An essential complementary role of NF-kappaB pathway to microbicidal oxidants in Drosophila gut immunity

In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-kappaB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. We used a novel in vivo approach to reveal the physiological role of gut NF-kappaB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-kappaB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-kappaB pathway mutants in the intestine. These results imply that the local 'NF-kappaB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity.     

Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila

BACKGROUND: Studies in Drosophila have taught us a great deal about how animals regulate the immediate innate immune response, but we still know little about how infections cause pathology. Here, we examine the pathogenesis associated with Mycobacterium marinum infection in the fly. M. marinum is closely related to M. tuberculosis, which causes tuberculosis in people. RESULTS: A microarray analysis showed that metabolism is profoundly affected in M. marinum-infected flies. A genetic screen identified foxo mutants as slower-dying after infection than wild-type flies. FOXO activity is inhibited by the insulin effector kinase Akt; we show that Akt activation is systemically reduced as a result of M. marinum infection. Finally, we show that flies infected with Mycobacterium marinum undergo a process like wasting: They progressively lose metabolic stores, in the form of fat and glycogen. They also become hyperglycemic. In contrast, foxo mutants exhibit less wasting. CONCLUSIONS: In people, many infections--including tuberculosis--can cause wasting, much as we see in Drosophila. Our study is the first examination of the metabolic consequences of infection in a genetically tractable invertebrate and gives insight into the metabolic consequences of mycobacterial infection, implicating impaired insulin signaling as a key mediator of these events. These results suggest that the fly can be used to study more than the immediate innate immune response to infection; it can also be used to understand the physiological consequences of infection and the immune response.     

Water loss through the integument in the desiccation-sensitive mutant, Parched, of Drosophila melanogaster

Two desiccation-sensitive mutants of Drosophila melanogaster were isolated. Genetic analysis showed that the phenotype is controlled by a recessive gene parched located in 1A1-8 of the X-chromosome. In a desiccated environment without any water supply, the survival time of the mutant flies was considerably shorter than that of the wild-type flies. The rate of water loss in the mutant flies was significantly higher than that of the wild-type flies, whether dead or alive. The survival time of the mosaic flies, which have the mutant and wild-type cuticle, was prolonged in proportion to the amount of wild-type cuticle which they possessed. These results suggest that the mutant has a defect in some waterproofing mechanism of the integument. The mutant flies drank much more water than the wild-type flies, to compensate for the rapid water loss. The hydrocarbons, which are the predominant constituent of cuticular lipids, were analyzed by gas-liquid chromatography, but there were no significant quantitative nor qualitative differences between the wild-type and the mutant flies.     

Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes

Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Delta(4,6)-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Delta(4,6)-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies.     

Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens.     

Prevention of apoptosis by mitochondrial phosphatase PGAM5 in the mushroom body is crucial for heat shock resistance in Drosophila melanogaster

The heat shock (HS) response is essential for survival of all organisms. Although the machinery of the HS response has been extensively investigated at the cellular level, it is poorly understood at the level of the organism. Here, we show the crucial role of the mushroom body (MB) in the HS response in Drosophila. Null mutants of the mitochondrial phosphatase Drosophila PGAM5 (dPGAM5) exhibited increased vulnerability to HS, which was reversed by MB-specific expression of the caspase inhibitor p35, and similar vulnerability was induced in wild-type flies by knockdown of MB dPGAM5. Elimination of the MB did not affect the HS response of wild-type flies, but did increase the resistance of dPGAM5-deficient flies to HS. Thus, the MB may possess an apoptosis-dependent toxic function, the suppression of which by dPGAM5 appears to be crucial for HS resistance.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Thioredoxin-2 affects lifespan and oxidative stress in Drosophila

Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotide-reductase. In Drosophila melanogaster there are three "classical" thioredoxins with the conserved active site: deadhead, ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene. Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the organism's oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental stress.     

Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and Gram-positive bacteria

The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G(+)) bacteria activate the Toll/Dif pathway and that Gram-negative (G(-)) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G(+) and G(-) bacteria. In Relish mutants, the Cecropin A gene was induced by the G(+) bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G(+) or G(-) bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G(+) bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G(+) bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G(+) bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. Here, we monitored phase response curves of cry(01) mutants and control flies to 1-hour 1000-lux light pulses. We found that cry(01) mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 significantly increased the magnitude of phase advances in cry(01) mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.     

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.     

Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Pathogenesis of listeria-infected Drosophila wntD mutants is associated with elevated levels of the novel immunity gene edin

Drosophila melanogaster mount an effective innate immune response against invading microorganisms, but can eventually succumb to persistent pathogenic infections. Understanding of this pathogenesis is limited, but it appears that host factors, induced by microbes, can have a direct cost to the host organism. Mutations in wntD cause susceptibility to Listeria monocytogenes infection, apparently through the derepression of Toll-Dorsal target genes, some of which are deleterious to survival. Here, we use gene expression profiling to identify genes that may mediate the observed susceptibility of wntD mutants to lethal infection. These genes include the TNF family member eiger and the novel immunity gene edin (elevated during infection; synonym CG32185), both of which are more strongly induced by infection of wntD mutants compared to controls. edin is also expressed more highly during infection of wild-type flies with wild-type Salmonella typhimurium than with a less pathogenic mutant strain, and its expression is regulated in part by the Imd pathway. Furthermore, overexpression of edin can induce age-dependent lethality, while loss of function in edin renders flies more susceptible to Listeria infection. These results are consistent with a model in which the regulation of host factors, including edin, must be tightly controlled to avoid the detrimental consequences of having too much or too little activity.     

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.