InteractiveFly: GeneBrief

Dual oxidase: Biological Overview | References

BIOLOGICAL OVERVIEW

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. This study reports that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras(V12) model. Together, these studies have identified a new function of Myo1D for AiP and tumorigenesis and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane (Amcheslavsky, 2018).

Under stress conditions, when a large number of cells are dying, there is a need for compensatory proliferation to replace the lost cells with new cells. Work using several model organisms has shown that, under these conditions, apoptotic cells can release mitogenic signals that induce proliferation of surviving cells for the replacement of dying cells. Because apoptotic cells are actively triggering this type of compensatory proliferation, this process has been termed apoptosis-induced proliferation (AiP) (Amcheslavsky, 2018).

Caspases are Cys proteases that are the main effectors of apoptosis. They are produced as inactive zymogens with a prodomain and after processing a large and small subunit. There are initiator and effector caspases. Initiator caspases carry protein/protein interacting motifs in their prodomains, which mediate their incorporation into large multimeric protein complexes. For example, the mammalian initiator caspase-9 is recruited into the Apaf-1 apoptosome, while its Drosophila ortholog Dronc forms the apoptosome with the Apaf-1 homolog Dark. Effector caspases such as mammalian caspase-3, or Drosophila DrICE and Dcp-1, are proteolytically processed by activated initiator caspases and mediate the apoptotic process (Amcheslavsky, 2018).

In addition to apoptosis, caspases are also mediating AiP. They trigger the release of Wnt, bone morphogenetic protein (BMP)/transforming growth factor β (TGF-β), epidermal growth factor (EGF), and Hedgehog mitogens for AiP. This has been best studied for the Drosophila initiator caspase Dronc using the 'undead' AiP model in which apoptotic signaling is induced by expression of upstream cell death factors such as hid, but the execution of apoptosis is blocked by co-expression of the effector caspase inhibitor p35, thus rendering cells in an undead condition. Because P35 inhibits apoptosis, but not Dronc, Dronc can still mediate non-apoptotic functions such as AiP. When hid and p35 are co-expressed using the ey-Gal4 driver (ey > hid,p35), which is expressed in epithelial cells of eye imaginal discs, Dronc continuously signals for AiP and triggers hyper-proliferation. Consequently, the discs are enlarged and the resulting heads of the adult flies are overgrown. In genetic screens, screening was carried out for suppressors of the overgrowth phenotype of undead (ey > hid,p35) adult heads to identify genes and mechanisms involved in AiP (Amcheslavsky, 2018).

Mechanistically, this study showed that, in undead cells, Dronc stimulates the NADPH-oxidase Duox for the production of extracellular reactive oxygen species (eROS). eROS recruits and activates hemocytes, Drosophila immune cells similar to macrophages, to the undead imaginal disc. In turn, hemocytes release the tumor necrosis factor-like ligand Eiger, which induces JNK activity in epithelial disc cells. JNK promotes the expression of the apoptotic genes reaper and hid, which initiate a positive feedback loop to maintain undead signaling (Fogarty, 2016). In addition, it induces the release of the mitogens Wingless (Wg), a Wnt-like gene in Drosophila, decapentaplegic, a BMP/TGF-β homolog, and Spitz, an EGF ligand, which all promote AiP (Amcheslavsky, 2018).

In addition to undead AiP, there is also 'genuine' AiP, during which dying cells complete the apoptotic process, and the response of the affected tissue to replace the dying cells is examined. In contrast to undead AiP, genuine AiP does not promote overgrowth. Therefore, although most genes identified in undead AiP also have important roles in genuine AiP, there must be differences between the two AiP models. In any case, genuine AiP is used as a model of tissue regeneration, while the hyper-proliferation of undead AiP serves as a tumorigenic model (Amcheslavsky, 2018).

Class I unconventional myosins are conserved actin-based motor proteins, composed of the N-terminal head (motor) region with an ATP binding motif (including P-, switch1-, and switch2 loops) and an actin-binding domain, a neck region characterized by two to three IQ motifs, and a C-terminal tail domain that interacts with phospholipids at membranes. Mammals have eight class I myosins, Drosophila has three, Myosin 1D (Myo1D, also known as Myo31DF), MyoIC (Myo61F), and Myo95E. While Myo1D and Myo1C are involved in left/right (L/R) development of visceral organs, the function of Myo95E is unknown (Amcheslavsky, 2018).

Although Drosophila is a bilateral organism, certain visceral organs such as the gut and the coiling of the spermiducts around the gut, which occurs in a morphogenetic movement termed male terminalia rotation, display L/R asymmetry. In Myo1D mutants, the chirality of these asymmetric organs and movements are reversed. For example, the male terminalia rotation during pupal development, which, in wild-type, occurs for 360° in clockwise (dextral) orientation, proceeds in Myo1D mutants sinistrally, defining Myo1D as dextral determinant. Myo1D engages the actin cytoskeleton and adherens junctions for this movement (Amcheslavsky, 2018).

Overexpression of Myo1C antagonizes the dextral activity of Myo1D by displacing it from adherens junctions. However, the loss-of-function phenotype of Myo1C did not confirm this antagonizing function. Instead, while Myo1C single mutants do not display any L/R defect, the Myo1C Myo1D double mutant has a stronger sinistral male terminalia phenotype than Myo1D mutants indicating that Myo1C has a partially redundant dextral activity with Myo1D (Amcheslavsky, 2018).

It has long been known that genes in the apoptosis pathway, such as hid, dronc, and drICE, are also involved in male terminalia rotation in Drosophila. Indeed, localized apoptotic activity is required for this L/R process. How Myo1D and the apoptosis pathway interact for male terminalia rotation is not very well understood. Interestingly, mutants of the JNK signaling pathway or overexpression of puckered, an inhibitor of JNK activity, also display defects in male terminalia rotation (Amcheslavsky, 2018).

This study reports that Myo1D is an essential component of AiP in the undead model. Genetic inactivation of Myo1D strongly suppresses ey > hid,p35-induced overgrowth of the head capsule, while overexpression of Myo1D enhances it. Myo1D promotes the generation of ROS by Duox for AiP signaling. Further mechanistic analysis reveals that Myo1D is required for membrane localization of Dronc, specifically to the basal side of the plasma membrane of undead epithelial disc and salivary gland cells. Here, Dronc exerts a non-apoptotic function resulting in Duox activation. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment that allows non-apoptotic processes of Dronc and potentially other caspases to occur. Therefore, in addition to the dextral activity of Myo1D, this study identified a second function of Myo1D for the control of apoptosis-induced proliferation (Amcheslavsky, 2018).

Mechanistically, it was found that Myo1D is involved in the localization of the initiator caspase Dronc to the basal side of the plasma membrane of undead DP disc and SG cells. Myo1D interacts with Dronc, suggesting that it may directly translocate Dronc to the plasma membrane. However, Myo1D does not appear to be a cleavage target of the caspase Dronc (Amcheslavsky, 2018).

The observed localization of Dronc to the basal side of the plasma membrane in undead DP cells is critical for the mechanism of AiP. Undead cells attract hemocytes to the discs in a Dronc- and Duox-dependent manner. However, that occurs at the basal side of DP cells of imaginal discs because the basal side is exposed to the hemolymph that contains circulating hemocytes, while the apical side faces the lumen between the DP and the PM. Consistently, there is also an enrichment of Duox at the basal side of the plasma membrane. Therefore, in order to be able to activate Duox for ROS generation and hemocyte activation, Dronc needs to be specifically present at the basal side of the plasma membrane (Amcheslavsky, 2018).

It has long been known that caspases, including Dronc, have non-apoptotic functions in addition to their well characterized role in apoptosis. This paper reveals one mechanism by which cells may activate a caspase (Dronc) without the detrimental consequences of apoptosis. The sequestration of Dronc to the basal side of the plasma membrane in a Myo1D-dependent manner and the low abundance of Dronc's apoptotic partner Dark at the plasma membrane may ensure localized and controlled apoptosome activity which is sufficient for AiP, but not for killing cells. Alternatively, apoptotic substrates needed for the execution of apoptosis may not be present at the plasma membrane or in insufficient amount to pass the apoptotic threshold (Amcheslavsky, 2018).

While this study addressed the role of membrane localization of Dronc under undead conditions, recently membrane-localized Dronc was shown in SGs under normal conditions, which explains the membrane localization of Dronc at control SGs. Here, membrane-localized Dronc is required for F-actin cytoskeleton dismantling at the end of larval development in a non-apoptotic manner. In addition to the plasma membrane, the outer mitochondrial membrane has been shown to provide a non-apoptotic platform for caspase activation, in this case during sperm maturation. Therefore, membranes in general may provide a local environment for non-apoptotic caspase activities (Amcheslavsky, 2018).

The membrane localization of Dronc in SGs is mediated by Tango7, which has previously been implicated in spermatid maturation. As mentioned above, membrane-localized Dronc is required for dismantling of the cortical F-actin cytoskeleton in SGs of late larvae. However, while Tango7 RNAi blocks actin dismantling, Myo1D RNAi does not, suggesting that the roles of Tango7 and Myo1D for membrane localization of Dronc are different from each other. That also explains why in undead SGs the membrane localization of Dronc strongly increases in a Myo1D-dependent manner. Unfortunately, it was not possible to test if Tango7 is involved in AiP. Tango7 RNAi in eye imaginal discs results in complete loss of the disc. Tango7 encodes the homolog of eukaryotic translation initiation factor 3m (eIF3m), suggesting that it may also have an important requirement for protein translation, explaining the loss of the eye disc by Tango7 RNAi (Amcheslavsky, 2018).

In addition to Myo1D and Tango7, there is at least one other factor, Crinkled (Ck), which directs Dronc to non-apoptotic functions. Ck bridges the interaction between Dronc and the kinase Shaggy/glycogen synthase kinase beta (GSK-β), resulting in the selective activation of Shaggy/GSK-β, which then promotes non-apoptotic activities such as the specification of scutellar bristles, border cell migration, and correct branching of the aristae. Interestingly, Ck encodes another unconventional myosin, a member of the class VII myosin family, potentially suggesting that other myosins may also direct non-apoptotic functions to caspases (Amcheslavsky, 2018).

Myo1D and the apoptotic machinery have been linked to male terminalia rotation, an L/R process during pupal development. Indeed, apoptosis is required for Myo1D-dependent male terminalia rotation. It is unknown how Myo1D interacts with the apoptotic machinery to direct this L/R movement. In future studies, it will be interesting to examine if the Myo1D-dependent mechanism identified here for AiP also applies to male terminalia rotation or whether a separate mechanism exists in this context (Amcheslavsky, 2018).

Myo1D not only localizes Dronc to the plasma membrane, it also stabilizes it. Dronc is activated in undead cells, and activated Dronc is subject of increased protein degradation. Thus, Myo1D prevents degradation of Dronc by changing its subcellular localization to the plasma membrane (Amcheslavsky, 2018).

Myo1D has a very strong requirement for AiP in the undead model, and a requirement in the scrib-/-RasV12 tumorigenesis model, yet it does not appear to play any significant role in genuine AiP. In fact, Myo1D is the first gene identified that is essential for the hyper-proliferation of undead AiP, but not required for the regeneration of genuine AiP. The mechanism revealed in this paper provides an explanation for this behavior. During genuine AiP, cells are allowed to undergo apoptosis, which requires cytosolic Dronc activity. Although ROS are generated during genuine AiP, the origin of these ROS has not been determined and may not require the plasma membrane-localized Duox. Therefore, a key difference between genuine AiP and undead AiP, and potentially between other regenerative versus tumorigenic models, may be the altered localization of Dronc to a non-apoptotic compartment at the plasma membrane, and a shift from balanced apoptosis and proliferation to dominant proliferation. The next big question will be to examine what exactly is prompting Myo1D to drive this re-localization of Dronc under sustained undead conditions, but not under the limited regenerative conditions of the genuine AiP models, and whether that answer provides any insight into the cancer versus wound healing models (Amcheslavsky, 2018).

In conclusion, in addition to its role in L/R development, this study identified a second function of Myo1D for AiP and tumorigenesis. The basal side of the plasma membrane was identified as a non-apoptotic environment for caspase function. In future work, it will be important to identify the mechanisms by which Dronc mediates its non-apoptotic functions at the plasma membrane for AiP and other cellular processes that require membrane localization of Dronc and other caspases (Amcheslavsky, 2018).

Shaka, M., Arias-Rojas, A., Hrdina, A., Frahm, D. and Iatsenko, I. (2022). Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster. PLoS Pathog 18(9): e1010825. PubMed ID: 36084158

Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster

Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. This study investigated the virulence factors of a representative Providencia species-P. alcalifaciens. A P. alcalifaciens transposon mutant library was generated, and an unbiased forward genetics screen was performed in vivo for attenuated mutants. The screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, it was found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. This reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism (Shaka, 2022)

The aim of this study was to dissect the host-pathogen interactions between Providencia and D. melanogaster. To achieve this aim, various genetic approaches were used that enabled determination of the contributions of both pathogen and host to the outcome of the infection. First, the responses of the fruit fly to Pa infection was characterized and, using mutant analysis, the Imd pathway and iron sequestration were identified as prominent defense mechanisms against Pa. Second, an unbiased forward genetics screen was performed using a transposon mutant library that were generated for this purpose, and Pa virulence factors necessary to infect the fly were identified. This mutant library has the potential to serve as a valuable resource for exploring the genetic basis for all Pa traits. Third, mutants of the major immune pathways in Drosophila were used, and they were infected with attenuated Pa mutants to identify pathogen virulence factors that allow the bacteria to respond to specific immune defenses and evade immune clearance. Thereby, this study dissected both sides of host-pathogen relationship in a Drosophila-Providencia model and provided the first insights into the molecular mechanisms of Pa virulence (Shaka, 2022)

To identify Pa virulence factors, an in vivo screen was performed which yielded 23 attenuated mutants. The majority of these mutants (15/23) had transposon insertions in genes involved in LPS biosynthesis and LPS modifications, pointing towards a vital role of intact LPS in Pa pathogenesis. This finding is consistent with a well-known role of LPS in host-pathogen interactions. At the mechanistic level, LPS protects Pa from Drosophila Imd pathway-dependent AMPs, particularly Drosocin. Consistent with this, Pa LPS mutants showed increased susceptibility in vitro to the cationic AMP polymyxin B and their virulence was restored in Relish and ΔAMP mutant flies deficient for Imd-dependent AMPs. The finding that Pa LPS mediates resistance to host AMPs complements numerous previous studies in diverse pathogens that reported a similar protective function of LPS against host innate defenses. Several studies that used Drosophila as an infection model also discovered LPS as an essential protective barrier against insect AMPs. For example, another study found that LPS O-antigen-deficient Serratia marcescens mutants were attenuated in wild-type flies but not in an Imd pathway mutant. A similar phenotype was reported for F. novicida mutants with affected LPS. These data demonstrate that a major determinant of virulence in several pathogens is the LPS-mediated ability to resist the systemic immune response. Additionally, LPS was shown to facilitate microbiota-host interactions. For instance, LPS biosynthesis mutants of Acetobacter fabarum, a Drosophila commensal, had a reduced ability to colonize the fruit fly intestine. While the mechanism behind this phenotype has not been investigated yet, increased sensitivity to intestinal AMPs is a likely reason, as shown for the human commensal Bacteroides thetaiotaomicron. Among the LPS mutants, ArnA (pmrA) (PL11H9) was found that encodes an enzyme that catalyzes the formation of modified arabinose UDP-L-4-formamido-arabinose (UDP-L-Ara4FN). The modified arabinose reduces the negative charge of lipid A and the binding of cationic AMPs. This is the most commonly observed LPS modification implicated in cationic AMP resistance. This modification is also crucial for Yersinia pestis resistance to the insect cecropin-like AMP cheopin (Shaka, 2022)

In addition to mutations affecting LPS, several were uncovered that disrupt lipoproteins, like OmpA (PL13H10), NlpI (PL7D10), and YbaY (PL5A4). While YbaY is poorly characterized, OmpA and NlpI were previously implicated in the virulence of different pathogens. Whereas OmpA contributes to virulence in various ways ranging from facilitating adhesion and invasion to conferring resistance to serum, NlpI function in virulence is less clear. The results suggest that all three lipoproteins mutants behave like LPS mutants–they are susceptible to polymyxin B and their virulence is rescued in an AMP mutant, indicating that their reduced virulence is due to an increased susceptibility to host AMPs. The mechanism behind this phenotype requires further investigation, however NlpI was shown to be essential for cell envelop integrity, which might contribute to increased sensitivity to AMPs. The screen uncovered two additional peptidoglycan-associated lipoproteins, TolB (PL2D4) and Pal (PL4B5), that are part of a multiprotein complex, the Tol-Pal system. It bridges between the peptidoglycan and the outer membrane and is important for proper structure and function of the outer membrane. Importantly, TolA and Pal are necessary for correct surface polymerization of O-antigen chains, likely explaining the sensitivity of tol and pal mutants to detergents and several antibiotics. Similar to the Pa tol and pal mutants, F. novicida mutants in these genes were attenuated in Drosophila infection and more sensitive to host AMPs (Shaka, 2022)

The second largest group of mutants with reduced virulence that were identified constitutes ROS-sensitive mutants. Since it was possible rescue the virulence of these mutants by chemical or genetic ROS scavenging, their attenuated virulence is likely due to an inability to resist host ROS produced in response to infection. Among such ROS-sensitive mutants, only the one lacking cytochrome oxidase (PL1A3) was previously shown to be required for virulence in other bacteria by enhancing the tolerance to oxidative stress. Some other genes, like dihydrolipoyl dehydrogenase (PL4F11) and typA (PL6B7), were also linked to virulence but not necessarily via ROS sensitivity. No previous evidence was found of the role of ATPase RavA stimulator ViaA (PL14C2) in virulence, however there seems to be a link to ROS response in E. coli. Therefore, further investigation of the identified genes is required to clarify their role in bacterial virulence and ROS sensitivity. While previous studies identified several sources of ROS in flies, including melanisation, hemocytes, Nox and, Duox, the results showed that Duox specifically in hemocytes is the major producer of ROS in case of Pa infection. Notably, in case of F. novicida melanisation played a prominent role as a source of ROS. An interesting avenue for future studies would be to understand the differences between Duox- and melanisation-derived ROS and their preferential activity against specific pathogens (Shaka, 2022)

The screen also identified several hypothetical proteins. Using ROS and polymyxin B sensitivity assays and rescue in AMP- and ROS-deficient flies, it was shown that PL4E6 and PL11H8 contribute to bacterial resistance to host AMPs, while PL6D10 is necessary to survive ROS exposure. Thus, with this approach a mechanism of virulence could be assigned to hypothetical proteins with unknown function. However, how those protein contribute to ROS or AMP sensitivity remains unknown (Shaka, 2022)

One Pa mutant (Sigma-E factor regulatory protein rseB, PL13C10), was identified that was not sensitive to ROS and polymyxin in vitro. However, the virulence of this mutant was rescued in Relish and AMP-deficient flies. Very likely the rescue phenotype could be due to sensitivity to additional antimicrobial peptides produced by flies. Such increased sensitivity to AMPs is possible given the role of Sigma-E factor in cell envelope integrity (Shaka, 2022)

Among all AMPs tested, Drosocin proved to be particularly important in controlling Pa infection. Consistent with the Pa LPS mutants, F. novicida mutants in LPS were particularly sensitive to Drosocin. Considering that Drosocin is known to bind bacterial LPS, alterations in LPS might promote Drosocin interactions with LPS and bacterial killing or make intracellular targets more accessible. A previous in vivo analysis of AMP specificity has shown that Drosocin plays a critical role in controlling Enterobacter cloacae infection. A recent study confirmed this finding, however additionally reported that the Drosocin gene encodes not one, but two AMPs: Drosocin and IM7 (newly named as Buletin). Buletin but not Drosocin contributes to host defense against Providencia burhodogranariea infection. Since the Drosocin mutant that was used lacked both Drosocin and Buletin and the Drosocin overexpression line similarly produced both peptides, it remains to be tested whether Drosocin or Buletin or both peptides together are involved in the defense against Pa (Shaka, 2022)

While in vivo experiments demonstrate that AMPs are the major Relish-regulated molecules controlling Pa LPS mutants, in vitro assays with synthesized Drosophila AMPs were not conclusive. None of the three Drosophila AMPs that were tested, Cecropin A, Cecropin B, and Diptericin B, showed activity against Pa. Considering the high specificity of some AMP-microbe interactions, it could be that the peptides that were tested have no effect on Pa. Indeed, based on in vivo results, Drosocin, which was not available for an in vitro test, is the primary AMP controlling Pa infection. Additionally, in vitro effects of AMPs can be different than in vivo effects of mutants or knockdowns for the same AMPs, suggesting that physiological context or interaction among peptides is important. Also, there are a number of technical reasons why in vitro assays may not reflect in vivo activities, including AMPs adhering to plastic assay plates, differences in salt concentrations or pH, stress on microbes, interactions among AMPs and between AMPs and other components of the immune system. These potential issues have to be considered when interpreting the results of in vitro antimicrobial tests performed with AMPs (Shaka, 2022)

Contrary to expectations, the screen did not hit any bacterial effectors, like toxins, that might be responsible for damaging the host. Since toxins are likely to be redundant, disruption of an individual toxin gene may not give a phenotype. Similarly, no mutants were identified in secretion systems, suggesting that Pa does not require effector translocation to infect Drosophila. The only toxin that was so far implicated in Pa pathogenesis is cytolethal distending toxin which blocks eukaryotic cell proliferation. Interestingly, Pa LPS was shown to cause epithelial barrier dysfunction by reducing occludin levels in Caco-2 cell monolayers and induced apoptosis in calf pulmonary artery endothelial cells. Thus, LPS might not only mediate resistance to host AMPs but also act as an effector-like molecule (Shaka, 2022)

By discovering the mechanisms of Pa resistance to host AMPs and ROS, this study opens the doors to potential strategies to exploit such Pa mechanisms and sensitize the pathogen to host defenses to improve infection treatment. To illustrate the feasibility of such an approach, polymyxin B treatment was used to disrupt Pa LPS in vivo, and it was found to be sufficient to improve Drosophila survival after infection. Such beneficial effect of polymyxin B required functional Imd pathway signalling and was independent of direct bactericidal activity, suggesting that disruption of the major barrier against AMPs sensitizes the pathogen to host defenses. These results suggest that affecting LPS function might be a useful strategy to treat Providencia infections, particularly those resistant to antibiotics (Shaka, 2022)

Sensitizing Pa to host ROS also appears to be an attractive anti-virulence strategy, considering that resistance to host ROS is one of the key Pa virulence mechanisms that was identified. Some compounds were shown to sensitize the pathogens to oxidative stress and immune clearance but in a species-specific manner. For example, 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC) treatment inhibits production of pyomelanin pigment and increases sensitivity of pyomelanogenic Pseudomonas aeruginosa strains to oxidative stress. Similarly, BPH-642 –cholesterol biosynthesis inhibitor, blocked biosynthesis of staphyloxanthin antioxidant pigment in S. aureus, resulting in increased immune clearance in a mouse infection model. However, to date there are no known compounds that would predispose Pa or generally any pathogen to ROS without being toxic to the host, thus limiting the development of ROS-potentiating anti-infectives (Shaka, 2022)

In summary, reciprocal analysis of interactions between D. melanogaster and P. alcalifaciens revealed that the host relies on Imd-dependent AMPs and hemocyte-derived ROS as major branches of immunity that are important for fighting infection with P. alcalifaciens. On the pathogen side, it was found that the ability to resist these host immune responses is the major virulence mechanism of P. alcalifaciens. Leveraging this knowledge has great potential to improve P. alcalifaciens infection treatment either by potentiating the host defenses or disrupting pathogen virulence (Shaka, 2022)

Inflammation-modulated metabolic reprogramming is required for DUOX-dependent gut immunity in Drosophila

DUOX, a member of the NADPH xidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, this study shows that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia (Lee, 2018).

Drosophila has been a successful model system for dissection of the molecular mechanisms of innate immunity. Two nuclear factor κB (NF-κB) signaling pathways, Toll and immune deficiency (IMD), operate to produce NF-κB-dependent antimicrobial peptides in response to systemic infection. However, unlike internal germ-free organs involved in systemic immunity, mucosal epithelia of metazoans, mostly gut epithelia, are in constant contact with different microorganisms. Most of these gut-associated microbes are considered as being commensal and/or symbiotic, whereas some of them may be pathogenic under certain circumstances. Despite the importance of gut-associated microbes, understanding of this gut strategy of the microbe-controlling system, i.e., pathogen elimination versus commensal protection, remains incomplete. Genetic analyses of Drosophila gut immunity demonstrated that dual oxidase (DUOX), a member of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, acts as the first line of host defense against invading pathogens by producing microbicidal reactive oxygen species (ROS) (Lee, 2018).

Due to the pivotal role of DUOX in gut immunity, the regulatory mechanism of the DUOX system has received considerable attention. Although the immunological roles of DUOX in mucosal epithelia seem to be conserved throughout the metazoans (e.g., Caenorhabditis elegans, insects, zebrafish, and rodents), the regulatory mechanisms governing DUOX regulation have been studied extensively in Drosophila. Genetic studies in Drosophila showed that the DUOX-activity pathway is composed of phospholipase Cβ (PLCβ)-Ca2+ signaling to control DUOX enzymatic activity, whereas the DUOX-expression pathway is composed of the MEKK1-p38 MAPK pathway to control DUOX gene expression (Ha, 2009a, Ha, 2009b). It has been demonstrated that, unlike symbiotic bacteria, pathogens release the uracil molecule, and DUOX has been shown to be activated by the pathogen-derived uracil molecule (Lee, 2013). Recently, it has been shown that bacterial uracil induces Hedgehog (Hh) signaling activation, which acts as an upstream regulator of the DUOX-activity pathway (Lee, 2015). Hh pathway activation is required for uracil-induced cadherin 99C (Cad99C) expression in the apical region of enterocytes. Uracil-induced Cad99C expression further induces the formation of Cad99C+ signaling endosomes to which PLCβ and protein kinase C (PKC) are recruited. Endosome formation was found to be necessary for PLCβ activity to increase intracellular calcium concentration for DUOX enzyme activation. Therefore, it is proposed that Hh-Cad99C pathway activation, Cad99C+ endosome formation, PLCβ activation, PLCβ-dependent calcium mobilization, and DUOX activation are sequential events in the DUOX-activity pathway for the production of DUOX-dependent ROS (Lee, 2015). Flies carrying any functional mutation in Hh-Cad99C, PLCβ-Ca2+, or MEKK1-p38 signaling pathways are highly susceptible to enteric infection due to impaired DUOX activity (Ha, 2009b, Lee, 2013, Lee, 2015). This highlights the importance of these signaling networks in DUOX-dependent gut immunity. The operation of such complex immune regulations may be energetically expensive. It has been recently suggested that infectious signals regulate cellular metabolic homeostasis to optimize the performance of the animal immune system. However, the immune-metabolic interactions, especially at an organism level, are currently poorly understood (Lee, 2018).

Despite the extensive research conducted, the elucidation of the DUOX regulatory mechanism in Drosophila innate immunity remains incomplete. The objective of the present study was to obtain a more complete picture of the DUOX regulatory mechanism by taking advantage of the Drosophila genetic tools available. During this analyses, it was found that bacterial infection acts as a pro-catabolic signal capable of initiating metabolic reprogramming of enterocytes toward lipid catabolism. Furthermore, it was revealed that complex intracellular kinase cascades are involved in infection-modulated metabolic reprogramming. Finally, the infection-modulated metabolic reprogramming was shown to be necessary for cellular NADPH homeostasis, sustained DUOX activity, and host resistance against enteric infection, highlighting the importance of immune-metabolic interactions at an organism level (Lee, 2018).

Complex interactions among bacterial infection, host immunity, and metabolism are frequently observed in animals ranging from Drosophila to humans. For example, chronic systemic infection with pathogens such as Mycobacterium or Listeria is known to induce metabolic disorders such as wasting phenotype, exhibiting extensive loss of lipids and carbohydrates in both humans and Drosophila. Similar host wasting was also observed in Drosophila in the case of enteric infection with Vibrio cholerae. However, it is unclear whether infection-induced metabolic wasting is a consequence of pathogen virulence or part of host immune response. Although immune-metabolic interactions are considered to be critical for host fitness during bacterial infection, the detailed signaling pathways by which pathogen infection regulates the host metabolism are not yet fully understood (Lee, 2018).

The present study was based on the well-characterized DUOX-dependent gut immunity in Drosophila. An unexpected link was found between the signaling pathways leading to DUOX-dependent intestinal immune activation and the pathways controlling lipid metabolism. Infection-induced DUOX-activating signaling exerted a pronounced effect on the metabolic requirement of enterocytes, leading to a metabolic shift from an energy-storing to an energy-consuming state. Genetic screening has identified four downstream kinases of TRAF3/NOPO (AMPK, WTS, ATG1, and MEKK1) capable of alleviating constitutive DUOX activation seen in NOPO knockdown or TRAF3-overexpressing conditions. AMPK, WTS, and ATG1 are representative metabolic signaling hubs known to be activated in response to nutritional and energy stress. This study revealed that these metabolic hubs are modulated by enteric infection, showing that activated TRAF3 signaling is found to be bifurcated into the WTS kinase and AMPK kinase pathways. What is the metabolic outcome of AMPK activation and WTS-induced AKT inhibition? It is well known that AMPK and AKT commonly phosphorylate TSC2 for activation and inactivation, respectively. AMPK induces the GTPase activity of TSC2 by phosphorylating its Ser1107 site, whereas AKT inhibits the GTPase activity of TSC2 by phosphorylating different sites of TSC2 (e.g., Ser924 and Thr1518). Therefore, enteric infection may lead to an increase in Ser1107 phosphorylation and a decrease in Ser924/Thr1518 phosphorylation, thereby resulting in a strong TSC2 activation. Therefore, enteric infection ultimately gave a signal for TOR inhibition via AMPK-WTS/AKT pathway-induced TSC2 activation. Infection-induced TOR inhibition leads to S6K inhibition for the suppression of NADPH-consuming lipogenesis while activating ATG1-dependent NADPH-yielding lipolysis. This infection-induced metabolic shift toward lipid catabolism is necessary to sustain DUOX activity by maintaining NADPH homeostasis, which is required for host resistance against enteric infection (Lee, 2018).

The relationship between inflammation and metabolism is poorly understood. The present study may provide an important conceptual framework for understanding the molecular crosstalk between gut immune activation and metabolic reprogramming. Previously, in a model of Toll/IMD-mediated systemic immunity, intracellular pathogens such as Mycobacterium or Listeria resulted in the wasting phenotype in Drosophila. In this case, inactivation of AKT activity produces pathological FOXO activation results in loss of anabolic activity, which is involved in the wasting phenotype. Recently, activation of the bacterial-induced Toll/IMD pathway was shown to antagonize S6K activity for the modulation of MEF2 activity, resulting in loss of anabolism. Although inactivation of AKT and S6K is observed during systemic inflammation, the relationship between S6K inhibition and AKT inhibition, and which signaling molecules act as upstream/downstream components of these kinases, are unclear. As inactivation of AKT and S6K in enterocytes is required for DUOX-dependent gut immunity, it is likely that infection-induced inactivation of AKT and S6K is commonly shared between Toll/IMD-based systemic immunity and DUOX-based gut immunity. This study further showed that WTS activation is upstream of AKT inhibition and that AKT inhibition led to S6K inhibition through TSC2 activation in DUOX-based gut immunity. It will be important to investigate whether the WTS-AKT-TSC2-S6K pathway plays a similar role in the fat body in Toll/IMD-based systemic immunity. In this regard, it is interesting to note that Toll activation can directly activate WTS in the fat body. Further studies are warranted to elucidate the shared aspects of immune-metabolic interactions between gut immunity and systemic immunity (Lee, 2018).

Cellular metabolism provides energy for all aspects of biological activities such as reproduction, development, and immunity. All of these biological activities require energy consumption; therefore, energy should be properly allocated to optimize the performance of animals. Operating the innate immune system is energetically expensive, which is believed to be controlled by metabolic homeostasis. Indeed, infection-modulated transcriptome analysis revealed that the functional category of 'metabolism' is mostly affected by enteric infection, suggesting dynamic energy allocation pattern changes in the intestine following enteric infection. Disruption of the infection-induced metabolic reprogramming as in the case of TRAF3-AMPK/WTS-ATG1 pathway-mutant flies can lead to high susceptibility of the animal to enteric infection. This highlights the importance of bacterial-modulated host metabolism in gut immunity. As metabolic dysregulation is believed to be closely associated with the pathogenesis of important inflammatory diseases of mucosal epithelia such as intestine, the discovery of signaling pathways governing inflammation-induced metabolic reprogramming will greatly advance our understanding of the etiology of different mucosal diseases arising from abnormal immune-metabolic interactions. Regulating the signaling pathways governing metabolic reprogramming at the tissue or organismal levels may provide a strategy for the treatment of these diseases (Lee, 2018).

Duox mediates ultraviolet injury-induced nociceptive sensitization in Drosophila larvae

Nociceptive sensitization is an increase in pain perception in response to stimulus. Following brief irradiation of Drosophila larvae with UV, nociceptive sensitization occurs in class IV multiple dendritic (mdIV) neurons, which are polymodal sensory nociceptors. Diverse signaling pathways have been identified that mediate nociceptive sensitization in mdIV neurons, including TNF, Hedgehog, BMP, and Tachykinin, yet the underlying mechanisms are not completely understood. This study reports that duox heterozygous mutant larvae, which have normal basal nociception, exhibit an attenuated hypersensitivity response to heat and mechanical force following UV irradiation. Employing the ppk-Gal4 line, which is exclusively expressed in mdIV neurons, this study further shows that silencing duox in mdIV neurons attenuates UV-induced sensitization. These findings reveal a novel role for duox in nociceptive sensitization of Drosophila larvae, and will enhance understanding of the mechanisms underlying this process in Drosophila sensory neurons (Jang, 2018).

This paper describes a novel role of duox in nociceptive sensitization in mdIV neurons. Firstly, the data show that duox heterozygous mutant larvae, which exhibit basal nociception, display defective hyperalgesia (pain amplification) to heat and mechanical force following UV irradiation. Secondly, duox silencing in mdIV neurons impairs induced hypersensitivity. Altogether, these genetic studies suggest that Duox is required in mdIV neurons to mediate UV irradiation-derived nociceptive sensitization (Jang, 2018).

It is of note that ~28% of larvae expressing either duox or ppk1 RNAi in mdIV neurons (ppk > Duox-RNAi and ppk > ppk1-RNAi) exhibited nociceptive response to 40°C heat, as opposed to ~12% of larvae expressing TrpA1 RNAi. This suggests that silencing of duox or ppk1 in mdIV neurons does not affect basal nociception against 40°C heat, while TrpA1 silencing reduces it. This makes sense in that Duox and Ppk1 are not heat sensors, while TrpA1 is. Notably, duox silencing abrogated heat hypersensitivity while ppk1 silencing did not, highlighting the role of Duox in nociceptive sensitization (Jang, 2018).

It has been shown that basal nociception against heat and harsh mechanical force is not affected by duox reduction, suggesting that mdIV neurons with reduced duox expression retain normal function in sensing nociceptive stimuli and in depolarization. To further confirm this notion, whether structural defects were present in duox heterozygous mutant larvae was determined. The dendrites of duox heterozygotes were examined using ppk-td-GFP lines that specifically expressed td-GFP in mdIV neurons. Confocal images showed that the dendrites of mdIV neurons in duox heterozygous mutant larvae were not reduced in comparison to those of control larvae (Jang, 2018).

It is proposed that UV irradiation either directly or indirectly activates Duox expression and/or Duox activation in mdIV neurons. Diverse signaling pathways including TNF, Hedgehog, BMP, and Tachykinin have been shown to mediate UV irradiation-induced nociceptive sensitization in mdIV neurons. These signaling pathways could induce the expression and/or activity of Duox, and further research should be done to determine whether they do so in mdIV neurons (Jang, 2018).

The genetic knockdown of heat sensors painless and TrpA1 abolishes not only basal nociception but also UV-induced nociceptive sensitization. This suggests that Painless and TrpA1 mediate nociceptive sensitization following UV irradiation. Duox is a member of the NADPH oxidase family, which produces reactive oxygen species (ROS) in a regulated manner. It is speculated that ROS produced by Duox following UV irradiation increase the gating of Painless and TrpA1 through direct oxidation (Jang, 2018).

Duox has been shown to catalyze dityrosine cross-links in epithelial cuticles, hormone synthesis, and mucosal immunity in Caenorhabditis elegans, D. melanogaster, and mammals. However, the role of Duox in pain signaling has not been addressed in any animal models. The current data uncover a novel role for Duox in the nociceptive sensitization of sensory nociceptors in Drosophila. Intriguingly, mammalian nociceptors employ a different member of the NADPH oxidase family in nociceptive sensitization. Thus, the findings support the notion that the underlying mechanisms of nociceptive sensitization are evolutionarily conserved from insects to mammals (Jang, 2018).

The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, whole-genome mRNAseq was performed of actively regenerating tissue from damaged Drosophila wing imaginal discs. Genetic tools to ablate the wing primordium to induce regeneration, and transcriptional profiling of the regeneration blastema was carried out by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes it was confirmed that they have roles in regeneration. This approach showed that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. mol encodes a co-factor for an enzyme, NADPH dual oxidase. JNK signaling was shown to upregulate mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue this study has identified a positive feedback loop that regulates the extent of regenerative growth (Khan, 2017).

This work has identified a novel mechanism that sustains regeneration signaling and ensures that regrowth of damaged tissue continues beyond the initial burst of damage signaling. While elevated ROS levels are sustained in other regeneration models such as amputated zebrafish fins and Xenopus tails, where they promote signaling and the later stages of regenerative growth, the mechanism through which elevated ROS levels are maintained has remained elusive. This work has provided insight into this puzzle in Drosophila by discovering a key positive feedback loop that uses JNK-induced upregulation of the Duox-maturation factor encoded by mol to sustain ROS production, JNK signaling, and late regeneration. Similar damage-induced regulation of the Duox-maturation factor may facilitate long-term regeneration signaling in many animals. This study identified this mechanism through generation of a transcriptional profile of actively regenerating tissue, made possible by a genetically induced tissue ablation system, and these technical advances enabling isolation of sufficient numbers of blastema cells (Khan, 2017).

This is the first report of upregulation of a Duox maturation factor as a key aspect of the regeneration response. Other cellular functions that are regulated by DUOXA/NIP have only recently been identified. For example, DUOXA/NIP affects differentiation in murine skeletal muscle myoblasts [murine thyroid hormone production and cerebellar development, and the response to bacterial infections in the murine gut, as well as development of the exoskeleton in C. elegans, and recruitment of hemocytes to wounds in the Drosophila embryo epidermis and neutrophils to airways in mice]. This study describes a role for mol during wing disc regeneration and shows that while mol is transcriptionally upregulated, Duox levels do not change according to a transcriptional profile, indicating that fine-tuning of ROS levels can be achieved by changes in expression of the maturation factor rather than the enzyme itself. This regulative strategy may be deployed in many other cases in which ROS act as crucial signaling molecules (Khan, 2017).

In addition to the transcriptional changes observed in regulators of ROS, many of the other changes in gene expression can be combined with the current understanding of tissue regeneration to identify novel and interesting relationships between developmental genes and signals and tissue regeneration. For example, the data indicated downregulation of the hormone receptor Hr78 in regenerating tissue. The expression of Hr78 in the wing disc appeared to be in some of the pro-vein regions. Tissue damage in the wing disc leads to a transient loss of cell-fate gene expression, including in the pro-veins, during regeneration. Thus, Hr78 may be a novel wing vein fate gene whose expression is downregulated along with the other known vein fate genes after tissue damage (Khan, 2017).

As an additional example, differential regulation of various nuclear hormone receptor genes was observed that are transcriptionally regulated by the hormone ecdysone. Regenerating animals delay metamorphosis to accommodate regrowth of the damaged tissue by regulating ecdysone signaling, which controls developmental transitions. Ecdysone targets that were found downregulated in regenerating wing discs include Hormone receptor 46 (Hr46/Hr3), Hormone receptor 4 (Hr4/CG42527), and Ecdysone-induced protein 78C (Eip78C). Interestingly, upregulation was seen of Cyp18a1, a cytochrome P450 enzyme that exerts negative feedback regulation on ecdysone signaling by decreasing intracellular levels of ecdysone. Thus, Cyp18a1 may be upregulated to ensure that ecdysone signaling stays low in the regenerating tissue to reinforce the developmental checkpoint induced by tissue damage (Khan, 2017).

Regeneration involves orchestration of various cellular processes to repair and replace the damaged body part. It requires coordination of proliferation, growth, patterning, and changes in cell architecture and movement in a highly regulated manner. These dramatic changes could be coordinated by key transcription factors. Several transcription factors are differentially expressed in the profile carried out in this study, including chinmo, Ets21C, AP-2/TfAP-2, fru, Atf3/A3-3, dve and Blimp-1. These transcription factors could lie at the center of regulatory networks that bring about key cellular changes. For example, Ets21C is a known downstream target of JNK signaling in wound healing, and EGFR signaling in the intestinal stem cells, and is also required as a co-factor for the JNK pathway transcription factor AP-1 in regulating transcriptional targets during tumor formation. Thus, its expression in the regenerating wing disc could result from integration of multiple signals, and its requirement in regeneration may be due to its role in promoting expression of JNK targets. Further investigation into the mechanisms of these transcription factors will lead to a better understanding of regeneration (Khan, 2017).

Regeneration is a tightly controlled process, requiring a balance between positive and negative regulators so that growth is stimulated but not deregulated. Indeed, our functional analysis demonstrated that several of the upregulated genes, including heartless and Nox, serve to restrict regeneration, as regeneration improved in heterozygous mutant animals. Therefore, functional analysis is critical for interpretation of gene expression data, as drawing conclusions based on differential expression alone can be misleading. Indeed, it was through functional analysis that mol, but not Nox, was identified as the critical regulator that promotes sustained ROS production and JNK signaling, completing the positive feedback loop that sustains regeneration. Further functional analysis of differentially expressed genes will likely reveal additional mechanisms that control tissue regeneration (Khan, 2017).

Interestingly, p38b has also been shown to provide tolerance to Salmonella typhimurium infections, promoting survival of the host without reducing bacterial burden. This study suggested that p38b contributes to tolerance by enabling hemocyte enlargement, and hence, engulfment of larger quantities of bacteria. In the context of IIV-6 infection, p38b could be acting to promote engulfment of infected and damaged cells, thereby providing a repair mechanism to enable the animals to better tolerate and limit virus infection. Future studies will be necessary to probe all the roles of p38b in antiviral defense (Khan, 2017).

Although the data presented in this study demonstrate that the JAK-STAT pathway is protective against IIV-6 infection, the protective mechanisms require further study. In the case of the RNA virus DCV, the JAK-STAT pathway is also protective, possibly through the induction of vir-1. However, the JAK-STAT pathway is not broadly antiviral and vir-1 was not induced by IIV-6. Curiously, a previous study examining the role of the JAK-STAT pathway during IIV-6 infection, using one particular hypomorphic allelic combination hopscotch (JAK), concluded that hopscotch (and by inference the JAK-STAT pathway) was not involved in protecting flies against IIV-6 infection. The current data, with multiple RNAi lines targeting stat92E, as well as the S2 cell based results with RNAi targeting domeless, hopscotch, and stat92E, demonstrate a consistent and reproducible role for this pathway in the response to and survival from IIV-6 infection. These contradictory outcomes may be due to differences in alleles used or dose delivered (Khan, 2017).

The Tots are intriguing candidates for JAK-STAT induced antivirals. They are rapidly evolving with evidence of positive selection, typical for immune effectors. However, the Tots have not yet been demonstrated to provide direct antimicrobial activity. To date, it has not been possible to demonstrate any antiviral activity for the Tots. In particular, over-expression of TotA resulted in reduced survival following IIV-6 infection and no change in viral titers, consistent with the previously reported general toxicity caused by over expression of this gene. Further studies, examining all six of the IIV-6 induced Tots, with both loss- and gain-of-function approaches, will be necessary to more fully examine this possibility (Khan, 2017).

The sensitivity of STAT knockdowns to IIV-6 infection argues that JAK-STAT signaling is an important antiviral target of p38b. However, other p38b targets are also possible. For example, an established target of p38b is the heat shock response. In the context of bacterial and fungal infections, p38b is known to regulate Heat shock factor (Hsf) expression and the induction of heat shock proteins (Hsps). In addition, another report has shown that Hsf protects flies against both RNA and DNA viral infections. Together, these results suggest that the antiviral effects of p38b could be mediated, at least in part, through Hsf and Hsps. Indeed, Hsf mutant flies display an increased rate of death after IIV-6 infection. It will be interesting to learn if the heat shock response is activated by p38b following IIV-6 infection, and how this response interacts with JAK-STAT dependent viral protection (Khan, 2017).

Successful host defenses detect multiple characteristics of an invading pathogen. For example, cellular damage is one common indicator of pathogenic infection that can be sensed by the innate immune system. In mammals, several danger-associated molecular patterns (DAMPs) have been characterized, including HMGB1, F-actin, and histones. Likewise, a recent report examining a Drosophila model of sterile injury demonstrated that extracellular actin activates JAK-STAT signaling. In this paradigm, detection of extracellular actin, via an unknown receptor, triggered Nox-dependent ROS generation, the activation of Src42A and Shark (Syk homolog), and induction of unpaireds and eventually Tots. This pathway is very similar to that reported in this study, although p38b was not examined in this actin-DAMP, and suggests that IIV-6 infection may cause cellular damage, rupture and the release of actin, which in turn triggers ROS production, unpaired expression, JAK-STAT signaling and the induction of Tots. Formally testing this model will be facilitated by the identification of an extracellular actin receptor (Khan, 2017).

In summary, this study has found a novel role for Drosophila p38b in protecting against DNA virus infection. Virus infection leads to p38b dependent responses, including the induction of the JAK-STAT activating cytokines, the Unpaireds, and the induction of downstream target genes such as the Tots. Based on the analysis of viral load, the p38b pathway appears to function primarily by increasing tolerance to IIV-6, as viral loads were not altered in the p38b strain. Whether the Tots contribute to this tolerance and, more generally, whether p38b induces a directly antiviral response, or relies entirely on the Unpaired and JAK-STAT signaling for its ability to tolerize against this viral infection will be probed in future studies (Khan, 2017).

A Mesh-Duox pathway regulates homeostasis in the insect gut

The metazoan gut harbours complex communities of commensal and symbiotic bacterial microorganisms. The quantity and quality of these microorganisms fluctuate dynamically in response to physiological changes. The mechanisms that hosts have developed to respond to and manage such dynamic changes and maintain homeostasis remain largely unknown. This study identified a dual oxidase (Duox)-regulating pathway that contributes to maintaining homeostasis in the gut of both Aedes aegypti and Drosophila melanogaster. A gut-membrane-associated protein, named Mesh, plays an important role in controlling the proliferation of gut bacteria by regulating Duox expression through an Arrestin-mediated MAPK JNK/ERK phosphorylation cascade. Expression of both Mesh and Duox is correlated with the gut bacterial microbiome, which, in mosquitoes, increases dramatically soon after a blood meal. Ablation of Mesh abolishes Duox induction, leading to an increase of the gut microbiome load. This study reveals that the Mesh-mediated signalling pathway is a central homeostatic mechanism of the insect gut (Xiao, 2017).

The intestinal tract of most metazoans harbors complex communities of microbes that contribute to maintaining homeostasis with the gut epithelia. These microbes manipulate a wide range of host physiology, including nutrition, development, differentiation and defense. Although these microbial communities maintain homeostasis with the gut epithelia, their quantity and composition dynamically fluctuate in response to the host dietary situations, environmental conditions and physical activities. The gut epithelia have developed mechanisms to tolerate commensal microorganisms, while the microorganisms have developed mechanisms to evade the host immune response. Nonetheless, the gut epithelia are required to mount a finely tuned immune response of proper strength and duration in response to microbial fluctuation in a timely and appropriate manner. The molecular mechanisms employed by the gut epithelia to manage the dynamic fluctuation of microbes and maintain homeostasis are not well understood (Xiao, 2017).

Similar to the mammalian intestinal tract, the insect gut constantly interacts with its microbial residents. Drosophila and mosquitoes are established models for deciphering the complex interactions between the gut and its microbes. Previous studies revealed that Dual oxidase (Duox)-mediated production of reactive oxygen species (ROS) is a major immune mechanism regulating insect gut-microbe homeostasis. In Drosophila, Duox-mediated ROS are required for routine control of Saccharomyces cerevisiae, an essential microbial food source. A reduction in ROS levels in the midgut of the major arboviral vector mosquito Aedes aegypti results in dysbiotic proliferation of the intestinal microbiota. Duox-mediated ROS also play a pivotal role in regulating homeostasis and the composition of the gut bacterial community in Bactrocera dorsalis and Phlebotomine sandflies. Indeed, under homeostatic conditions, both the expression and activity of Duox are tightly restricted to a level that allows healthy gut-microbe interactions, thereby precluding any pathophysiological effects on the gut epithelia. Induction of Duox gene expression is limited in an off-state status by phospholipase Cβ (PLCβ)-mediated mitogen-activated protein kinase (MAPK) P38 dephosphorylation in the Drosophila gut epithelia. Modest ROS levels are achieved by activation of basal Duox expression through intracellular Ca2+ mobilization by a G-protein α subunit q protein (Gαq)-PLCβ signal cascade. Thus, the basal expression and activation of Duox are essential to manage symbiotic microbes under healthy conditions in the insect gut (Xiao, 2017).

The complement control protein (CCP) domain is an evolutionarily conserved module essential for complement-mediated immune functions. Previous work has demonstrated that the CCP domain plays an important role in insect-microbe interactions. This study shows that a CCP-containing protein named Mesh regulates commensal bacterial proliferation through regulation of Duox expression in the gut of both Drosophila and A. aegypti, via a signaling cascade involving Arrestin-mediated MAPK JNK/ERK phosphorylation. In both insects, Mesh expression correlates with the gut commensal bacterial load, enabling Duox abundance to be dynamically regulated by microbial fluctuation. Since generation of Duox-mediated ROS is a major gut immune response in maintenance of insect gut homeostasis, this study reveals a fine-tuning mechanism for Duox expression to manage healthy gut-microbe interactions in insects (Xiao, 2017).

The quantity and quality of the gut symbiotic bacteria fluctuate dynamically with gut activities such as alimentary flow, food ingestion and other physiological changes. The host senses these changes and fine-tunes its gut immune system to respond with proper strength and duration to the dynamic changes in commensal microbes. In mammals, ROS plays an important role in controlling the normal gut microbiota. The previous studies indicated that Duox2, an important source of hydrogen peroxide, can be induced by normal gut microbiota in mice. In addition to Duox2-induced ROS generation, NADPH oxidase 1 (Nox1) also acts as a key player for ROS responses in the murine intestine, particularly induced by members of the genus Lactobacillus, revealing an important role of commensal bacteria-mediated ROS generation in maintenance of homeostasis in the mammalian intestine (Xiao, 2017).

It is largely unknown how the insect gut epithelium might sense the dynamic changes of the microbiome and adjust its response. Several studies in Drosophila and other insects have revealed that the gut epithelia rely on basal production of Duox-dependent ROS for maintaining gut-microbe homeostasis. This study identified Mesh as an indispensable factor of the A. aegypti and D. melanogaster gut, which fine-tunes the expression of Duox and production of ROS, thereby regulating the gut bacterial load. The expression level of Mesh correlates with the load of gut bacteria, which in mosquitoes are induced dramatically after a blood meal. Mechanistic studies have demonstrated that Mesh mediates constitutive Duox expression in the gut via an Arrestin-MAP kinase signaling cascade. First, silencing Mesh abolishes bacterially induced Duox expression, while its ectopic overexpression increases Duox levels. Second, depletion of the gut microbiome reduces Mesh expression, while reconstitution of the microbiome recovers Mesh and Duox expression level in a microbial burden-dependent manner. Third, silencing either of the Arrestins, JNK or ERK abolishes Mesh-mediated Duox expression. Taken together, these results suggest that Mesh, directly or indirectly, senses the dynamic fluctuation of the gut microbiome thereby transmitting a signal to an intracellular signaling cascade to fine-tune the antibacterial immune responses and restore homeostasis (Xiao, 2017).

Mesh has a complement control protein (CCP) domain, which is an evolutionarily conserved immune module that recognizes microbial ligands. Scavenger Receptor-C (SR-C), a Drosophila membrane receptor that also contains 2 CCP domains, is capable of recognizing both Gram-positive and Gram-negative bacteria acting as a pattern recognition receptor for phagocytosis in hemocytes. Moreover, the CCP module of various A. aegypti immune factors mediates direct recognition of Dengue viral particles restricting viral infection. Preliminary data suggest that neither of Aedes or Drosophila Mesh (expressed as full-length proteins in Drosophila S2 cells) can directly interact with C. testosteroni, C. meningosepticum or A. thailandicus, which are common members of the insect microbiome. In addition to surface bacterial components, metabolites generated by gut microbes can also contribute to the regulation of gut immunity. It is known that bacterial-derived uracil acts as a modulator to boost the ROS activity in Drosophila. Future work should aim to investigate bacterial ligands for the Mesh-mediated Duox expression (Xiao, 2017).

RNA-Seq and in-depth analysis of immune-related genes identified that 5 genes were consistently down-regulated in both AaMesh-silenced and AaMesh-immuno-blockaded mosquito guts. In these 5 genes, LYSC9 is an enzyme with bactericidal activity. Duox is a member of the ROS-generating NADPH oxidases. Besides the Duox gene that this study focused on, expression of a C-type lysozyme 9 (LYSC9) was also significantly impaired by immuno-blocking/silencing AaMesh, suggesting expression of LYSC9 might be controlled by Mesh-mediated signaling. Recent studies showed that a C. elegans lysozyme, known as invertebrate-type lysozyme-3 (ilys-3), can be up-regulated by ERK-MAPK-dependent signaling in the worm intestine, during challenge with Gram-positive pathogens. Indeed, the current studies have demonstrated that Mesh mediates down-stream gene (Duox) expression via the ERK/JNK MAPK signaling cascade. It is therefore speculated that LYSC9 might also be counted as one of the Mesh-mediated MAPK signaling-regulated genes, and contribute to fine-tuning the gut-microbe homeostasis in insects. The role of LYSC9 in the regulation of microbiota will be investigated in a further study (Xiao, 2017).

In addition to commensal gut bacteria in Drosophila and mosquitoes, the gut epithelia also face invasion from allochthonous microorganisms with pathogenic properties. The proliferation of these pathogens and their metabolites stimulates the epithelia to generate ROS at a much higher level than under conventional conditions. Induction of the Duox in the Drosophila gut is mediated by the MAP kinase kinase kinase (MEKK1)-MAP kinase kinase 3 (MKK3)-P38-ATF2 pathway. Gαq-mediated PLC-β directly activates MEKK1, thereby leading to the MAPK P38-dependent Duox induction. However, under healthy conditions, the expression of Duox is offset by MAPK P38 dephosphorylation via PLC-β-MAP kinase phosphatase-3 (MKP3) signaling, indicating that the MAPK P38 signaling cascade plays a central role in regulating Duox expression during pathogenic microbial infections. Moreover, MAPK P38 pathway deficient flies can survive with conventional rearing, suggesting that the MAPK P38-mediated cascade is dispensable for the maintenance of basal Duox expression in healthy gut-microbe interactions. This study shows that indeed Mesh constitutively regulates the basal Duox expression via phosphorylation of the MAP kinases JNK and ERK but not P38. This study also shows that these phosphorylation events are mediated by Arrestins, the expression of which is also controlled by the pathway. Indeed, previous studies have shown that the mammalian β-arrestin acts as a signal transduction scaffold for MAP kinases, and is thereby essential for the MAPK signaling cascade (Xiao, 2017).

The current findings suggest that Mesh-mediated Duox induction is a bacterium-specific response. However, three important questions remain unanswered: 1) is this pathway responsive to all the commensal bacteria or specific classes? 2) how does Mesh sense bacteria, the bacterial surface PAMPs or secreted metabolites? and 3) is this pathway applicable to the mammalian system? Addressing these questions in future endeavors may not only provide a complete picture of delicate interactions between microbiota and host, but also a conceptual advancement in general (Xiao, 2017).

Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. This study shows that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. It is proposed that in an immortalized ('undead') model of AiP, in which the activity of the effector caspases is blocked, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer (Fogarty, 2016).

The role of ROSs as a regulated form of redox signaling in damage detection and damage response is becoming increasingly clear. This study has shown that in Drosophila, extracellular ROSs generated by the NADPH oxidase Duox drive compensatory proliferation and overgrowth following hid-induced activation of the initiator caspase Dronc in developing epithelial tissues. At least one consequence of ROS production is the activation of hemocytes at undead epithelial disc tissue. Furthermore, the work implies that extracellular ROS and hemocytes are part of the feedback amplification loop between Hid, Dronc, and JNK that occurs during stress-induced apoptosis. Finally, hemocytes release the TNF ligand Eiger, which promotes JNK activation in epithelial disc cells (Fogarty, 2016).

This work helps to understand why JNK activation occurs mostly in apoptotic/undead cells but occasionally also in neighboring surviving cells. Because the data indicate that hemocytes trigger JNK activation in epithelial cells, the location of hemocytes on the imaginal discs determines which epithelial cells receive the signal for JNK activation. Nevertheless, the possibility is not excluded that there is also an autonomous manner of Dronc-induced JNK activation in undead/apoptotic cells (Fogarty, 2016).

In the context of apoptosis, hemocytes engulf and degrade dying cells. However, there is no evidence that hemocytes have this role in the undead AiP model. No Caspase-3 (CC3) material is observed in hemocytes attached to undead tissue. Therefore, the role of hemocytes in driving proliferation is less clear and likely context dependent. In Drosophila embryos, hemocytes are required for epidermal wound healing, but this is a nonproliferative process. With respect to tumor models in Drosophila, much of the research to date has focused on the tumor-suppressing role of hemocytes and the innate immune response. However, a few reports have implicated hemocytes as tumor promoters in a neoplastic tumor model. Consistently, in the undead model of AiP, this study found that hemocytes have an overgrowth- and tumor-promoting role. Therefore, the state of the damaged tissue and the signals produced by the epithelium may have differential effects on hemocyte response (Fogarty, 2016).

In a recent study, ROSs were found to be required for tissue repair of wing imaginal discs in a regenerative (p35-independent) model of AiP, consistent with the current work. Although a role of hemocytes was not investigated in this study, it should be noted that p35-independent AiP models do not cause overgrowth, whereas undead ones such as the ey>hid-p35 AiP model do. It is therefore possible that ROSs in p35-independent AiP models are necessary for tissue repair independent of hemocytes, whereas ROSs in conjunction with ROS-activated hemocytes in undead models mediate the overgrowth of the affected tissue. Future work will clarify the overgrowth-promoting function of hemocytes. These considerations are reminiscent of mammalian systems, where many solid tumors are known to host alternatively activated (M2) tumor-associated macrophages, which promote tumor growth and are associated with a poor prognosis (Fogarty, 2016).

Because tumors are considered 'wounds that do not heal', the undead model of AiP is seen as a tool to probe the dynamic interactions and intercellular signaling events that occur in the chronic wound microenvironment. Future studies will investigate the specific mechanisms of hemocyte-induced growth and the tumor-promoting role of inflammation in Drosophila as well as roles of additional tissue types, such as the fat body, on modulating tumorous growth (Fogarty, 2016).

Curly encodes Dual Oxidase, which acts with Heme Peroxidase Curly Su to shape the adult Drosophila wing

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. This study shows that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), this study demonstrated that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, this study identified a novel heme peroxidase, Curly Su (Cysu; CG5873) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. This work adds to a growing number of studies suggesting that Duox's primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases (Hurd, 2015).

Over 90 years ago, Lenore Ward first described a dominant mutation, Curly, that causes the wings of Drosophila melanogaster to bend upwards. Since then, Curly has become a ubiquitous second chromosomal marker used by Drosophila geneticists on a daily basis to follow and track mutations. Despite its widespread use, how Curly mutations dominantly alter wing curvature has remained obscure. Waddington first proposed that Curly causes an unequal contraction of the dorsal and ventral wing surfaces during the drying period shortly after flies emerge from their pupal cases. Others have subsequently demonstrated that comparable alterations in wing curvature can be caused by differential growth of the dorsal and ventral epithelia. Irrespective of the mechanism, that similar wing phenotypes have been described for D. pseudoobscura and D. montium mutants suggests the underlying cause of curly wing formation is evolutionarily conserved among Drosophilids. The major factor limiting understanding of Curly's function in wing morphogenesis, however, is the fact that its molecular identity has remained unknown (Hurd, 2015).

This study has uncover the long unknown molecular nature of Curly. Mutations in the gene duox cause the Curly wing phenotype. Duox is a member of a highly conserved group of transmembrane proteins collectively referred to as NADPH oxidases. These enzymes function to transfer electrons across biological membranes to generate ROS by transferring electrons from NADPH to oxygen through flavin adenine dinucleotide (FAD) and heme cofactors. Several biological functions have been described for Duox. Perhaps the best studied of these in Drosophila is its role in host defense where it is thought to generate ROS to kill pathogens. However, Duox also plays an important role in providing ROS, specifically hydrogen peroxide, for heme peroxidases to catalyze the formation of covalent bonds between biomolecules. In mammals, Duox generates hydrogen peroxide for thyroid peroxidase to catalyze the iodination and crosslinking of tyrosine residues in the formation of thyroid hormones. Duox is also expressed in tissues other than the thyroid, such as the gastrointestinal tract, where its function is less clear. In insects, worms and sea urchins, Duox participates in the formation of extracellular structures through the crosslinking of tyrosine residues. Indeed, instead of its function in generating bactericidal ROS, the tyrosine crosslinking activity of Duox may be the primary ancestral function, as it appears to be conserved across phyla (Hurd, 2015).

This study shows that specific mutations in the NADPH binding-domain encoding region of duox cause a Curly wing phenotype. Using Curly, this study demonstrated that duox is required during the last day of pupal development to stabilize the wing. Furthermore, through suppression experiments, a novel heme peroxidase, Curly Su (Cysu), was identified that works with Duox to adhere the dorsal surface of the wing to the ventral one. Uncovering the molecular identity of Curly not only provides an entry point for the functional understanding of this prominent wing mutant phenotype, but also will allow for the discovery of novel duox interacting genes and regulators through unbiased genetic screens. Only through these approaches can an understanding be gained of the precise molecular function of Duox in the myriad biological processes in which it is involved (Hurd, 2015).

This study has shown that the Curly mutation arises in the NADPH-binding pocket encoding region of duox. Using Curly mutations and duox RNAi, it was shown that Duox is required within the wing to maintain its shape beginning on the last day of pupal development. Results from these genetic studies suggest Duox does this by supplying hydrogen peroxide to the heme peroxidase Cysu to facilitate the bonding of the two wing cuticle surfaces, likely by physically crosslinking them, during wing formation (Hurd, 2015).

In all Curly mutants sequenced, a glycine residue, 1505, in the NADPH-binding pocket of Duox is mutated. This glycine is present in all NADPH oxidases from microbial eukaryotes to humans, and more broadly in oxidoreductase and ferric reductase NAD-binding domains (PFAM PF00175 and PF08030, respectively). Though mutagenesis studies have not been conducted on this residue itself, it sits beside an equally conserved cysteine residue, which has been studied in detail because mutations in it cause chronic granulomatous disease in humans. This cysteine residue does not appear to be important for NADPH oxidase assembly or binding NADPH. Instead it is thought to be required for orienting bound NADPH for efficient electron transfer (via hydride) to FAD, and eventually oxygen. Given glycine 1505's proximity, it is possible that mutations in it similarly affect the transfer of electrons from NADPH to FAD. Consistent with this is the observation that Curly mutants are neither homozygous viable nor viable over a deficiency, suggesting that mutation of glycine 1505 causes a reduction in Duox's normal function (Hurd, 2015).

Although Curly mutations reduce Duox's normal function, they also endow it with a new function. Precisely what this new function is remains obscure, however it likely requires a source of electrons because altering the NADPH/NADP+ by removing niacinamide from the food or knocking down NAD+ kinase suppressed the wing phenotype. It is known that the expressivity of the Curly wing phenotype can be suppressed by larval crowding and/or starving larvae. Given this, it is possible that reduced uptake of niacinamide is a cause of the decreased expressivity of the Curly wing phenotype in starved larvae. Riboflavin shortage during the larval stage has also been suggested to be a cause of this suppression. Since riboflavin is a precursor of FAD, a co-factor also necessary for the Duox function, it too may suppress the wing phenotype by reducing endogenous FAD and in turn reducing the Duox activity. Regardless, Curly mutations are likely neomorphic and their sensitivity to environmental factors is likely mediated by changes in substrate availability (Hurd, 2015).

Duox is required autonomously for wing stabilization. Results from this study and another strongly support this assertion. Expression of duoxCyK or knockdown of duox on the last day prior to eclosion, but not earlier, caused defects in wing morphogenesis. This suggests that Duox and Curly do not influence growth or proliferation of the wing epithelia because these processes are complete by this time. Instead, ultrastructural analysis suggests that Duox plays an important role in forming the cuticle of the wing. In duox knockdowns, frequent gaps between the two wing cuticle surfaces were observed, in contrast to the wild-type wings. Defects in adhesion of the two cuticle surfaces were also apparent in Curly mutants. Unlike wings from duox knockdowns, however, the cuticle surfaces in Curly wings were most often tightly apposed with occasional bunching of the dorsal surface. It is possible that in the Curly mutants this aberrant pinching of the dorsal surface decreases its area relative to the ventral surface causing the wing to bend, as first intimated by Waddington 75 years ago However, it is not known whether this is the cause of the curling or just a consequence of it (Hurd, 2015).

Duox is known to be involved in the formation of extracellular matrices and cuticles. Typically, it does this by supplying hydrogen peroxide to heme peroxidases, which use the hydrogen peroxide to perform crosslinking reactions. Consistent with Duox playing a role in crosslinking the cuticle this study found that the heme peroxidase Cysu was essential for Duox function in the wing. Duox is unusual among NADPH oxidases in that it contains its own peroxidase homology domain, which in Caenorhabditis elegans and D. melanogaster has been proposed to fulfill the function of heme peroxidases, thereby obviating their need. However, given that the peroxidase homology domain of Drosophila Duox lacks many amino acid residues, including the proximal and distal histidines, essential for efficient peroxidase function it is unclear how well it functions in this capacity. Indeed, the results suggest that in D. melanogaster, Duox requires the heme peroxidase Cysu not only for stabilizing the wing cuticle, but also in the formation of the notum and scutellum. These findings point to a more general role for Duox and Cysu in cuticle formation (Hurd, 2015).

In Drosophila, Duox has been intensely studied in the context of host defense and gut immunity. In the gut, Duox is thought to generate ROS to kill pathogens; flies that have reduced Duox activity have increased susceptibility to infection. Upon infection ROS generated by Duox kill pathogens, and possibly signal intestinal epithelial cells to proliferate and renew. The results, as well as others, demonstrate that Duox is also critical in the formation of cuticle structures and extracellular matrices. It is possible that Duox performs a similar function in the Drosophila intestine, perhaps by forming extracellular barriers or structures to protect against infection. Indeed, Duox in conjunction with heme peroxidases has been shown to form such barriers in guts of ticks and mosquitos. It would therefore be interesting to explore whether Duox and possibly Cysu are also involved in forming barriers to protect against infection in the Drosophila intestine (Hurd, 2015).

Duox is an important protein that has a number of diverse functions, which we are only beginning to understand. Curly mutations provide an excellent opportunity to further explore Duox's functions by identifying unknown interactors and regulators through unbiased genetic suppressor screens. The identification of Cysu through such an approach demonstrates its feasibility and utility. Such approaches will not only tell us about Duox's function in the wing, but also about its role in immunity and beyond (Hurd, 2015).

Bacterial uracil modulates Drosophila DUOX-dependent gut Immunity via Hedgehog-induced signaling endosomes

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. This study shows that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCβ/PKC/Ca(2+)-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection (Lee, 2015).

Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila

All metazoan guts are subjected to immunologically unique conditions in which an efficient antimicrobial system operates to eliminate pathogens while tolerating symbiotic commensal microbiota. However, the molecular mechanisms controlling this process are only partially understood. This study shows that bacterial-derived uracil acts as a ligand for dual oxidase (DUOX)-dependent reactive oxygen species generation in Drosophila gut and that the uracil production in bacteria causes inflammation in the gut. The acute and controlled uracil-induced immune response is required for efficient elimination of bacteria, intestinal cell repair, and host survival during infection of nonresident species. Among resident gut microbiota, uracil production is absent in symbionts, allowing harmonious colonization without DUOX activation, whereas uracil release from opportunistic pathobionts provokes chronic inflammation. These results reveal that bacteria with distinct abilities to activate uracil-induced gut inflammation, in terms of intensity and duration, act as critical factors that determine homeostasis or pathogenesis in gut-microbe interactions (Lee, 2013).

The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine

The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. This study analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Dual oxidase (Duox) is reduced in p38c mutant flies. This genetic analysis shows that p38c functions in a pathway with Mekk1 and Licorne (Mkk3) to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, this study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c (Chakrabarti, 2014).

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. A variety of fluorescent reporters were developed that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. Four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-β-cyclodextrin, and hydrogen peroxide) were found to be sufficient to globally activate epidermal wound response genes in Drosophila embryos. The epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals were examined. The results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration (Juarez, 2011).

Drosophila wound healing is an example of a regenerative process, which requires localized epidermal cytoskeletal changes, and localized wound-induced changes in epidermal transcriptional activity. This genetic screen with wound-dependent reporters has allowed identification of novel components that regulate the localized transcriptional response to wounding in epidermal cells. This research identifies seven genes that are required to either activate (Duox and ghost/stenosis) or localize (Flo-2, Src42A, wurst, varicose, and Drosophila homolog of yeast Mak3) the expression patterns of epidermal wound reporters. The number of new functions involved in the delimitation of epidermal wound response near wound sites was unexpected, but indicates that considerable genetic effort is devoted to localizing the activity of transcriptional wound responses during regeneration (Juarez, 2011).

One of the genes that limits the spread of epidermal wound reporters after clean epidermal punctures is Flo-2, as mutants of this gene show a broad expansion of epidermal wound gene activation. Drosophila Flo-2 is itself transcriptionally activated around epidermal wound sites, consistent with an evolutionarily conserved role in regeneration after wounding. In vertebrates, reggie-1/Flo-2 gene expression is activated in wounded fish optic neurons, and reggie-1/Flo-2 and reggie-2/Flo-1 morpholino knockdowns in wounded zebrafish retinal explants reduced axon outgrowth compared to controls. Flo-2 transcriptional activation around Drosophila epidermal wound sites is dependent on the grh genetic function, which is required to activate at least a few other epidermal wound response genes. Flo-2 thus appears to act in the same pathway as grh, although it may act both downstream and upstream of grh, since overexpression of Flo-2 can inhibit the activation of other grh-dependent wound response genes. In this respect, Flo-2 resembles stit receptor tyrosine kinase gene (Wang, 2009), which is both transcriptionally activated by Grh, as well as required for grh-dependent activation of other downstream wound genes. Amazingly, overexpression of Flo-2 can even inhibit the global activation of the Ddc and ple-WE1 wound reporters that are induced by the serine protease trypsin, or by hydrogen peroxide. The inhibitory function of overexpression of Flo-2 on wound induced transcription is cell non-autonomous, at least over the range of a few cell diameters, as shown by the ability of striped overexpression of Flo-2 to silence puncture or trypsin-induced gene activation throughout the epidermis (Juarez, 2011).

The only animal where Flo-2 null mutants have so far been characterized is Drosophila, where Flo-2 has been shown to regulate the spread of Wingless (Wg) and Hedgehog (Hh) signals in the wing imaginal discs. In the wing discs, both the secretion rate and the diffusion rate of these two lipid-modified morphogens were increased when Flo-2 was overexpressed, and decreased when Flo-2 and Flo-1 proteins were not expressed. Despite the reduced spread of Wg and Hh morphogen proteins in Flo-2 mutant imaginal discs, adult morphology of mutants was normal, presumably because of compensatory mechanisms that occur later in development. Whereas a reduced range of activation of wg and hh long range transcription target genes was observed in Flo-2 mutant imaginal discs, a greatly increased range of wound-induced gene activation was observed in Flo-2 mutant embryos. This apparent discrepancy could be explained if one invokes of a long-range wound-induced inhibitory signal that in wild type embryos diffuses faster and farther than a wound activating signal, and thereby functions to limit the wound response to nearby epidermal cells, and that in Flo-2 mutants this potential inhibitory signal has reduced secretion, concentration, and/or diffusion range. This notion is consistent with the cell non-autonomous effect of overexpressed Flo-2 on inhibiting wound- or trypsin-induced gene activation. A similar scheme of controlling signal spreading has been seen in the way that Mmp2 acts cell non-autonomously to limit FGF signaling during Drosophila tracheal development and branch morphogenesis. It's also possible that Flo-2 normally is required to set a global threshold that wound-induced signals must overcome in order to activate wound transcription, for example via Flo-2-dependent endocytosis/degradation of a diffusible wound signal and its receptor (perhaps the Stit RTK), and that signal strength normally surpasses the Flo-2 threshold only in the vicinity of a wound. In this model, loss of Flo-2 would result in all epidermal cells being able to exceed the wound signal threshold, and overexpression of Flo-2 would prevent any cells from exceeding the wound signal threshold. The cell non-autonomous effects of Flo-2 overexpression under this model might be explained by an increase in Flo-2-dependent endocytosis/degradation that rapidly depletes an activating signal from the extracellular space (Juarez, 2011).

Many previous studies have documented biochemical, molecular biological, and cell biological interactions between Src family kinases and Flotillins. In Drosophila, lack of Src42A and Flo-2 leads to expanded spread of wound gene activation, and overexpression of Flo-2 or activated Src42A can inhibit wound gene activation, which is consistent with an interaction between the two functions during the process of wound gene regulation. In cultured mammalian cells, Flo-2 can be phosphorylated by Src family kinases in an extracellular signal-dependent fashion. This phosphorylation is associated with changes in the normal intracellular trafficking of Flotillin-containing membrane microdomains and vesicles. Since overexpressed Flo-2 in Drosophila can act in a cell non-autonomous fashion to inhibit wound gene activation, and overexpressed Src42A acts in a cell autonomous fashion to inhibit wound gene activation, one interpretation is that Flo-2 lies genetically upstream of Src42A in the epidermal wound response. This hypothesis appears to be inconsistent with the vertebrate biochemical data indicating that Src kinases phosphorylate Flotillins to activate their diverse functions. However, an observation that is consistent with Src42A activating Flo-2 protein function, is that even when Flo-2 is overexpressed, addition of chemical inhibitors of Src family kinases to wounded embryos, results in widespread Ddc .47 or ple-WE1 wound reporter activation. One interpretation of this suggests Flo-2 protein, no matter the level of expression, is inactive in the absence of Src42A function. Complex feedback loops involving signaling proteins being regulated by a transcription factor, while the activity of the same transcription factors is regulated by the same signaling pathway, have been observed in the control of Drosophila epidermal wound gene expression and reepithelialization, so there may be similar dynamic cross-regulatory interactions between Flo-2 and Src42A in the localization of the epidermal wound response, interactions not easily captured in linear genetic pathway diagrams (Juarez, 2011).

The inhibitory effect of Src42A on wound gene activation suggests that it might antagonize a signaling cascade that leads to the epidermal wound response. A good candidate for such a signaling cascade is the RTK pathway involving the Stit kinase. Stit is a RET-family RTK that is required for robust activation of the Ddc and stit wound reporter genes in wounded embryos (Wang, 2009). Other evidence consistent with RTK pathway importance in wound gene activation is that phosphotyrosine accumulates persistently around wound sites, and that ERK kinase function is required for robust activation of the Ddc wound reporter gene. Interestingly, Src42A has been shown to act as an inhibitor of some Drosophila RTK proteins (those encoded by the torso, Egfr, and sevenless genes) in a few different tissues during Drosophila development. The Flo-2 and Src42A functions in epidermal wound localization after clean wounding are reminiscent of the role of Drosophila WntD during infectious wounding. WntD mutants show higher levels of some antimicrobial peptide genes after septic injury of adults (Juarez, 2011).

Previous evidence suggested that H₂O₂ and Duox could provide wound-induced inflammatory signals and antimicrobial activities. The current studies show that Duox is required to activate wound reporter genes after epidermal wounding, and that injected exogenous H₂O₂ is sufficient to activate widespread epidermal wound gene expression. Overexpression of either Flo-2 or Src42A.CA can inhibit the H₂O₂ -dependent wound reporter expression, suggesting that all of these components are in a common pathway controlling the activation of epidermal wound reporters. However, the ability of trypsin injection to activate the Ddc .47 and ple-WE1 wound reporters in Duox mutants suggests that a serine protease might act downstream of, or in parallel to, H₂O₂-dependent wound signals. A recent report showed that in cultured mammalian cells, a Src kinase phosphorylates and inhibits a Flo-2-associated enzyme, peroxiredoxin-1, which results in increased stability of H₂O₂. This is consistent with the results placing Flo-2, Src42A, and H₂O₂ in a common wound signaling pathway (Juarez, 2011).

Like H₂O₂, the injection of methyl-β-cyclodextrin (MβCD) into wounded embryos triggers a global wound response in the epidermis. MβCD strongly depletes cholesterol and other sterols from membranes and disrupt lipid rafts, but was also shown to remove sphingolipid-associated proteins such as Src-Family Kinases. The effects of MβCD, in combination with the effects of loss of Flo-2, suggests that the integrity of lipid rafts and associated proteins are required to inhibit epidermal wound signals. In cultured cells, MβCD treatments trigger a release of EGF receptors from membrane microdomains, which increases EGFR, and perhaps other RTK, signaling in a ligand-independent manner. Interestingly, in cultured keratinocytes, MβCD treatment can induce the expression of involucrin, which encodes a protein, analogous to Drosophila Ple/tyrosine hydroxylase, which is required for the formation of an epidermal barrier. Similarly, MβCD injections into Drosophila embryos might also cause an increase the levels of a wound signal produced or released from cells adjacent to the wound site, allowing more widespread transcriptional activation of wound reporter genes. The observations that overexpression of Src42A or Flo-2 can inhibit the MβCD -triggered activation of epidermal wound reporter genes suggest that high levels of these proteins might overcome lipid raft-inhibitory effects on wound signaling pathways (Juarez, 2011).

Other genes (wurst and varicose) identified in the screen have phenotypes similar to Flo-2 and Src42A mutants. wurst encodes an evolutionarily conserved trans-membrane protein, containing a heat shock cognate protein 70 binding domain and a clathrin binding motif. wurst is ubiquitously expressed in embryonic epithelial cells, strongly up-regulated during endocytosis-dependent luminal clearance, and mislocalized in mutants with endocytosis defects. wurst mutant embryos have tortuous tracheal tubes, due to a failure to properly endocytose matrix material from the tracheal lumen. varicose encodes an evolutionarily-conserved septate junction scaffolding protein, in the Membrane Associated GUanylate Kinase (MAGUK) family. varicose is expressed in epidermally-derived cells (including the hindgut and trachea) and co-localizes with the septate junction proteins, Coracle and Neurexin4. varicose mutant embryos develop permeable tracheal tubes and paracellular barrier defects in epithelia. Like wurst mutants, varicose mutants also have abnormal matrix composition in the tracheal lumen, and may also have abnormal extracellular matrix composition produced by other epidermal cells (Juarez, 2011).

Another gene (ghost), also known as stenosis) identified in this screen is required for wound reporter activation like Duox or grh. ghost encodes the Drosophila Sec24CD homolog, a coat protein of COPII vesicles in the ER/Golgi trafficking pathway. Transport of cargo from the ER to the Golgi via COPII vesicles is required to achieve normal amounts of secretion of extracellular matrix proteins into the developing Drosophila tracheae and normal apical-basal localization of membrane proteins. Presumably, similar secretion and membrane localization defects occur in non-tracheal epidermal cells, which account for the severe cuticle deposition defects in ghost (Sec24CD) mutants. It is fascinating to note that the finding that ghost (Sec24CD) is required for transcriptional activation of epidermal wound reporter genes is consistent with the finding that RNAi knockdowns of Sec24C in a planaria (Schmidtea mediterranea) interfered with normal regeneration after amputation wounds. It is possible that the ghost mutants do not secrete enough wound signals, or the protein matrix necessary for the propagation of a wound signal (Juarez, 2011).

Another gene required for the activation of wound reporters is shroud (sro). It is believed sro to be an allele in the Drosophila Fos-D isoform, and it was hypothesized that one of the Drosophila kayak/Fos transcription factors was required for the activation of some epidermal wound gene reporters. However, has been recently discovered that sro[1] and other sro point mutant alleles do not map in the kayak/Fos gene, but in an immediately adjacent transcription unit (Nm-g/sro) that encodes an enzyme in the sterol metabolic pathway that is necessary for production of ecdysone hormone. At first glance, the requirement of sro to activate some wound reporters suggested that these reporters rely on ecdysone signaling. This is possible, although deletions were tested that eliminate zygotic functions of the ecdysone receptor gene, as well as of the phantom gene (which encodes another enzyme in the ecdysone synthesis pathway), and embryos that are zygotic mutants in either gene show normal activation of the ple-WE1 wound reporter after puncture wounding (Juarez, 2011).

In summary, though this large unbiased screen, several genes were identified that add to the understanding of the complex pathways that control the signals that activate wound response transcription near puncture wounds. At the cellular level, there appears to be a correlation between genetic functions required to localize wound-induced gene activation, and cellular functions required for endocytosis and/or apical-basal polarity. For example, one function of Flo-2 is in signal-dependent endocytosis, although Flo-2 also plays other roles in vesicular trafficking. There have been many studies showing that endocytosis can regulate extracellular signaling strength and duration. For example, one study found that tagged-FGF8 showed increased accumulation, spread, and target gene activation when Rab-5-mediated endocytosis was reduced in zebrafish embryos. It is believed that further studies on wound response signaling may provide new insights into how membrane microdomains, endocytosis of membrane receptors, and the composition and organization of the extracellular matrix, regulates the transmission of wound signals (Juarez, 2011).

Invasive and indigenous microbiota impact intestinal stem cell activity through JAK-STAT and JNK pathways in Drosophila

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. This study addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. The increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, evidence is provided that the JAK-STAT and JNK pathways are both required for bacteria-induced stem cell proliferation. Similarly, it was demonstrated that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, it was shown that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity (Buchon, 2009).

The JAK-STAT and JNK signaling pathways are required to maintain gut homeostasis upon exposure to a broad range of bacteria. In normal conditions, low levels of the indigenous gut microbiota and transient environmental microbes maintain a basal level of epithelium renewal. The increase in gut microbes in old or Imd-deficient flies is associated with a chronic activation of the JNK and JAK-STAT pathways, leading to an increase in intestinal stem cells (ISC) proliferation and gut disorganization. The impact of pathogenic bacteria can have different outcomes on gut homeostasis, depending on the degree of damage they inflict on the host. Damage to the gut caused by infection with E. carotovora is compensated for by an increase in epithelium renewal. Infection with a high dose of P. entomophila disrupts the homeostasis normally maintained by epithelium renewal and damage is not repaired, contributing to the death of the fly (Buchon, 2009).

Previous studies have shown that the NADPH oxidase Duox plays an essential role in Drosophila gut immunity by generating microbicidal effectors such as ROS to eliminate both invasive and dietary microbes. Ecc15 is a potent activator of Duox, which in turn is important in the clearance of this bacterium. This oxidative burst is coordinated with the induction of many genes involved in ROS detoxification upon Ecc15 ingestion. This study provides evidence that the observed increase in epithelium renewal upon Ecc15 infection is a compensatory mechanism that repairs the damage inflicted to the gut by this oxidative burst. This is supported by the observation that reducing ROS levels by either the ingestion of antioxidants or silencing the Duox gene reduces epithelium renewal. Although ISC proliferation could be directly triggered by ROS, it is more likely a consequence of signals produced by stressed enterocytes. A number of data support this hypothesis: (1) Ingestion of corrosive agents can also induce ISC proliferation, and (2) physical injury is sufficient to induce local activation of the cytokine Upd3, which promotes epithelium renewal. Interestingly, a significant increase in epithelium renewal was observed in Duox RNAi flies at late time points following infection, correlating with damage attributed to the proliferation of Ecc15 in the guts of Duox-deficient flies. While the increase in epithelium renewal observed with Ecc15 is clearly linked to the damage induced by the host immune response, it is likely that effects on epithelium renewal by other pathogens could be more direct and mediated by virulence factors, such as the production of cytolytic toxins (Buchon, 2009).

The data indicate that the JAK-STAT and JNK pathways synergize to promote ISC proliferation and epithelium renewal in response to the damage induced by infection. The JAK-STAT pathway is implicated in the regulation of stem cells in multiple tissues and is proposed to be a common regulator of stem cell proliferation. The data extend this observation by showing that the JAK-STAT pathway is also involved in ISC activation upon bacterial infection. The cytokine Upd3 is produced locally by damaged enterocytes and subsequently stimulates the JAK-STAT pathway in ISCs to promote their proliferation. The results globally agree with a recent study showing that the JAK-STAT pathway is involved in ISC proliferation upon infection with a low dose of P. entomophila (Jiang, 2009). This work and the current study clearly demonstrate that the JAK-STAT pathway adjusts the level of epithelium renewal to ensure proper tissue homeostasis by linking enterocyte damage to ISC proliferation. The study by Jiang also uncovered an additional role of this pathway in the differentiation of enteroblasts during basal gut epithelium turnover. The implication of the JAK-STAT pathway in differentiation could explain the accumulation of the small-nucleated escargot-positive cells observed in the gut of flies with reduced JAK-STAT signaling in ISCs. The JAK-STAT pathway was also shown previously to control the expression of some antimicrobial peptides such as Drosomycin 3 (Dro3). Therefore, the JAK-STAT pathway has a dual role in the gut upon infection, controlling both the immune response and epithelium renewal (Buchon, 2009).

The data show that the lack of JNK pathway activity in ISCs results in the loss of ISCs in guts infected with Ecc15, thus preventing epithelium renewal. The findings are consistent with the attributed function of JNK at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. This cytoprotective role against ROS would protect ISCs from the oxidative burst induced upon Ecc15 infection, explaining why ISCs die by apoptosis when JNK activity is reduced. It is likely that JNK signaling is required not only to protect ISCs from oxidative stress, but also to induce stem cell proliferation to replace damaged differentiated cells. This is supported by the observation that overexpression of the JNKK Hep in ISCs is sufficient to trigger an epithelium renewal in the absence of infection. In addition, increased JNK activity in ISCs of old flies has been linked to hyperproliferative states and age-related deterioration of the intestinal epithelium. This study shows that JNK signaling is also required for epithelium renewal upon Ecc15 infection. Thus, infection with Ecc15 recapitulates in an accelerated time frame the impacts of increased stress observed in guts of aging flies (Buchon, 2009).

The inhibition of the dJun transcription factor in ISCs leads to a loss of stem cells in the absence of infection, suggesting that this transcription factor plays a critical role in ISC maintenance in the gut. There is no definitive explanation for why the dJun-IR construct behaves differently than the basket and hep-IR constructs. It is speculated that this could be due to (1) differences in the basal activity of the JNK pathway, which would be blocked only with the dJun-IR that targets a terminal component of the pathway; (2) effects of Jun in ISCs independent of the JNK pathway; or (3) side effects of the dJun-IR construct (Buchon, 2009).

In contrast to the requirement of the JNK pathway upon Ecc15 infection, it has been reported that oral ingestion with a low dose of P. entomophila still induced mitosis in the JNK-defective mutant hep¹. In agreement, this study found that inhibiting the JNK pathway in ISCs did not block the induction of epithelium renewal by a low dose of P. entomophila. This difference in the requirement of the JNK pathway may be explained by the nature of these two pathogens. Whereas Ecc15 damages the gut through an oxidative burst that activates the JNK pathway, the stimulation of epithelium renewal by P. entomophila could be due to a more direct effect of this bacterium on the gut. Altogether, this work points to an essential role of the JAK-STAT pathway in modulation of epithelium renewal activity, while the role of JNK may be dependent on the infectious agent and any associated oxidative stress. While it is known that the JNK pathway is activated by a variety of environmental challenges including ROS, the precise mechanism of activation of this pathway has not been elucidated. Similarly, the molecular basis of upd3 induction in damaged enterocytes is not known. Future work should decipher the nature of the signals that activate these pathways in both ISCs and enterocytes, as well as the possible cross-talk between the JNK and JAK-STAT pathways in ISC control (Buchon, 2009).

The observation that flies unable to renew their gut epithelium eventually succumb to Ecc15 infection highlights the importance of this process in the gut immune response. It is striking that defects in epithelium renewal are more detrimental to host survival than deficiency in the Imd pathway, even though this pathway controls most of the intestinal immune-regulated genes induced by Ecc15. The results are in agreement with a previous study indicating that, in the Drosophila gastrointestinal tract, the Imd-dependent immune response is normally dispensable to most transient bacteria, but is provisionally crucial in the event that the host encounters ROS-resistant microbes. However, this study demonstrates that efficient and rapid clearance of bacteria in the gut by Duox is possible only when coordinated with epithelium renewal to repair damage caused by ROS. This finely tuned balance between bacterial elimination by Duox activity and gut resistance to collateral damage induced by ROS is likely the reason why flies normally survive infection by Ecc15. Yet, this calibration also exposes a vulnerability that could easily be manipulated or subverted by other pathogens. Along this line, this work also exposes the range of impact different bacteria can have on stem cell activation. It was observed that infection with high doses of P. entomophila led to a loss of gut integrity, including the loss of stem cells. Moreover, the ability of P. entomophila to disrupt epithelium renewal correlates with damage to the gut and the death of the host. Since both JNK and JAK-STAT pathways are activated upon infection with P. entomophila, this suggests that this bacterium activates the appropriate pathways necessary to repair the gut, but ISCs are unable to respond accordingly. Interestingly, a completely avirulent P. entomophila mutant (gacA) does not persist in the gut and does not induce epithelium renewal. In contrast, an attenuated mutant (aprA) somewhat restores epithelium renewal. These observations, along with the dose response analysis using P. entomophila and corrosive agents, suggest that the virulence factors of this entomopathogen disrupt epithelium renewal through excessive damage to the gut. Of note, recent studies suggest that both Helicobacter pylori and Shigella flexneri, two bacterial pathogens of the human digestive tract, interfere with epithelium renewal to exert their pathological effects. This suggests that epithelium renewal could be a common target for bacteria that infect through the gut. In this respect, the host defense to oral bacterial infection could be considered as a bimodular response, composed of both immune and homeostatic processes that require strict coordination. Disruption of either process results in the failure to resolve the infection and impedes the return to homeostasis (Buchon, 2009).

In contrast to the acute invasion by pathogenic bacteria, indigenous gut microbiota are in constant association with the gut epithelium, and thus may impact gut homeostasis. Using axenically raised flies, it was established that indigenous microbiota stimulate a basal level of epithelium renewal that correlates with the level of activation of the JAK-STAT and JNK pathways. This raises the possibility that both indigenous and invasive bacteria, such as Ecc15, are capable of triggering epithelium renewal by the same process. Additionally, the data support a novel homeostatic mechanism in which the density of indigenous bacteria is coupled to the level of epithelium renewal. This is the first report that gut microbiota affect stem cell activation and epithelium renewal, concepts proposed previously in mammalian systems but never fully demonstrated. This also implies that variations in the level of epithelium renewal observed in different laboratory contexts could actually be due to impacts from gut microbes (Buchon, 2009).

Importantly, in this context, it was shown that lack of indigenous microbiota reverts most age-related deterioration of the gut. Aging of the gut is usually marked by both hyperproliferation of ISCs and differentiation defaults that lead to disorganization of the gut epithelium. These alterations have been shown to be associated with activation of the PDGF- and VEGF-related factor 2 (Pvf2)/Pvr and JNK signaling pathways directly in ISCs. Accordingly, inhibition of the JNK pathway in ISCs fully reverts the epithelium alterations that occur with aging. This raises the possibility that gut microbiota could exert their effect through prolonged activation of the JNK pathway. Interestingly, immune-deficient flies, lacking the Imd pathway, also display hyperproliferative guts and have higher basal levels of activation of the JNK and JAK-STAT pathways. The observation that these flies also harbor higher numbers of indigenous bacteria further supports a model in which failure to control gut microbiota leads to an imbalance in gut epithelium turnover. Future work should analyze the mechanisms by which gut microbiota affect epithelium renewal and whether this is due to a direct impact of bacteria on the gut or is mediated indirectly through changes in fly physiology. Moreover, the correlation between higher numbers of indigenous bacteria and increased disorganization of the gut upon aging in flies lacking the Imd pathway raises the possibility that a main function of this pathway is to control gut microbiota. This is in agreement with concepts emerging in mammals that support an essential role of the gut immune response in maintaining the beneficial nature of the host-microbiota association. This function also parallels the theory of 'controlled inflammation' described in mammals, where a low level of immune activation is proposed to maintain gut barrier integrity (Buchon, 2009).

In conclusion, this study unravels some of the complex interconnections between the immune response, invasive and indigenous microbiota, and stem cell homeostasis in the gut of Drosophila. Based on the evolutionary conservation of transduction pathways such as JNK and JAK-STAT between Drosophila and mammals, it is likely that similar processes occur in the gut of mammals during infection. Interestingly, stimulation of stem cell activity by invasive bacteria is proposed to favor the development of hyperproliferative states found in precancerous lesions. Thus, Drosophila may provide a more accessible model to elucidate host mechanisms to maintain homeostasis and the impact of bacteria on this process (Buchon, 2009).

Regulation of dual oxidase activity by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity

All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. This study shows that phospholipase C-β (PLCβ) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCβ through Gαq, which in turn mobilizes intracellular Ca²⁺ through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCβ mutant flies have a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCβ restores efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Gαq-PLCβ-Ca²⁺-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle (Ha, 2009a).

All organisms are in constant contact with a large number of different types of microbes. This is especially true in the case of the gut epithelia, which control life-threatening pathogens as well as food-borne microbes. In addition to this microbe-eliminating capacity, gut epithelia also need to protect normal commensal microbes which are in a mutually beneficial relationship. Therefore, gut epithelia must be equipped to differentially operate innate immunity in order to efficiently eliminate life-threatening microbes while protecting beneficial microbes. Studies using Drosophila as a genetic model have greatly enhanced understanding of the microbe-controlling mucosal immune strategy in gut epithelia. Previous studies in a gut infection model using oral ingestion of pathogens revealed that the redox system has an essential role in host survival by generating microbicidal effectors such as reactive oxygen species (ROS) (Ha, 2005a; Ha, 2005b). In this redox system, dual oxidase (DUOX), a member of the nicotinamide adenine dinucleotide phosphate (NADP)H oxidase family, is responsible for the production of ROS in response to gut infection (Ha, 2005a). Following microbe-induced ROS generation, ROS elimination is assured by immune-regulated catalase (IRC), thereby protecting the host from excessive oxidative stress (Ha, 2005b). In addition to the redox system, the mucosal immune deficiency (IMD)/NF-κB signaling pathway, which leads to the de novo synthesis of microbicidal effector molecules such as antimicrobial peptides (AMPs), has an essential complementary role to the redox system when the host encounters ROS-resistant pathogenic microbes. These findings indicate that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the versatility necessary for Drosophila gut immunity to control microbial infections. Furthermore, in the absence of gut infection, a selective repression of IMD/NF-κB-dependent AMPs is mediated by the homeobox gene Caudal, which is required for protection of the resident commensal community and host health. Therefore, fine-tuning of different gut immune systems appears to be essential for both the elimination of pathogens and the preservation of commensal flora (Ha, 2009a).

Most studies evaluating gut immunity have been performed in an oral infection model in which the pathogens are ingested. However, the gut epithelia constitute the interface between the host and the microbial environment; therefore, it is likely that animals in nature have already been subjected to continuous microbial contact, even in the absence of oral infection. Thus, it is essential to determine the mechanism by which this natural and continuous microbial interaction produces ROS at a tightly controlled, yet adequate level that allows for healthy gut-microbe interactions and gut homeostasis, because deregulated generation of ROS is believed to lead to a pathophysiologic condition in the gut epithelia. Although the DUOX system is of central importance in gut immunity, the signaling pathway(s) by which gut epithelia regulate DUOX-dependent microbicidal ROS generation are poorly understood (Ha, 2009a).

Drosophila feed on microbes, and one of their most essential microbial food sources is baker's yeast, Saccharomyces cerevisiae. As early as 1930, yeast was discovered to be an essential nutrient source for Drosophila and is now used as a major ingredient in standard laboratory Drosophila food recipes. Further, Drosophila-Saccharomyces interaction occurs in wild-captured Drosophila, which suggests that this interaction is an evolutionarily ancient natural phenomenon. Although many studies have investigated the effect of yeast on Drosophila metabolism and aging, very few works have been reported on the effect of yeast in terms of the host immunity. Specifically, it has previously been shown that dietary yeast contributes to the cellular immune responsiveness of Drosophila against a larval parasitoid, Leptopilina boulardi. However, the relationship between yeast and Drosophila gut immunity during the normal life cycle has never been closely examined. Therefore, in this study, a Drosophila-yeast model was used to investigate the intracellular signaling pathway by which the host mounts mucosal antimicrobial immunity, as well as the in vivo value of this pathway in the host's natural life. Through biochemical and genetic analyses, this study revealed that the Gαq-mediated phospholipase C-β (PLCβ) pathway is involved in the routine control of dietary yeast in the Drosophila gut. PLCβ is dynamically activated in the presence of ingested yeast and subsequently mobilizes the intracellular Ca²⁺ to produce ROS in a DUOX-dependent manner. The presence of all of these signaling components of the Gαq-PLCβ-Ca²⁺-DUOX-ROS pathway in the gut is essential to ensure routine control of dietary yeast and host fitness, highlighting the importance of this immune signaling as a bona fide first line of defense in Drosophila (Ha, 2009a).

This study demonstrates that the Gαq-PLCβ-Ca²⁺ signaling pathway controls the mucosal gut epithelial defense system through DUOX-dependent ROS generation, which is responsible for routine microbial interactions in the gut epithelia in the absence of infection. The PLCβ pathway impacts a wide variety of biological processes through the generation of a lipid-derived second messenger. In this process, the hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, by PLCβ generates two intracellular messengers, IP₃ and diacylglycerol. This process is one of the earliest events through which more than 100 extracellular signaling molecules regulate functions in their target cells. It has been shown that Gαq-PLCβ signaling is essential for the activation of the phototransduction cascade in Drosophila. This study revealed a physiological role of PLCβ wherein it is involved in the regulation of DUOX enzymatic activity, which leads to the generation of microbicidal ROS in the mucosal epithelia (Ha, 2009a).

PLCβ signaling is very rapid, with only a few seconds necessary to activate Ca²⁺ release and ROS production. This rapid response may be advantageous for the host and may be the mechanism by which dynamic and routine control of microbes in the gut epithelia is achieved. Because the gut is in continuous contact with microbes such as dietary microorganisms, it is conceivable that under normal conditions routine microbial contact dynamically induces a certain level of basal Gαq-PLCβ activity that varies depending on the local microbe concentration. This basal Gαq-PLCβ-DUOX activity seems to be sufficient for host survival. In such conditions of low bacterial burden, NF-κB-dependent AMP expression is known to be largely repressed by Caudal repressor for the preservation of commensal microbiota (Ryu, 2008). However, in the case of high bacterial burden (e.g., gut infection condition), the DUOX-ROS system would be strongly activated for full microbicidal activity. Furthermore, all of the flies that contained impaired signaling potentials for the Gαq-PLCβ-Ca²⁺-DUOX pathway were totally intact following septic injury but short-lived under natural rearing conditions or under gut infection conditions, indicating that the mucosal immune pathway is distinct from the systemic immune pathway (Ha, 2009a).

It is not clear how Gαq- and PLCβ-induced Ca²⁺ modulates DUOX enzymatic activity. Because the DUOX lacking Ca²⁺-binding EF hand domains is unable to rescue the DUOX-RNAi flies (Ha, 2005a), it is plausible that Ca²⁺ directly modulates the enzymatic activity of DUOX through binding to the EF hand domains (Ha, 2009a).

It is also important to determine what pathogen-associated molecular patterns (PAMPs) are responsible for the activation of PLCβ signaling. In Drosophila, peptidoglycan and β-1,3-glucan are the only two PAMPs known to induce the NF-κB signaling pathway in the systemic immunity. The results showed that neither peptidoglycan nor β-1,3-glucan was able to induce ROS in S2 cells, which suggests that a previously uncharacterized type(s) of PAMP is involved in the mucosal immunity. Because the Gαq protein acts as an upstream signaling component of the PLCβ-Ca²⁺ pathway, a microbe-derived ligand capable of activating G protein coupled receptor(s) and/or Gαq protein may be the best candidate for the Gαq-PLCβ-Ca²⁺-DUOX signaling pathway. Given the broad spectrum of microbes that activate the response, it remains possible that the unknown upstream sensors resemble a stress response more than a PAMP response. Elucidation of the molecular nature of such agonists will greatly enhance understanding of bacteria-modulated redox signaling in the gut epithelia. In conclusion, this study demonstrates that mucosal epithelia have evolved an innate immune strategy, which is functionally distinct from the NF-κB-dependent systemic innate immune system. The rapid Gαq-PLCβ-Ca²⁺-DUOX signaling is adapted to the routine and dynamic control of gut-associated microbes and may impact the long-term physiology of the intestine and host fitness (Ha, 2009a).

Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut

All metazoan guts are in permanent contact with the microbial realm. However, understanding of the exact mechanisms by which the strength of gut immune responses is regulated to achieve gut-microbe mutualism is far from complete. This study identify a signaling network composed of complex positive and negative mechanisms that controlled the expression and activity of dual oxidase (DUOX), which 'fine tuned' the production of microbicidal reactive oxygen species depending on whether the gut encountered infectious or commensal microbes. Genetic analyses demonstrated that negative and positive regulation of DUOX was required for normal host survival in response to colonization with commensal and infectious microbes, respectively. Thus, the coordinated regulation of DUOX enables the host to achieve gut-microbe homeostasis by efficiently combating infection while tolerating commensal microbes (Ha, 2009b).

Search PubMed for articles about Drosophila Duox

Amcheslavsky, A., Wang, S., Fogarty, C. E., Lindblad, J. L., Fan, Y. and Bergmann, A. (2018). Plasma membrane localization of apoptotic caspases for non-apoptotic functions. Dev Cell 45(4): 450-464.e453. PubMed ID: 29787709

Buchon, N., Broderick, N. A., Chakrabarti, S. and Lemaitre, B. (2009). Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 23(19): 2333-44. PubMed Citation: 19797770

Chakrabarti, S., Poidevin, M. and Lemaitre, B. (2014). The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine. PLoS Genet 10(9): e1004659. PubMed ID: 25254641

Fogarty, C. E., Diwanji, N., Lindblad, J. L., Tare, M., Amcheslavsky, A., Makhijani, K., Bruckner, K., Fan, Y. and Bergmann, A. (2016). Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages. Curr Biol 26(5): 575-584. PubMed ID: 26898463

Ha, E. M., et al. (2009a). Regulation of DUOX by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity. Dev. Cell 16(3): 386-97. PubMed Citation: 19289084

Ha, E. M., Lee, K. A., Seo, Y. Y., Kim, S. H., Lim, J. H., Oh, B. H., Kim, J. and Lee, W. J. (2009b). Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut. Nat Immunol 10(9): 949-957. PubMed ID: 19668222

Hurd, T. R., Liang, F. X. and Lehmann, R. (2015). Curly encodes Dual Oxidase, which acts with Heme Peroxidase Curly Su to shape the adult Drosophila wing. PLoS Genet 11: e1005625. PubMed ID: 26587980

Jang, W., Baek, M., Han, Y. S. and Kim, C. (2018). Duox mediates ultraviolet injury-induced nociceptive sensitization in Drosophila larvae. Mol Brain 11(1): 16. PubMed ID: 29540218

Juarez, M. T., Patterson, R. A., Sandoval-Guillen, E. and McGinnis, W. (2011). Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila. PLoS Genet. 7(12): e1002424. PubMed ID: 22242003

Khan, S. J., Abidi, S. N. F., Skinner, A., Tian, Y. and Smith-Bolton, R. K. (2017). The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling. PLoS Genet 13(7): e1006937. PubMed ID: 28753614

Lee, K. A., Kim, S. H., Kim, E. K., Ha, E. M., You, H., Kim, B., Kim, M. J., Kwon, Y., Ryu, J. H. and Lee, W. J. (2013). Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell 153(4): 797-811. PubMed ID: 23663779

Lee, K. A., Kim, B., Bhin, J., Kim, D. H., You, H., Kim, E. K., Kim, S. H., Ryu, J. H., Hwang, D. and Lee, W. J. (2015). Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes. Cell Host Microbe 17(2): 191-204. PubMed ID: 25639794

Lee, K. A., Cho, K. C., Kim, B., Jang, I. H., Nam, K., Kwon, Y. E., Kim, M., Hyeon, D. Y., Hwang, D., Seol, J. H. and Lee, W. J. (2018). Inflammation-modulated metabolic reprogramming is required for DUOX-dependent gut immunity in Drosophila. Cell Host Microbe 23(3): 338-352 PubMed ID: 29503179

Xiao, X., Yang, L., Pang, X., Zhang, R., Zhu, Y., Wang, P., Gao, G. and Cheng, G. (2017). A Mesh-Duox pathway regulates homeostasis in the insect gut. Nat Microbiol 2: 17020. PubMed ID: 28248301

date revised: 5 August 2023

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