Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

The peptidoglycan recognition protein PGRP-LE regulates the Drosophila immune response against the pathogen Photorhabdus.

Photorhabdus bacteria are potent pathogens of insects and humans. To elucidate the infection strategies Photorhabdus employs to subvert the host innate immune response, it is critical to use model organisms that permit the genetic dissection of the dynamics involved in host-pathogen interactions. Here, we employed the fruit fly Drosophila melanogaster to interrogate the role of the immune deficiency (Imd) pathway receptor peptidoglycan recognition protein LE (PGRP-LE) in the regulation of the fly's response to the insect pathogen Photorhabdus luminescens and the insect/human pathogen P. asymbiotica. We show that PGRP-LE is upregulated in response to injection of Photorhabdus bacteria in background control flies, and that loss-of-function PGRP-LE mutant flies are more sensitive specifically to P. luminescens infection and harbor a higher bacterial burden of this species compared to background controls. Also, our results indicate that the absence of functional PGRP-LE alters the transcriptional pathway activity of Imd and Jnk signaling upon infection with P. asymbiotica, while infection with P. luminescens modifies the activity of Jak/Stat signaling. These findings denote the participation of the PGRP-LE receptor in the response of D. melanogaster to Photorhabdus challenge and contribute to a better understanding of pathogen detection and host immune regulation against virulent microbial invaders.

RNA-seq profiles of putative genes involved in specific immune priming in Bombyx mori haemocytes.

BACKGROUND: The immune system of many invertebrates, including insects, has been shown to comprise memory, or specific immune priming. However, knowledge of the molecular mechanisms especially the candidate immune-related genes mediated the specificity of the immune priming are still very scarce and fragmentary. We therefore used two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) as the priming agents and employed Illumina/Solexa platform to investigate the transcriptional changes of the haemocytes of Bombyx mori larvae after priming. RESULTS: In total, 23.0 Gbp of sequence data and 153,331,564 reads were generated, representing 10,496 genes. Approximately 89% of the genes or sequenced reads could be aligned to the silkworm reference genome. The differentially expressed genes (DEGs) of PBS-vs-TT01 (up-regulated expression of TT01 relative to PBS), PBS-vs-H06 (up-regulated expression of H06 relative to PBS) and TT01-vs-H06 (up-regulated expression of H06 relative to TT01) were 707, 159 and 461 respectively. In addition, expression patterns of 25 selected DEGs derived from quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with their transcript abundance changes obtained by transcriptomic analyses. The DEGs are mainly related to pattern recognition receptors (PRRs), antimicrobial peptides (AMPs), signaling molecular, effector molecules, phagosome and spliceosome, indicating that they have participated in the regulation of the specific immune priming in the B. mori larvae. CONCLUSIONS: The transcriptome profiling data sets from this study will provide valuable resources to better understand the molecular and biological mechanisms regulating the specificity of invertebrates' immune priming. All these will shed light on controlling insect pests or preventing epidemic of infectious diseases in economic invertebrates.

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.

Allatostatin C modulates nociception and immunity in Drosophila.

Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. In this study, we investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, we found robust upregulation of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. We further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, we demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. We posit that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation.

Differential immunosuppression by inhibiting PLA2 affects virulence of Xenorhabdus hominickii and Photorhabdus temperata temperata.

Immunity negatively influences bacterial pathogenicity. Eicosanoids mediate both cellular and humoral immune responses in insects. This study tested a hypothesis that differential bacterial virulence of Xenorhabdus/Photorhabdus is dependent on their inhibitory activity against phospholipase A2 (PLA2) activity. P. temperata subsp. temperata ('Ptt') was more than 40 times more potent than X. hominickii ('Xh'). Although both bacteria suppressed cellular immune responses, Ptt infection suppressed hemocyte nodule formation much more than Xh infection. Their differential immunosuppression appeared to be induced by their secondary metabolites because organic extracts of Ptt-cultured broth exhibited higher inhibitory activities against cellular immune responses than Xn-cultured broth extracts. Humoral immune responses were analyzed by measuring expression levels of 11 antimicrobial peptide (AMP) genes. Among inducible AMPs in hemocytes and fat body, higher number and more kinds of AMPs exhibited lower expression levels in Ptt infection than those in Xh infection. Suppressed immune responses induced by Ptt or Xh infection were significantly rescued by the addition of a catalytic product of PLA2, suggesting that PLA2 was a common inhibitory target. In fact, Ptt infection inhibited PLA2 activity more strongly than Xh infection. RNA interference of a PLA2 gene decreased its expression and significantly increased bacterial virulence. Moreover, addition of PLA2 inhibitor to Xh infection enhanced its virulence, similar to virulence level of Ptt infection. These results suggest that variation in Xenorhabdus/Photorhabdus bacterial virulence can be explained by their differential inhibitory activities against host insect PLA2.

Changes in Caenorhabditis elegans gene expression following exposure to Photorhabdus luminescens strain TT01.

Photorhabdus bacteria enter into a mutualistic symbiosis with Heterorhabditis nematodes to infect insect larvae. However, they rapidly kill the model nematode Caenorhabditis elegans. One hypothesis for these divergent outcomes is that the nematode defense responses differ. To begin testing this hypothesis, we have systematically analyzed available data on the transcriptional response of C. elegans to P. luminescens strain Hb. From a starting pool of over 7000 differentially expressed genes, we carefully chose 21 Heterorhabditis-conserved genes to develop as comparative markers. Using newly designed and validated qRT-PCR primers, we measured expression of these genes in C. elegans exposed to the sequenced TT01 strain of P. luminescens, on two different media types. Almost all (18/21) of the genes showed a significant response to P. luminescens strain TT01. One response is dependent on media type, and a subset of genes may respond differentially to distinct strains. Overall, we have established useful resources and generated new hypotheses regarding how C. elegans responds to P. luminescens infection.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

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.

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.

Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses.

Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1×10(4), 1×10(5) or 1×10(6) cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heat-killed P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response.

A Novel Formulation of Bacillus thuringiensis for the Control of Brassica Leaf Beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

Cabbage is a major vegetable crop over the world. Various insect pests can affect cabbage production. Excessive spray of chemical insecticides can lead to the development of insecticide resistance with various adverse effects on the environment and humans. Brassica leaf beetle, Phaedon brassicae Baly, is a coleopteran pest. Both larvae and adults cause damages to cabbage. The objective of this study was to develop an effective microbial insecticide against P. brassicae by adding an immunosuppressive agent to Bacillus thuringiensis (Bt). The immunosuppressive agent was chosen from bacterial cultured broth of Photorhabdus temperata subsp. temperata (Ptt). Reverse phase HPLC revealed that Ptt-cultured broth possessed at least two eicosanoid biosynthesis inhibitors (oxindole and indole) in its hexane extract. The bacterial cultured broth exhibited potent immunosuppressive activity against P. brassicae. Based on toxicity results, B. thuringiensis subsp. tenebrionis (BtT) was selected from four strains of Bts. When Ptt-cultured broth was added to spore-producing BtT cells, the insecticidal activities of BtT against both larvae and adults of P. brassicae were significantly increased. This bacterial mixture applied to develop a "Bt-Plus," which was formulated by mixing BtT cells (10(11) spores per ml) and 48-h Ptt-cultured broth along with additives (surfactant and preservative). When Bt-Plus was sprayed to cabbage infested by P. brassicae at 1,000-fold dilution, the mixture exhibited much higher control efficacy than BtT treatment alone, suggesting it could be used as a novel Bt insecticide for the control of P. brassicae.

No evidence for priming response in Galleria mellonella larvae exposed to toxin protein PirA2B2 from Photorhabdus luminescens TT01: An association with the inhibition of the host cellular immunity.

There is accumulating evidence that many invertebrates including insects can acquire enhanced immune protection against subsequently pathogens infection through immune priming. However, whether the toxin protein from pathogenic bacteria can induce such priming response remains unclear. Here we cloned, expressed and purified the toxin Photorhabdus insect-related proteins A2B2 (PirA2B2) from Photorhabdus luminescens TT01. We primed Galleria mellonella with sublethal dose of PirA2B2 and then challenged the larvae with viable P. luminescens TT01 at 48 h after priming. We found no evidence for immune priming in G. mellonella larvae exposed to PirA2B2. Priming the larvae with PirA2B2 did not improve their resistance in a subsequent challenge with P. luminescens TT01. Whereas a robust priming response was observed when the larvae exposed to lipopolysaccharide (LPS) extracted from P. luminescens TT01. Because the larvae primed with LPS showed significant higher resistance against P. luminescens TT01 infection than those of the PBS and BSA controls. Furthermore, we investigated the changes of the cellular immune parameters, such as hemocyte counts, phagocytic activity and encapsulation ability of the hemocytes, after priming. We found that the toxin PirA2B2 significantly decreased the cellular immunity of the larvae, whereas the LPS significantly increased them. These results indicated that the degree of priming response in G. mellonella correlated positively to the levels of cellular immune parameters, and the underlying mechanism in regulating the immune priming of invertebrates was not homologous to that of the immunological memory of vertebrates.

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.

The lipopolysaccharide (LPS) of Photorhabdus luminescens TT01 can elicit dose- and time-dependent immune priming in Galleria mellonella larvae.

In this work, we primed Galleria mellonella larvae by haemocoel injection of lipopolysaccharide (LPS) extracted from Photorhabdus luminescens TT01 to determine whether bacterial LPS can induce enhanced immune protection (recently called immune priming). We also analyzed the relationship between changes in the levels of innate immune elements and the degree of enhanced immune protection in the larvae at designated time points after priming. The larvae that received experimental doses (20.0, 10.0 and 5.0µg per larva) of LPS demonstrated increased resistance against lethal challenge with P. luminescens TT01; the degree and period of protection correlated positively with the priming dose. These results indicated that the P. luminescens TT01 LPS could induce typical immune priming in G. mellonella. Moreover, the levels of innate immune parameters (i.e. haemocyte density, phagocytosis, haemocyte encapsulation ability, and antibacterial activity of cell-free haemolymph) and endogenous enzyme activities (i.e. acid phosphatase, ACP; alkaline phosphatase, AKP; superoxide dismutase, SOD and lysozyme, LSZ) were significantly increased following priming of the larvae with LPS, whereas the activities of peroxidase (POD) and catalase (CAT) were significantly decreased. All of the parameters examined changed in a dose- and time-dependent manner. This study demonstrated that G. mellonella larvae could modulate their immune responses based on different doses of LPS used for priming, and that priming phenomenon in G. mellonella larvae elicited by LPS was mediated by the innate immune elements and enzyme activity.

Structure of a zwitterionic O-polysaccharide from Photorhabdus temperata subsp. cinerea 3240.

A phosphorylated O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. cinerea 3240 and studied by sugar analysis, dephosphorylation, and (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit of the O-polysaccharide was established: →3)-ß-D-GalpNAc4PEtN-(1→4)-ß-D-GlcpA-(1→3)-ß-D-FucpNAc4N-(1→ where GlcA indicates glucuronic acid, FucNAc4N 2-acetamido-4-amino-2,4,6-trideoxygalactose, and PEtN 2-aminoethyl phosphate.

Nitric oxide levels regulate the immune response of Drosophila melanogaster reference laboratory strains to bacterial infections.

Studies on the innate immune response against microbial infections in Drosophila melanogaster involve mutant strains and their reference strains that act as experimental controls. We used five standard D. melanogaster laboratory reference strains (Oregon R, w1118, Canton-S, Cinnabar Brown, and Yellow White [YW]) and investigated their response against two pathogenic bacteria (Photorhabdus luminescens and Enterococcus faecalis) and two nonpathogenic bacteria (Escherichia coli and Micrococcus luteus). We detected high sensitivity among YW flies to bacterial infections and increased bacterial growth compared to the other strains. We also found variation in the transcription of certain antimicrobial peptide genes among strains, with Oregon and YW infected flies showing the highest and lowest gene transcription levels in most cases. We show that Oregon and w1118 flies possess more circulating hemocytes and higher levels of phenoloxidase activity than the other strains upon infection with the nonpathogenic bacteria. We further observed reduced fat accumulation in YW flies infected with the pathogenic bacteria, which suggests a possible decline in physiological condition. Finally, we found that nitrite levels are significantly lower in infected and uninfected YW flies compared to w1118 flies and that nitric oxide synthase mutant flies in YW background are more susceptible to bacterial infection compared to mutants in w1118 background. Therefore, increased sensitivity of YW flies to bacterial infections can be partly attributed to lower levels of nitric oxide. Such studies will significantly contribute toward a better understanding of the genetic variation between D. melanogaster reference strains.

Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.

f57f4.4p::gfp as a fluorescent reporter for analysis of the C. elegans response to bacterial infection.

Host defense mechanisms are multi-layered and involve constitutive as well as inducible components. The dissection of these complex processes can be greatly facilitated using a reporter gene strategy with a transparent animal. In this study, we use Caenorhabditis elegans as a model host and introduce a new pathogen-inducible fluorescent reporter involving the promoter of f57f4.4, a gene encoding a putative component of the glycocalyx. We show that this reporter construct does not respond to heavy metal or hypertonic environments, but is specifically and locally induced in the intestine upon Photorhabus luminescens and Pseudomonas aeruginosa infections. We further demonstrate that its upregulation requires live pathogens as well as elements of the nematode p38 MAP kinase and TGF-beta pathways. In addition to introducing a new tool for the study of the interactions between C. elegans and a pathogen, our results suggest a role for the glycocalyx in gut immunity.

Vitellogenins increase stress resistance of Caenorhabditis elegans after Photorhabdus luminescens infection depending on the steroid-signaling pathway.

Resistance against environmental stress is a crucial factor in determining the lifespan of organisms. A central role herein has been recently attributed to the transport and storage of lipids with the vitellogenin family emerging as a potential key factor. Here we show that the knockdown of one out of five functional vitellogenin genes, encoding apolipoprotein B homologues, results in a reduced survival of the nematode Caenorhabditis elegans at 37 °C subsequent to infection with the bacterial pathogen Photorhabdus luminescens. An active steroid-signaling pathway, including supply of cholesterol by vitellogenins, steroid ligand formation by the cytochrome P450 dependent DAF-9, and activation of the nuclear hormone receptor DAF-12, in the presence of pathogenic bacteria was associated with reduced nuclear translocation of the forkhead transcription factor DAF-16 and increased antioxidative capacity. Taken together, the study provides functional evidence for a crucial role of vitellogenins and the steroid-signaling pathway in determination of resistance against bacteria.