Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens.     

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

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

An antioxidant system required for host protection against gut infection in Drosophila

A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. Here, we show that in Drosophila an extracellular immune-regulated catalase (IRC) mediates a key host defense system that is needed during host-microbe interaction in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-kappaB pathway plays in eliciting antimicrobial responses, NF-kappaB pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interaction in the gastrointestinal tract.     

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis.     

The receptor guanylate cyclase Gyc76C and a peptide ligand, NPLP1-VQQ, modulate the innate immune IMD pathway in response to salt stress

Receptorguanylate cyclases (rGCs) modulate diverse physiological processes including mammalian cardiovascular function and insect eclosion. The Drosophila genome encodes several receptor and receptor-like GCs, but no ligand for any Drosophila rGC has yet been identified. By screening peptide libraries in Drosophila S2 cells, the Drosophila peptide NPLP1-VQQ (NLGALKSSPVHGVQQ) was shown to be a ligand for the rGC, Gyc76C (CG42636, previously CG8742, l(3)76BDl, DrGC-1). In the adult fly, expression of Gyc76C is highest in immune and stress-sensing epithelial tissues, including Malpighian tubules and midgut; and NPLP1-VQQ stimulates fluid transport and increases cGMP content in tubules. cGMP signaling is known to modulate the activity of the IMD innate immune pathway in tubules via activation and nuclear translocation of the NF-kB orthologue, Relish, resulting in increased anti-microbial peptide (AMP) gene expression; and so NPLP1-VQQ might act in immune/stress responses. Indeed, NPLP1-VQQ induces nuclear translocation of Relish in intact tubules and increases expression of the anti-microbial peptide gene, diptericin. Targeted Gyc76C RNAi to tubule principal cells inhibited both NPLP1-VQQ-induced Relish translocation and diptericin expression. Relish translocation and increased AMP gene expression also occurs in tubules in response to dietary salt stress. Gyc76C also modulates organismal survival to salt stress - ablation of Gyc76C expression in only tubule principal cells prevents Relish translocation, reduces diptericin expression, and reduces organismal survival in response to salt stress. Thus, the principal-cell localized NPLP1-VQQ/Gyc76C cGMP pathway acts to signal environmental (salt) stress to the whole organism.     

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

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

Ovarian tumors in Rbp9 mutants of Drosophila induce an immune response

The Drosophila protein, Rbp9, is homologous to human Hu, which is reported to be involved in small cell lung cancer. Rbp9 functions in cystocyte differentiation, and mutations in Rbp9 cause ovarian tumors. Here we show that the antimicrobial peptide, Attacin, is upregulated in Rbp9 mutants, especially in ovaries where tumors form. Upregulation seems to result from activation of the NF-kappaB pathway since we detected nuclear localization of Relish in Rbp9 mutant ovaries but not in wild type ovaries. Inactivation of NF-kappaB in the Rbp9 mutant allows prolonged survival of malformed egg chambers. We conclude that Drosophila initiates an anti-tumor defense response via activation of NF-kappaB.     

Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.