can phagocytose bacteria (Choe, 2002). The current study found that ird7 mutants are able to phagocytose both E. coli and S. aureus particles. This finding suggested that phagocytosis of Gram– bacteria may require other receptors in addition to PGRP-LC in vivo. In contrast, it was found that seml mutants are specifically impaired in their ability to phagocytose S. aureus but not E. coli. 94% of the seml mutant flies were able to phagocytose E. coli, but only 25% were able to phagocytose S. aureus. This finding suggests that PGRP-SA may be important for recognition of Gram+ bacteria for phagocytosis in addition to its role in activating the Toll pathway. In contrast, flies with mutations affecting other Toll pathway components, spatzle (the Toll ligand) and Dif (an NF-kappaB) were still able to phagocytose S. aureus, indicating that the phagocytosis defect is not a secondary effect from loss of Toll signaling (Garver, 2006).
From a pilot screen, one mutation was identified that was named picky eaterZ2–4761 (picky) because the mutants fail to phagocytose S. aureus particles, but can still phagocytose E. coli and Saccharomyces cerevisiae zymosan particles. Only 25% of picky mutant flies showed any phagocytosis response to S. aureus, but 83% of those same flies were still able to phagocytose E. coli. To investigate whether the recognition involved the difference in peptidoglycan between Gram+ and Gram– bacteria, phagocytosis was examined of a B. subtilis-GFP strain. picky mutants are able to phagocytose B. subtilis. This finding suggests that the picky gene product is important for the specific recognition of the Lys-type peptidoglycans, typical of most Gram+ bacteria, but not found in Bacillus spp (Garver, 2006).
Because picky flies appear to be impaired in the recognition of S. aureus for phagocytosis and survival, it was possible that the mutation might also affect activation of the Toll or Imd pathways. The induction of Drosomycin and Diptericin AMP genes is often used to assess activation of the Toll/Gram+ and Imd/Gram– signaling pathways, respectively. The AMP responses in picky were compared with those of the known Toll pathway components, seml and Dif, and to an Imd pathway component, ird7. picky flies showed no induction of Drosomycin in response to S. aureus, E. coli, or Micrococcus luteus at either 2 or 24 h. All of the S. aureus-infected picky flies were dead at 24 h, so the Drosomycin expression in this sample was not assessed. seml and Dif both show some induction of Drosomycin at 2 and 24 h. In comparison, picky has a more consistent and dramatic effect on Drosomycin expression and likely encodes an essential component of the Toll pathway. In contrast, the expression of Diptericin in response to bacterial infection in picky mutants was higher than that seen in wild type. Therefore, the defect in picky appears to be selective for the Toll pathway. To determine where in the Toll pathway picky might lie, the picky mutation was crossed into a Toll10b gain of function mutant. picky was not able to suppress the constitutive expression of Drosomycin in a Toll10b mutant. This epistasis analysis places picky either upstream of Toll, which would be consistent with a role in bacterial recognition or in a parallel pathway (Garver, 2006).
The peptidoglycan recognition protein family is important for allowing the Drosophila immune response to distinguish between Gram+ and Gram– bacteria for the activation of specific AMP signaling pathways (Choe, 2002; Werner, 2003). The current data indicate that phagocytosis also relies on PGRPs to distinguish between Gram+ and Gram– bacterial peptidoglycan. A catalytic PGRP, PGRP-SC1a, is essential for the phagocytosis of S. aureus and the activation of AMP responses. The fact that PGRP-SC1a mutants have a striking phagocytosis defect indicates that it is absolutely required and that other PGRPs are not able to substitute for its loss of function. PGRP-SC1a differs from the existing PGRP mutants in that it has catalytic activity that is essential for efficient phagocytosis and ultimately limiting the infection process. Hence, noncatalytic PGRPs, such as PGRP-SA, may not be able to substitute for PGRP-SC1a function. Because PGRP-SC1a is likely present in the hemolymph, it may be acting as an opsonin to bind bacteria, with the PGRP-bacterial complex afterwards being recognized by transmembrane phagocytosis receptors that complete the uptake into the phagocyte. An alternative model is that PGRP-SC1a may generate peptidoglycan cleavage products that function as immune modulators to stimulate phagocytic activity (Garver, 2006).
For S. aureus, PGRP-SC1a is necessary for recognition of bacteria for both the phagocytosis and AMP responses. However, for E. coli, PGRP-SC1a is similar to PGRP-LC in that it is not necessary for recognition for phagocytosis, but is necessary for the activation of an AMP response. This finding raises the interesting issue of the role PGRPs play in recognizing Gram– bacteria. In Gram– bacteria, the peptidoglycan is buried under an outer membrane, so it has been proposed that the small amounts of PGN that are shed may be sufficient to trigger the AMP response (Leulier, 2003). Because the cell wall PGN is not easily accessible, it makes sense that the PGRPs are not playing a direct role in recognition for phagocytosis. An alternate possibility is that the exposure of bacterial peptidoglycan occurs after the initial uptake into phagocytes, and at that later point, is then recognized by PGRP-LC and PGRP-SC1a to trigger AMP responses. There is precedence for intracellular recognition in the phagosome in mammalian immune responses, with studies indicating that mammalian short PGRPs function in the phagosome to inhibit bacterial growth and the observation that Toll-like receptor recognition of bacteria in the phagosome can influence the rate of phagosome maturation. A related possibility is that some basal level of activation of PGRP-SC1a and the Toll pathway may be required for any expression of Drosomycin. picky mutants show much lower expression of Drosomycin than wild type in the absence of infection, so perhaps an induction may not be detectable (Garver, 2006).
It was also found that seml mutants are defective in the phagocytosis of S. aureus but not E. coli. Recent reports have indicated that the processing of peptidoglycan into monomers, lactyltetrapeptides, or muropeptides, can yield potent activators of the AMP response for both the Imd and the Toll pathway. It was further suggested for the Toll pathway that processing of peptidoglycan may be a prerequisite step upstream of PGRP-SA/seml recognition and activation of the Toll AMP pathway. This model would be consistent with catalytic PGRPs, such as PGRP-SC1a, playing a supporting role in the processing of peptidoglycan for initiating recognition events (Garver, 2006).
Since many PGRPs are clearly required for activation of immune effector responses, the issue arises as to the specificity of PGRPs for their substrates. Work from several groups indicates that alternative splice variants (in PGRP-LC) or small differences in amino acid sequences (from PGRP-LB and human PGRP-Ialpha structural analyses) may result in the ability of different PGRPs to have distinct recognition potential. It will be interesting to explore the range of bacterial types recognized by the 17 Drosophila PGRP proteins and to determine how subtle differences in recognition may be reflected by specific amino acid sequences. There may also be genetic interactions or antagonism in their function, so using the genetic tools available in Drosophila may be necessary for ultimately determining both their unique and redundant roles (Garver, 2006).
This work demonstrates the utility of a forward genetic approach to understanding the recognition of pathogen types for phagocytosis and activation of AMP responses. It is hoped that identification of genes important for recognition of bacteria in Drosophila should increase understanding of how the process works the human immune system (Garver, 2006).
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