Virulent Secondary Metabolites of Entomopathogenic Bacteria, Xenorhabdus and Photorhabdus, Inhibit PLA2 to Suppress Host Insect Immunity


 Background: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates of Xenorhabdus and Photorhabdus. Results: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. Conclusion: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.


Background
Xenorhabdus and Photorhabdus are entomopathogenic bacteria that exhibit mutualistic symbiosis with entomophagous nematodes Steinernema and Heterorhabditis, respectively [1]. These nematodes can carry and release these bacteria into insect hemocoel by infecting susceptible insect larvae, in which these bacteria can kill insects and convert the cadaver into a food source suitable for nematode growth and development [2]. When nematode population increases to a certain carrying level in a speci c insect host, these nematodes are re-associated with speci c symbiotic bacteria before emerging from the insect cadaver to search for a new host [3]. There are complex chemical communications in tripartite interactions of bacteria-nematode for symbiosis, bacteria-insect for pathogenicity, and nematode-insect for host recognition. Pathogenic interactions between bacteria and susceptible insects have been relatively well studied regarding the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) [4]. However, there are secondary compounds other than NRPS-PKS produced from synthetic machineries of bacteria [5].
Virulence of entomopathogenic bacteria exhibits variations among species and strains [6,7]. Bacteria can secrete several virulence factors in insect hemocoel to suppress insect immune responses and cause fatal septicemia [8,9]. To induce immunosuppression, both bacterial genera commonly inhibit phospholipase A 2 (PLA 2 ) activity of insects [10]. PLA 2 is known to catalyze the release of arachidonic acid from phospholipids, which is a committed step to produce various eicosanoids [11]. Eicosanoids mediate cellular and humoral immune responses against various microbial pathogens in insects [12].
Indeed, X. nematophila can secrete at least eight secondary metabolites to suppress insect immunity by inhibiting PLA 2 [13]. Inter-speci c variations in Xenorhabdus bacterial virulence have been explained by variations in their inhibitory activities against PLA 2 activities [14]. Intra-speci c variations in bacterial virulence of X. nematophila have also been reported and explained by differences in immunosuppression due to differential inhibitory effects on PLA 2 catalytic activity [15]. Park et al. [16] have explained that virulence variations of different bacterial phases are associated with the expression of a speci c virulence factor. This hypothesis is further supported by the nding that the expression level of leucineresponsive protein (Lrp), a global transcriptional factor, can modulate bacterial pathogenicity [17]. Expression levels of Lrp and other transcriptional factors can modulate secondary metabolite production [18], suggesting that the production of secondary metabolites might be positively correlated with bacterial pathogenicity.
Secondary metabolites produced by NRPS and PKS of different species of Xenorhabdus and Photorhabdus are different in their compositions [19]. Different secondary metabolites can inhibit diverse physiological molecules of susceptible insects to induce immunosuppression. For example, rhabducin, an isocyanide-containing compound produced from BGC, can inhibit the activity of phenoloxidase (PO) in Galleria mellonella [20]. More than 70 kinds of rhabdopeptide/xenortide peptides derived from NRPS are structurally similar to protease inhibitors. They might degrade various proteins associated with immunity [21,22]. Phurealipids produced from NRPS/PKS can prevent the expression of antimicrobial peptide genes [23]. Thus, diverse secondary metabolites produced by entomopathogenic bacteria might effectively suppress insect immune responses to induce septicemia.
The aim of this study was to determine virulent secondary metabolites produced by Xenorhabdus or Photorhabdus based on their inhibitory activities against insect PLA 2 . To this end, this study compared virulence and secondary metabolites of 14 different bacterial isolates of Xenorhabdus and Photorhabdus.

Comparative analysis of bacterial extracts for insect immunosuppression
Variations in insecticidal activities among entomopathogenic bacteria might be caused by their differential immunosuppressive capabilities against target insects. To test this hypothesis, effects of bacterial extracts on cellular immune responses were assessed using hemocyte-spreading behaviour and nodulation assays after bacterial infection. All extracts signi cantly (p < 0.05) inhibited the hemocytespreading behavior ( Fig. 2A) and nodulation (Fig. 2B). Immunosuppression was signi cantly (p < 0.05) rescued by the addition of arachidonic acid (a catalytic product of PLA 2 ), suggesting that the immunosuppression was caused by the inhibition of PLA 2 . However, inhibitory activities of these bacterial extracts were different among bacterial species. When bacterial insecticidal activities were compared with immunosuppressive activities, these two parameters showed high positive correlations for all extracts (Fig. 2C).
To determine whether immunosuppressive activities of these bacterial extracts were caused by inhibition of insect PLA 2 , these extracts were incubated with PLA 2 extract obtained from S. exigua hemocytes. All bacterial extracts signi cantly inhibited sPLA 2 activity, although they showed variations in inhibiting sPLA 2 (Fig. 3A). For cPLA 2 , some extracts failed to inhibit its enzyme activity (Fig. 3B). Compared to HEX and CX, EAX and BX appeared to show higher inhibitory activities. Ptt and Xh extracts showed signi cantly higher inhibitory activities than the other bacterial extracts. There were positive correlations between bacterial insecticidal activities and PLA 2 inhibition except HEX against cPLA 2 (Fig. 3C).

Prediction of virulent secondary metabolites produced by Xenorhabdus and Photorhabdus
Immunosuppressive activities of bacterial extracts suggested that their secondary metabolites contained inhibitory compounds. To predict functional secondary metabolites, all extracts were assessed by TLC to con rm the presence of compounds. These compounds were then analyzed by GC-MS (Fig. S1). Predicted compounds of four extracts for each bacterial species were combined and resulting 182 compounds were compared between bacterial isolates (Table S2). In addition to different compositions among bacterial isolates, the total number of secondary metabolites varied from 32 compounds of X. nematophila SK1 to 63 compounds of Ptt. Interestingly, there was a signi cant (p < 0.05) negative correlation between the total number of secondary metabolites and target insect immune responses measured by nodulation, sPLA 2 activity, or cPLA 2 (Fig. 4A). In addition, if bacteria had more secondary metabolites, they exhibited higher insecticidal activities.
To select virulent bacterial compounds, two most potent bacteria, Ptt and Xh, were compared with the least potent Xe for their secondary metabolites (Fig. 4B). Ptt and Xh shared over 44% metabolites. Almost 82% metabolites of Xe were detected in the culture broth of Ptt or Xh. Thus, Ptt-(29 compounds) or Xh-(23 compounds) speci c compounds and overlapping (17 compounds) compounds were chosen as possible virulent compounds (Table 3). Validation of virulent secondary metabolites for their immunosuppression and insecticidal activities Twelve compounds were selected from 70 virulent candidates produced by Ptt or Xh (Table S3). As a reference, one compound (2-mercaptobenzothiazole: 'MT') was randomly selected from metabolites produced by Xe as well as Ptt and Xh. All 12 virulent compounds highly suppressed hemocytic nodulation in response to bacterial infection (Fig. 5A). MT also suppressed immune responses, although it exhibited much lower inhibitory activity than those 12 virulent compounds. Compounds that showed the highest inhibitory activities were indole ('IND'), ethoxymethoxyphenol ('EMP'), and dimethylhydroxyfuranone ('DHF'). These twelve virulent compounds signi cantly (p < 0.05) inhibited sPLA 2 activity (Fig. 5B) and cPLA 2 activity (Fig. 5C) whereas MT did not. IND, EMP, and DHF also showed high inhibitory activities against PLA 2 . These twelve virulent compounds also showed high insecticidal activities (Table 3). In contrast, MT had relatively low toxicities to the test insect species. However, IND, EMP, and DHF exhibited high insecticidal activities, with LD 50 ranging from 4.29 to 5.12 µg/larva.

Discussion
Bacterial species in genera Xenorhabdus and Photorhabdus can produce various secondary metabolites to maintain their mutualistic symbiosis with host entomopathogenic nematodes [24]. Some of metabolites are required for their colonization to speci c nematode hosts while others are crucial for pathogenesis speci cally targeting insect's immune system [3]. This study assessed all metabolites extracted with four organic solvents from bacterial culture broth of Xenorhabdus and Photorhabdus to identify their virulent secondary metabolites against insect immune responses.
Organic extracts of bacterial culture broth of seven different species classi ed in Xenorhabdus and Photorhabdus showed immunosuppressive and insecticidal activities. However, pathogenic activities of these extracts were different among 14 bacterial isolates and highly correlated with their bacterial insecticidal activities. These results suggest that these bacteria can induce immunosuppression of target insects via their secondary metabolites, supporting the hypothesis that immunosuppression is required for bacterial pathogenicity. Protein toxins that can kill target insects are known in Xenorhabdus and Photorhabdus [25]. However, acute attack by a target insect's immune system should be avoided and actively suppressed by the secondary metabolites [26].
Immunosuppression of target insects by Xenorhabdus and Photorhabdus infection is caused by inhibition of PLA 2 by bacterial secondary metabolites [10]. The rst identi ed PLA 2 inhibitor produced by bacteria was benzylideneacetone (BZA) produced by X. nematophila [27]. Subsequently, eight different PLA 2 inhibitors were identi ed from bacterial culture broths of X. nematophila and P. temperata temperata [13]. On the other hand, these PLA 2 inhibitors induced apoptosis-associated cytotoxicity and insecticidal activities [28]. This explains why bacterial metabolites containing PLA 2 inhibitors in our current study showed insecticidal activities. Indeed, the degree of PLA 2 inhibition by bacteria has been found to be highly correlated with bacterial pathogenicity [15]. These ndings suggested that bacterial metabolites that inhibited PLA 2 activities in this current study contained virulent secondary metabolites.
Virulent secondary metabolites produced by Xenorhabdus and Photorhabdus were chosen based on comparative analysis of secondary metabolites among bacterial isolates. GC-MS analyses for bacterial culture broths of 14 isolates predicted 182 compounds. Most of them were phenol derivatives, tryptophan derivatives, peptides, and fatty acid derivatives presumably synthesized by NRPS-PKS [4]. In general, predicted and identi ed compounds from NRPS-PKS of Xenorhabdus and Photorhabdus genomes are associated with bacterial pathogenicity [19]. This is supported by the high correlation between the number of predicted compounds in each bacterial isolate and bacterial pathogenicity found in the current study. This also suggests that these compounds predicted by GC-MS might contain virulent secondary metabolites. To select virulent compounds, predicted metabolites of highly pathogenic bacterial isolates (Ptt and Xh) were compared with those of a low pathogenic isolate (Xe

Conclusions
Bacterial isolates of Xenorhabdus and Photorhabdus were pathogenic by suppressing insect immune responses via PLA 2 inhibition using their secondary metabolites. Such immunosuppression was highly correlated with insecticidal activity of bacteria. Based on this correlation, 70 virulent compounds were predicted by comparative analysis of secondary metabolites between high and low pathogenic bacteria.

Insect and bacteria
Larvae of Spodoptera exigua were reared at temperature of 25 ± 2 o C and relative humidity of 65 ± 5% under 16 h light and 8 h dark condition with an arti cial diet [29]. Under these conditions, larvae underwent ve instars (L1-L5). Adults were provided with 10% sucrose solution. For bioassays to determine bacterial pathogenicity, larvae were taken from a cohort.

Hemocyte-spreading behavior assay
Hemolymph (~ 250 µL) was collected from L5 larvae of S. exigua by cutting prolegs. It was mixed with 750 µL ACB. The hemolymph suspension was centrifuged at 800 × g for 5 min and 800 µL supernatant was mixed with lter-sterilized 200 µL TC100 insect culture medium (Hyclone, Daegu, Korea). This hemocyte suspension (9 µL) and each bacterial metabolite (1 µL) were mounted on a glass slide to assess hemocyte-spreading behavior. For rescue experiment with AA (10 µg/µL in DMSO), 8 µL of cell suspension, 1 µL of bacterial metabolite, and 1 µL of AA were placed on a glass slide. After incubating the mixture at room temperature under darkness for 40 min, hemocytes were observed under a phase contrast microscope (Olympus) at 800 × magni cation. As control, the dilution solvent (DMSO) of metabolites was used. Spread hemocytes were recognized based on cytoplasmic extension out of cell boundary. Each treatment was replicated three times with separately prepared suspension mixture. In each replication, 100 hemocytes were randomly chosen to assess hemocyte-spreading behavior.

Nodulation assay
Hemocyte nodule formation was observed using three days old L5 larvae of S. exigua after injecting 3 µL of overnight grown E. coli Top 10 (10 4 cells/larva) and 2 µL of DMSO to the hemocoel through prolegs using a microsyringe (Hamilton, Reno, NV, USA). To assess any inhibitory effect of bacterial extracts or secondary metabolites, 1 µL (1 µg/µL in DMSO) of test sample was co-injected with 3 µL of E. coli and 1 µL of DMSO. For AA rescue experiment, 1 µL of AA (10 µg/µL in DMSO) was co-injected with 1 µL of bacterial metabolite and 3 µL of E. coli. After incubation at 25 o C for 8 h, injected larvae were dissected to count melanized nodules under a stereomicroscope (Stemi SV11, Zeiss, Jena, Germany) at 50 × magni cation. Each treatment was replicated three times using ve larvae per replication. PLA 2 enzyme assay PLA 2 activity was assessed using processes previously described by Vatanparast et al. [34]. Brie y, enzymes were extracted after grinding hemocytes of L5 larvae of S. exigua with PBS followed by centrifugation at 12,500 ⋅ g for 5 min. The resulting supernatant containing the cytosol and membrane mixture was used as enzyme source. The reaction mixture (225 µL) contained 10 µL of PLA 2 enzyme, 10 µL of Ellman's reagent (5,5-di-thio-bis-(2-nitrobenzoicacid)), 5 µL of test inhibitor, and 200 µL of sPLA 2 or cPLA 2 substrate. DMSO was used instead of test inhibitor as control. Change in absorbance of reaction product was measured at wavelength of 405 nm using a microplate reader (Victor, PerkinElmer, Waltham, MA, USA). Each treatment was replicated three times with biologically independent samples.

Bacterial pathogenicity test
To determine insecticidal activities of 14 bacterial isolates, bacterial cells after 24 h of culture were centrifuged at 10,000 × g for 2 min at 4 °C. Cell pellet was re-suspended in PBS for injection. Bacterial suspension was diluted with PBS to obtain different concentrations (0, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 and 10 6 colony-forming unit (cfu)/larva) and injected to hemocoel of L5 S. exigua larvae using a microsyringe. Before injection, larvae were surface sterilized with 70% ethanol. Bacterial dose was measured after plating the suspension onto LB agar medium followed by culturing at 28 o C for 48 h. Mortality was counted at 24 h after bacterial injection. As control bacterial treatment, E. coli was used. Injected larvae were incubated at room temperature with su cient diet. Larvae were considered to be dead if there was no movement upon touching. Each treatment consisted of three replications using 10 larvae for each replication.

Toxicity tests of bacterial metabolites and synthetic compounds
To determine toxicities of bacterial extracts and predicted metabolites, hemocoelic injection was performed. Dried bacterial extracts and metabolites were weighed and dissolved in DMSO to prepare different concentrations (0.001, 0.01, 0.1, 1 and 10 µg/µL) for toxicity assays. A 10 µL microsyringe was used for hemocoelic injection. Each test sample (3 µL) was injected to L5 larvae of S. exigua. DMSO was injected as control. Treated larvae were placed in 9 cm diameter dishes and incubated at room temperature. Mortality was determined every 24 h for 96 h after treatment. Larvae were considered as dead if there was no movement upon touching. Each test concentration was replicated three times using 10 larvae per replication.

Statistical analysis
All assay data for continuous variables were subjected to one-way analysis of variance (ANOVA) using PROG GLM in SAS program [35]. For ANOVA, mortality data were subjected to arcsine transformation. (LSD test). (C) Correlations (r) between insecticidal activities of bacterial extracts (Fig. 1) and their immunosuppressive activities. Lines represent the best-t regression.  (Fig. 1) and their inhibitory activities against PLA2 enzyme. Lines represent the best-t regression.  Validation of virulence activities of 12 virulent candidate secondary metabolites produced by two highly potent bacterial isolates of Photorhabdus temperata subsp. temperata ('Ptt') and Xenorhabdus hominickii ('Xh'). A reference metabolite was selected from compounds produced by a low virulent isolate of X. ehlersii ('Xe'). For virulence tests, suppression of hemocyte nodulation, inhibition of sPLA2, and inhibition of cPLA2 were determined. To induce hemocyte nodulation, 4 × 104 cfu/larva of E. coli was injected to L5 larvae of S. exigua. Bacterial metabolites (10 µg/larvae) were injected to inhibit nodule formation. For each treatment, 15 larvae were used. To measure sPLA2 and cPLA2 enzyme activities, a commercial assay kit was used with PLA2-speci c substrate as described in Materials and methods. As control ('CON'), DMSO was used. Each treatment was independently replicated three times.