Pre-exposure to non-pathogenic bacteria does not protect Drosophila against the entomopathogenic bacterium Photorhabdus.

Immune priming in insects involves an initial challenge with a non-pathogenic microbe or exposure to a low dose of pathogenic microorganisms, which provides a certain degree of protection against a subsequent pathogenic infection. The protective effect of insect immune priming has been linked to the activation of humoral or cellular features of the innate immune response during the preliminary challenge, and these effects might last long enough to promote the survival of the infected animal. The fruit fly Drosophila melanogaster is a superb model to dissect immune priming processes in insects due to the availability of molecular and genetic tools, and the comprehensive understanding of the innate immune response in this organism. Previous investigations have indicated that the D. melanogaster immune system can be primed efficiently. Here we have extended these studies by examining the result of immune priming against two potent entomopathogenic bacteria, Photorhabdus luminescens and P. asymbiotica. We have found that rearing D. melanogaster on diet containing a non-pathogenic strain of Escherichia coli alone or in combination with Micrococcus luteus upregulates the antibacterial peptide immune response in young adult flies, but it does not prolong fly life span. Also, subsequent intrathoracic injection with P. luminescens or P. asymbiotica triggers the Immune deficiency and Toll signaling pathways in flies previously exposed to a live or heat-killed mix of the non-pathogenic bacteria, but the immune activation fails to promote fly survival against the pathogens. These findings suggest that immune priming in D. melanogaster, and probably in other insects, is determined by the type of microbes involved as well as the mode of microbial exposure, and possibly requires a comprehensive and precise alteration of immune signaling and function to provide efficient protection against pathogenic infection.

Thioester-Containing Proteins 2 and 4 Affect the Metabolic Activity and Inflammation Response in Drosophila.

Drosophila melanogaster is an outstanding model for studying host antipathogen defense. Although substantial progress has been made in understanding how metabolism and immunity are interrelated in flies, little information has been obtained on the molecular players that regulate metabolism and inflammation in Drosophila during pathogenic infection. Recently, we reported that the inactivation of thioester-containing protein 2 (Tep2) and Tep4 promotes survival and decreases the bacterial burden in flies upon infection with the virulent pathogens Photorhabdus luminescens and Photorhabdus asymbiotica Here, we investigated physiological and pathological defects in tep mutant flies in response to Photorhabdus challenge. We find that tep2 and tep4 loss-of-function mutant flies contain increased levels of carbohydrates and triglycerides in the presence or absence of Photorhabdus infection. We also report that Photorhabdus infection leads to higher levels of nitric oxide and reduced transcript levels of the apical caspase-encoding gene Dronc in tep2 and tep4 mutants. We show that Tep2 and Tep4 are upregulated mainly in the fat body rather than the gut in Photorhabdus-infected wild-type flies and that tep mutants contain decreased numbers of Photorhabdus bacteria in both tissue types. We propose that the inactivation of Tep2 or Tep4 in adult Drosophila flies results in lower levels of inflammation and increased energy reserves in response to Photorhabdus, which could confer a survival-protective effect during the initial hours of infection.

Evolution and Function of Thioester-Containing Proteins and the Complement System in the Innate Immune Response.

The innate immune response is evolutionary conserved among organisms. The complement system forms an important and efficient immune defense mechanism. It consists of plasma proteins that participate in microbial detection, which ultimately results in the production of various molecules with antimicrobial activity. Thioester-containing proteins (TEPs) are a superfamily of secreted effector proteins. In vertebrates, certain TEPs act in the innate immune response by promoting recruitment of immune cells, phagocytosis, and direct lysis of microbial invaders. Insects are excellent models for dissecting the molecular basis of innate immune recognition and response to a wide range of microbial infections. Impressive progress in recent years has generated crucial information on the role of TEPs in the antibacterial and antiparasite response of the tractable model insect Drosophila melanogaster and the mosquito malaria vector Anopheles gambiae. This knowledge is critical for better understanding the evolution of TEPs and their involvement in the regulation of the host innate immune system.

The Drosophila Thioester containing Protein-4 participates in the induction of the cellular immune response to the pathogen Photorhabdus.

The function of thioester-containing proteins (TEPs) in the immune defense of the fruit fly Drosophila melanogaster is yet mostly unexplored. Recently, we showed the involvement of TEP4 in the activation of humoral and phenoloxidase immune responses of Drosophila against the pathogenic bacteria Photorhabdus luminescens and Photorhabdus asymbiotica. Here, we investigated the participation of Tep4 in the cellular defense of Drosophila against the two pathogens. We report significantly lower numbers of live and dead plasmatocytes in the tep4 mutants compared to control flies in response to Photorhabdus infection. We also find fewer crystal cells in the control flies than in tep4 mutants upon infection with Photorhabdus. Our results further suggest that Drosophila hemocytes constitute a major source for the transcript levels of Tep4 in flies infected by Photorhabdus. Finally, we show that Tep4 participates in the phagocytic function in Drosophila adult flies. Collectively our data support the protective role for TEP4 in the cellular immune response of Drosophila against the entomopathogen Photorhabdus.

The distinct function of Tep2 and Tep6 in the immune defense of Drosophila melanogaster against the pathogen Photorhabdus.

Previous and recent investigations on the innate immune response of Drosophila have identified certain mechanisms that promote pathogen elimination. However, the function of Thioester-containing proteins (TEPs) in the fly still remains elusive. Recently we have shown the contribution of TEP4 in the antibacterial immune defense of Drosophila against non-pathogenic E. coli, and the pathogens Photorhabdus luminescens and P. asymbiotica. Here we studied the function of Tep genes in both humoral and cellular immunity upon E. coli and Photorhabdus infection. We found that while Tep2 is induced after Photorhabdus and E. coli infection; Tep6 is induced by P. asymbiotica only. Moreover, functional ablation of hemocytes results in significantly low transcript levels of Tep2 and Tep6 in response to Photorhabdus. We show that Tep2 and Tep6 loss-of-function mutants have prolonged survival against P. asymbiotica, Tep6 mutants survive better the infection of P. luminescens, and both tep mutants are resistant to E. coli and Photorhabdus. We also find a distinct pattern of immune signaling pathway induction in E. coli or Photorhabdus infected Tep2 and Tep6 mutants. We further show that Tep2 and Tep6 participate in the activation of hemocytes in Drosophila responding to Photorhabdus. Finally, inactivation of Tep2 or Tep6 affects phagocytosis and melanization in flies infected with Photorhabdus. Our results indicate that distinct Tep genes might be involved in different yet crucial functions in the Drosophila antibacterial immune response.

The Global Transcription Factor Lrp Is both Essential for and Inhibitory to Xenorhabdus nematophila Insecticidal Activity.

In the entomopathogenic bacterium Xenorhabdus nematophila, cell-to-cell variation in the abundance of the Lrp transcription factor leads to virulence modulation; low Lrp levels are associated with a virulent phenotype and suppression of antimicrobial peptides (AMPs) in Manduca sexta insects, while cells that lack lrp or express high Lrp levels are virulence attenuated and elicit AMP expression. To better understand the basis of these phenotypes, we examined X. nematophila strains expressing fixed Lrp levels. Unlike the lrp-null mutant, the high-lrp strain is fully virulent in Drosophila melanogaster, suggesting that these two strains have distinct underlying causes of virulence attenuation in M. sexta Indeed, the lrp-null mutant was defective in cytotoxicity against M. sexta hemocytes relative to that in the high-lrp and low-lrp strains. Further, supernatant derived from the lrp-null mutant but not from the high-lrp strain was defective in inhibiting weight gain when fed to 1st instar M. sexta These data suggest that contributors to the lrp-null mutant virulence attenuation phenotype are the lack of Lrp-dependent cytotoxic and extracellular oral growth inhibitory activities, which may be particularly important for virulence in D. melanogaster In contrast, the high-Lrp strain was sensitive to the antimicrobial peptide cecropin, had a transient survival defect in M. sexta, and had reduced extracellular levels of insecticidal activity, measured by injection of supernatant into 4th instar M. sexta Thus, high-lrp strain virulence attenuation may be explained by its hypersensitivity to M. sexta host immunity and its inability to secrete one or more insecticidal factors.IMPORTANCE Adaptation of a bacterial pathogen to host environments can be achieved through the coordinated regulation of virulence factors that can optimize success under prevailing conditions. In the insect pathogen Xenorhabdus nematophila, the global transcription factor Lrp is necessary for virulence when injected into Manduca sexta or Drosophila melanogaster insect hosts. However, high levels of Lrp, either naturally occurring or artificially induced, cause attenuation of X. nematophila virulence in M. sexta but not D. melanogaster Here, we present evidence suggesting that the underlying cause of high-Lrp-dependent virulence attenuation in M. sexta is hypersensitivity to host immune responses and decreased insecticidal activity and that high-Lrp virulence phenotypes are insect host specific. This knowledge suggests that X. nematophila faces varied challenges depending on the type of insect host it infects and that its success in these environments depends on Lrp-dependent control of a multifactorial virulence repertoire.

Thioester-Containing Protein-4 Regulates the Drosophila Immune Signaling and Function against the Pathogen Photorhabdus.

Despite important progress in identifying the molecules that participate in the immune response of Drosophila melanogaster to microbial infections, the involvement of thioester-containing proteins (TEPs) in the antibacterial immunity of the fly is not fully clarified. Previous studies mostly focused on identifying the function of TEP2, TEP3 and TEP6 molecules in the D. melanogaster immune system. Here, we investigated the role of TEP4 in the regulation and function of D. melanogaster host defense against 2 virulent pathogens from the genus Photorhabdus, i.e. the insect pathogenic bacterium Photorhabdus luminescens and the emerging human pathogen P. asymbiotica. We demonstrate that Tep4 is strongly upregulated in adult flies following the injection of Photorhabdus bacteria. We also show that Tep4 loss-of-function mutants are resistant to P. luminescens but not to P. asymbiotica infection. In addition, we find that inactivation of Tep4 results in the upregulation of the Toll and Imd immune pathways, and the downregulation of the Jak/Stat and Jnk pathways upon Photorhabdus infection. We document that loss of Tep4 promotes melanization and phenoloxidase activity in the mutant flies infected with Photorhabdus. Together, these findings generate novel insights into the immune role of TEP4 as a regulator and effector of the D. melanogaster antibacterial immune response.

The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila.

BACKGROUND: Molecular and genetic studies in model organisms have recently revealed a dynamic interplay between immunity and ageing mechanisms. In the fruit fly Drosophila melanogaster, inhibition of the insulin/insulin-like growth factor signaling pathway prolongs lifespan, and mutations in the insulin receptor substrate Chico extend the survival of mutant flies against certain bacterial pathogens. Here we investigated the immune phenotypes, immune signaling activation and immune function of chico mutant adult flies against the virulent insect pathogen Photorhabdus luminescens as well as to non-pathogenic Escherichia coli bacteria. RESULTS: We found that D. melanogaster chico loss-of-function mutant flies were equally able to survive infection by P. luminescens or E. coli compared to their background controls, but they contained fewer numbers of bacterial cells at most time-points after the infection. Analysis of immune signaling pathway activation in flies infected with the pathogenic or the non-pathogenic bacteria showed reduced transcript levels of antimicrobial peptide genes in the chico mutants than in controls. Evaluation of immune function in infected flies revealed increased phenoloxidase activity and melanization response to P. luminescens and E. coli together with reduced phagocytosis of bacteria in the chico mutants. Changes in the antibacterial immune function in the chico mutants was not due to altered metabolic activity. CONCLUSIONS: Our results indicate a novel role for chico in the regulation of the antibacterial immune function in D. melanogaster. Similar studies will further contribute to a better understanding of the interconnection between ageing and immunity and lead to the identification and characterization of the molecular host components that modulate both important biological processes.

Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.

BACKGROUND: Symbiotic interactions between microbes and animals are common in nature. Symbiotic organisms are particularly common in insects and, in some cases, they may protect their hosts from pathogenic infections. Wolbachia and Spiroplasma endosymbionts naturally inhabit various insects including Drosophila melanogaster fruit flies. Therefore, this symbiotic association is considered an excellent model to investigate whether endosymbiotic bacteria participate in host immune processes against certain pathogens. Here we have investigated whether the presence of Wolbachia alone or together with Spiroplasma endosymbionts in D. melanogaster adult flies affects the immune response against the virulent insect pathogen Photorhabdus luminescens and against non-pathogenic Escherichia coli bacteria. RESULTS: We found that D. melanogaster flies carrying no endosymbionts, those carrying both Wolbachia and Spiroplasma, and those containing Wolbachia only had similar survival rates after infection with P. luminescens or Escherichia coli bacteria. However, flies carrying both endosymbionts or Wolbachia only contained higher numbers of E. coli cells at early time-points post infection than flies without endosymbiotic bacteria. Interestingly, flies containing Wolbachia only had lower titers of this endosymbiont upon infection with the pathogen P. luminescens than uninfected flies of the same strain. We further found that the presence of Wolbachia and Spiroplasma in D. melanogaster up-regulated certain immune-related genes upon infection with P. luminescens or E. coli bacteria, but it failed to alter the phagocytic ability of the flies toward E. coli inactive bioparticles. CONCLUSION: Our results suggest that the presence of Wolbachia and Spiroplasma in D. melanogaster can modulate immune signaling against infection by certain insect pathogenic and non-pathogenic bacteria. Results from such studies are important for understanding the molecular basis of the interactions between endosymbiotic bacteria of insects and exogenous microbes.

An improved method for generating axenic entomopathogenic nematodes.

BACKGROUND: Steinernema carpocapsae are parasitic nematodes that invade and kill insects. The nematodes are mutualistically associated with the bacteria Xenorhabdus nematophila and together form an excellent model to study pathogen infection processes and host anti-nematode/antibacterial immune responses. To determine the contribution of S. carpocapsae and their associated X. nematophila to the successful infection of insects as well as to investigate the interaction of each mutualistic partner with the insect immune system, it is important to develop and establish robust methods for generating nematodes devoid of their bacteria. FINDINGS: To produce S. carpocapsae nematodes without their associated X. nematophila bacteria, we have modified a previous method, which involves the use of a X. nematophila rpoS mutant strain that fails to colonize the intestine of the worms. We confirmed the absence of bacteria in the nematodes using a molecular diagnostic and two rounds of an axenicity assay involving appropriate antibiotics and nematode surface sterilization. We used axenic and symbiotic S. carpocapsae to infect Drosophila melanogaster larvae and found that both types of nematodes were able to cause insect death at similar rates. CONCLUSION: Generation of entomopathogenic nematodes lacking their mutualistic bacteria provides an excellent tool to dissect the molecular and genetic basis of nematode parasitism and to identify the insect host immune factors that participate in the immune response against nematode infections.

Drosophila anti-nematode and antibacterial immune regulators revealed by RNA-Seq.

BACKGROUND: Drosophila melanogaster activates a variety of immune responses against microbial infections. However, information on the Drosophila immune response to entomopathogenic nematode infections is currently limited. The nematode Heterorhabditis bacteriophora is an insect parasite that forms a mutualistic relationship with the gram-negative bacteria Photorhabdus luminescens. Following infection, the nematodes release the bacteria that quickly multiply within the insect and produce several toxins that eventually kill the host. Although we currently know that the insect immune system interacts with Photorhabdus, information on interaction with the nematode vector is scarce. RESULTS: Here we have used next generation RNA-sequencing to analyze the transcriptional profile of wild-type adult flies infected by axenic Heterorhabditis nematodes (lacking Photorhabdus bacteria), symbiotic Heterorhabditis nematodes (carrying Photorhabdus bacteria), and Photorhabdus bacteria alone. We have obtained approximately 54 million reads from the different infection treatments. Bioinformatic analysis shows that infection with Photorhabdus alters the transcription of a large number of Drosophila genes involved in translational repression as well in response to stress. However, Heterorhabditis infection alters the transcription of several genes that participate in lipidhomeostasis and metabolism, stress responses, DNA/protein synthesis and neuronal functions. We have also identified genes in the fly with potential roles in nematode recognition, anti-nematode activity and nociception. CONCLUSIONS: These findings provide fundamental information on the molecular events that take place in Drosophila upon infection with the two pathogens, either separately or together. Such large-scale transcriptomic analyses set the stage for future functional studies aimed at identifying the exact role of key factors in the Drosophila immune response against nematode-bacteria complexes.

Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals.

In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies.

Intrinsic TGF-ß signaling promotes age-dependent CD8+ T cell polyfunctionality attrition.

Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell-extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-ß1. Furthermore, TGF-ß depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-ß signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-ß1 promote apoptosis of CD8+ effector T cells and high TGF-ß1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-ß levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-ß represents an evolutionarily conserved negative regulator of the immune response in aging organisms.

Implication of microsatellite instability in human gastric cancers.

Microsatellite instability, one of the phenomena implicated in gastric cancer, is mainly associated with the expansion or contraction of microsatellite sequences due to replication errors caused most frequently by mutations in the mismatch repair (MMR) and tumour suppressor genes. Tumours exhibiting microsatellite instability are proven to have truncated products resulting from frequent mutations in mononucleotide or dinucleotide runs in coding and non-coding regions of the targeted genes. Epigenetic changes like hypermethylation of the promoter region of MMR genes as well as gene silencing are also responsible for the microsatellite instability phenotypes. Assessing microsatellite instability in tumours has proved to be an efficient tool for the prognosis of various cancers including colorectal and gastric cancers. Such tumours are characterized by distinct clinicopathological profiles. Biotic agents like Epstein Barr Virus and H. pylori along with other factors like family history, diet and geographical location also play an important role in the onset of gastric carcinogenesis. Instability of mitochondrial DNA has also been investigated and claimed to be involved in the occurrence of gastric cancers in humans. Development of simplified but robust and reproducible microsatellite instability based molecular tools promises efficient prognostic assessment of gastric tumours.

A novel method for infecting Drosophila adult flies with insect pathogenic nematodes.

Drosophila has been established as an excellent genetic and genomic model to investigate host-pathogen interactions and innate immune defense mechanisms. To date, most information on the Drosophila immune response derives from studies that involve bacterial, fungal or viral pathogens. However, immune reactions to insect parasitic nematodes are still not well characterized. The nematodes Heterorhabditis bacteriophora live in symbiosis with the entomopathogenic bacteria Photorhabdus luminescens, and they are able to invade and kill insects. Interestingly, Heterorhabditis nematodes are viable in the absence of Photorhabdus. Techniques for infecting Drosophila larvae with these nematodes have been previously reported. Here, we have developed a method for infecting Drosophila adult flies with Heterorhabditis nematodes carrying (symbiotic worms) or lacking (axenic worms) their associated bacteria. The protocol we present can be readily adapted for studying parasitic strategies of other insect nematodes using Drosophila as the host infection model.