Partner-specific induction of Spodoptera frugiperda immune genes in response to the entomopathogenic nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophila.

The Steinernema carpocapsae-Xenorhabdus nematophila association is a nematobacterial complex used in biological control of insect crop pests. The infection success of this dual pathogen strongly depends on its interactions with the host's immune system. Here, we used the lepidopteran pest Spodoptera frugiperda to analyze the respective impact of each partner in the induction of its immune responses. First, we used previously obtained RNAseq data to construct the immunome of S. frugiperda and analyze its induction. We then selected representative genes to study by RT-qPCR their induction kinetics and specificity after independent injections of each partner. We showed that both X. nematophila and S. carpocapsae participate in the induction of stable immune responses to the complex. While X. nematophila mainly induces genes classically involved in antibacterial responses, S. carpocapsae induces lectins and genes involved in melanization and encapsulation. We discuss putative relationships between these differential inductions and the pathogen immunosuppressive strategies.

Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production.

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15-30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.

Identification of an entomopathogenic bacterium, Xenorhabdus ehlersii KSY, from Steinernema longicaudum GNUS101 and its immunosuppressive activity against insect host by inhibiting eicosanoid biosynthesis.

Steinernema longicaudum GNUS101, an entomopathogenic nematode, was isolated from soils in Korea. Its internal transcribed space sequence was highly similar to the known S. longicaudum species. Infective juveniles (IJs) of S. longicaudum were highly virulent to lepidopteran and coleopteran insects. Two different bacteria were isolated from the hemolymph of lepidopteran larvae infected with S. longicaudum. They exhibited blue and red colonies on nutrient bromothymol blue agar. The red-colored bacterium was identified as Enterococcus mundtii KHY while the blue-colored bacterium was identified as Xenorhabdus ehlersii KSY based on 16S rRNA sequencing and biochemical characters. The bacterial species showed different growth rates, with X. ehlersii KSY growing more slowly than E. mundtii KHY. Both bacteria were entomopathogenic, but showed differences in suppressing host immune responses. X. ehlersii KSY, but not E. mundtii KHY, showed inhibitory activity against cellular immune responses of Spodoptera exigua larvae including hemocyte-spreading behavior and nodule formation in bacteria-cultured broth. Its immunosuppressive activity was reversed by adding arachidonic acid, an eicosanoid biosynthesis precursor. Furthermore, organic extracts of X. ehlersii KSY using hexane or ethyl acetate showed inhibitory activity against cellular immune responses of S. exigua larvae. Arachidonic acid addition to S. exigua larvae infected with X. ehlersii significantly rescued the survival rate of target insect. Of the two bacteria isolated from S. longicaudum GNUS101, only X. ehlersii induced immunosuppression of target insect by inhibiting eicosanoid biosynthesis.

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.

Nitric Oxide Mediates Insect Cellular Immunity via Phospholipase A2 Activation.

After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids, and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Separate treatments with an insect cytokine, 2 biogenic amines, and an eicosanoid lead to a single result, hemocyte spreading, understood in terms of intracellular cross-talk among these signaling systems. This study focuses on the cross-talk between NO and eicosanoid signaling in our model insect, Spodoptera exigua. Bacterial injection increased NO concentrations in the larval hemocytes and fat body, and RNA interference (RNAi) of the S. exigua NO synthase (NOS) gene suppressed NO concentrations. RNAi treatment also led to a significant reduction in hemocyte nodulation following bacterial injection. Similar RNAi treatments led to significantly reduced PLA2 activities in the hemocytes and fat body compared to control larvae. Injection of L-NAME also prevented the induction of PLA2 activity following bacterial challenge. An injected NO donor, S-nitroso-N-acetyl-DL-penicillamine, increased PLA2 activity in a dose-dependent manner. However, eicosanoids did not influence NO concentrations in immune-challenged larvae. We infer that NO and eicosanoid signaling operate via cross-talk mechanisms in which the elevated NO concentrations activate PLA2 and eicosanoid biosynthesis, which finally mediates various immune responses.

A functional genomic screen for evolutionarily conserved genes required for lifespan and immunity in germline-deficient C. elegans.

The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species that diverged from C. elegans 250-400 MYA.

An entomopathogenic bacterium, Xenorhabdus nematophila, suppresses expression of antimicrobial peptides controlled by Toll and Imd pathways by blocking eicosanoid biosynthesis.

Immune-associated genes of the beet armyworm, Spodoptera exigua, were predicted from 454 pyrosequencing transcripts of hemocytes collected from fifth instar larvae challenged with bacteria. Out of 22,551 contigs and singletons, 36% of the transcripts had at least one significant hit (E-value cutoff of 1e-20) and used to predict immune-associated genes implicated in pattern recognition, prophenoloxidase activation, intracellular signaling, and antimicrobial peptides (AMPs). Immune signaling and AMP genes were further confirmed in their expression patterns in response to different types of microbial challenge. To discriminate the AMP expression signaling between Toll and Imd pathways, RNA interference was applied to specifically knockdown each signal pathway; the separate silencing treatments resulted in differential suppression of AMP genes. An entomopathogenic bacterium, Xenorhabdus nematophila, suppressed expression of most AMP genes controlled by Toll and Imd pathways, while challenge with heat-killed X. nematophila induced expression of all AMPs in experimental larvae. Benzylideneacetone (BZA), a metabolite of X. nematophila, suppressed the AMP gene inductions when it was co-injected with the heat-killed X. nematophila. However, arachidonic acid, a catalytic product of PLA2 , significantly reversed the inhibitory effect of BZA on the AMP gene expression. This study suggests that X. nematophila suppresses AMP production controlled by Toll and Imd pathways by inhibiting eicosanoid biosynthesis in S. exigua.

An insecticidal protein from Xenorhabdus ehlersii stimulates the innate immune response in Galleria mellonella.

The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxic proteins that interfere with the immune system of insects. Recently, we purified the insecticidal protein XeGroEL from Xenorhabdus ehlersii and discovered that injection of XeGroEL into larvae of Galleria mellonella triggers strong immune responses. In this study, we determined the level of induction of several immune-responsive proteins that were secreted into the hemolymph using comparative proteomic analyses of hemolymph proteins from XeGroEL-challenged larvae. Additionally, quantitative real-time reverse transcription-PCR analyses demonstrated increased transcriptional rates of immune-related genes at 5 h post-challenge with purified XeGroEL. Our results help to understand anti-microbial immune responses in G. mellonella, suggesting that the immune system recognizes exogenous proteins and pathogen-associated molecular patterns.

Role of D535 and H538 in endogenous toxicity of xenocin from Xenorhabdus nematophila.

Bacteriocins are the toxic proteins produced by bacteria under stress condition to inhibit the growth of closely related bacterial strain(s). In our earlier study, purified recombinant xenocin-immunity protein complex from Xenorhabdus nematophila showed detrimental effect on six different insect gut residing bacteria. In this study, endogenous toxicity assay with xcinA and its catalytic domain under tightly regulated ara promoter was performed. Multiple sequence alignment and homology modelling revealed six conserved amino acid residues in the catalytic domain of xenocin. Site-directed mutagenesis was performed in all the conserved residues, followed growth profile analysis of all the mutants by endogenous toxicity assay. Among the six different conserved sites in catalytic domain of xenocin, we have identified one position where mutation resulted in no measurable reduction in the endogenous toxicity (K564), three positions with measurable reduction in the endogenous toxicity (E542, H551 and R570) and two positions where mutation caused a significant reduction in the toxicity (D535 and H538). Endogenous toxicity assay is validated by in vitro RNA degradation assay. Structural integrity of purified recombinant proteins was confirmed through circular dichroism and fluorescence spectroscopy. Our results indicate that D535 and H538 act as the acid-base pair for RNA hydrolysis.

Small molecule perimeter defense in entomopathogenic bacteria.

Two gram-negative insect pathogens, Xenorhabdus nematophila and Photorhabdus luminescens, produce rhabduscin, an amidoglycosyl- and vinyl-isonitrile-functionalized tyrosine derivative. Heterologous expression of the rhabduscin pathway in Escherichia coli, precursor-directed biosynthesis of rhabduscin analogs, biochemical assays, and visualization using both stimulated Raman scattering and confocal fluorescence microscopy established rhabduscin's role as a potent nanomolar-level inhibitor of phenoloxidase, a key component of the insect's innate immune system, as well as rhabduscin's localization at the bacterial cell surface. Stimulated Raman scattering microscopy visualized rhabduscin at the periphery of wild-type X. nematophila cells and E. coli cells heterologously expressing the rhabduscin pathway. Precursor-directed biosynthesis created rhabduscin mimics in X. nematophila pathway mutants that could be accessed at the bacterial cell surface by an extracellular bioorthogonal probe, as judged by confocal fluorescence microscopy. Biochemical assays using both wild-type and mutant X. nematophila cells showed that rhabduscin was necessary and sufficient for potent inhibition (low nM) of phenoloxidases, the enzymes responsible for producing melanin (the hard black polymer insects generate to seal off microbial pathogens). These observations suggest a model in which rhabduscin's physical association at the bacterial cell surface provides a highly effective inhibitor concentration directly at the site of phenoloxidase contact. This class of molecules is not limited to insect pathogens, as the human pathogen Vibrio cholerae also encodes rhabduscin's aglycone, and bacterial cell-coated immunosuppressants could be a general strategy to combat host defenses.

Cecropins as a marker of Spodoptera frugiperda immunosuppression during entomopathogenic bacterial challenge.

An antimicrobial peptide (AMP) of the cecropin family was isolated by HPLC from plasma of the insect pest, Spodoptera frugiperda. Its molecular mass is 3910.9 Da as determined by mass spectrometry. Thanks to the EST database Spodobase, we were able to describe 13 cDNAs encoding six different cecropins which belong to the sub-families CecA, CecB, CecC and CecD. The purified peptide identified as CecB1 was chemically synthesized (syCecB1). It was shown to be active against Gram-positive and Gram-negative bacteria as well as fungi. Two closely related entomopathogenic bacteria, Xenorhabdus nematophila F1 and Xenorhabdus mauleonii VC01(T) showed different susceptibility to syCecB1. Indeed, X. nematophila was sensitive to syCecB1 whereas X. mauleonii had a minimal inhibitory concentration (MIC) eight times higher. Interestingly, injection of live X. nematophila into insects did not induce the expression of AMPs in hemolymph. This effect was not observed when this bacterium was heat-killed before injection. On the opposite, both live and heat-killed X. mauleonii induced the expression of AMPs in the hemolymph of S. frugiperda. The same phenomenon was observed for another immune-related protein lacking antimicrobial activity. Altogether, our data suggest that Xenorhabdus strains have developed different strategies to supplant the humoral defense mechanisms of S. frugiperda, either by increasing their resistance to AMPs or by preventing their expression during such host-pathogen interaction.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.

Imd pathway is involved in the interaction of Drosophila melanogaster with the entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens.

Xenorhabdus nematophila/Steinernema carpocapsae and Photorhabdus luminescens/Heterorhabditis bacteriophora are nemato-bacterial complexes highly pathogenic for insects. Using a syringe as artificial vector, we have analyzed the effects of the two bacteria, X. nematophila and P. luminescens on the genetic tool insect, Drosophila melanogaster. Both bacteria were found to kill adult flies in a dose dependent manner with X. nematophila being the fastest. On the other hand, when an injection of non-pathogenic bacteria, Escherichia coli, is performed 1 day before challenge with the entomopathogenic bacteria, then the survival of Drosophila flies was prolonged by at least 20h. After injection of entomopathogenic bacteria, Drosophila mutant Dif(1), affected on the Toll pathway, showed a similar phenotype than wild-type flies whereas Drosophila mutant Dredd(D55), affected on the imd pathway, was not protected by a prior injection of E. coli. This suggested that members of the imd pathway might be targets of these entomopathogenic bacteria albeit synthesis of antimicrobial peptides through this signaling pathway was induced by X. nematophila as well as P. luminescens. Finally, P. luminescens phoP mutant, an avirulent mutant in the Lepidopteran insect, Spodoptera littoralis, was found poorly virulent for D. melanogaster. phoP mutant partially protected D. melanogaster flies if injected 1 day before the injection of P. luminescens wild-type TT01 to the same extent than the E. coli-induced protection. However, phoP recovered a level of pathogenicity comparable to P. luminescens wild-type TT01 when injected to Drosophila flies affected on the imd pathway.

Transcriptional analysis and functional characterization of a gene pair encoding iron-regulated xenocin and immunity proteins of Xenorhabdus nematophila.

We describe a two-gene cluster encoding a bacteriocin, xenocin, and the cognate immunity protein in the insect-pathogenic bacterium Xenorhabdus nematophila, which infects and kills larval stages of the common crop pest Helicoverpa armigera. The two genes, xcinA and ximB, are present in the genome as a single transcriptional unit, which is regulated under SOS conditions. The stress-inducible promoter was activated by mitomycin C, glucose, and Fe(3+) depletion and at an elevated temperature when it was tested in Escherichia coli cells. Expression of the xenocin protein alone in E. coli inhibited the growth of this organism. The growth inhibition was abolished when the immunity protein was also present. A recombinant xenocin-immunity protein complex inhibited the growth of E. coli indicator cells when it was added exogenously to a growing culture. Xenocin is an endoribonuclease with an enzymatically active C-terminal domain. Six resident bacterial species (i.e., Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas species) from the H. armigera gut exhibited sensitivity to recombinant xenocin when the organisms were grown under iron-depleted conditions and at a high temperature. Xenocin also inhibited the growth of two Xenorhabdus isolates. This study demonstrates that Fe(3+) depletion acts as a common cue for synthesis of xenocin by X. nematophila and sensitization of the target strains to the bacteriocin.

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph.

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2).

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.

A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions.

We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naive or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50 to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression.

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.

Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens.

Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.

Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses.

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.