Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 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 (species or strains) 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.

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

Xenorhabdus nematophila bacteria shift from mutualistic to virulent Lrp-dependent phenotypes within the receptacles of Steinernema carpocapsae insect-infective stage nematodes.

Xenorhabdus nematophila bacteria are mutualists of Steinernema carpocapsae nematodes and pathogens of insects. Xenorhabdus nematophila exhibits phenotypic variation between insect virulence (V) and the mutualistic (M) support of nematode reproduction and colonization initiation in the infective juvenile (IJ) stage nematode that carries X. nematophila between insect hosts. The V and M phenotypes occur reciprocally depending on levels of the transcription factor Lrp: high-Lrp expressors are M+V- while low-Lrp expressors are V+M-. We report here that variable (wild type) or fixed high-Lrp expressors also are optimized, relative to low- or no-Lrp expressors, for colonization of additional nematode stages: juvenile, adult and pre-transmission infective juvenile (IJ). In contrast, we found that after the bacterial population had undergone outgrowth in mature IJs, the advantage for colonization shifted to low-Lrp expressors: fixed low-Lrp expressors (M-V+) and wild type (M+V+) exhibited higher average bacterial CFU per IJ than did high-Lrp (M+V-) or no-Lrp (M-V-) strains. Further, the bacterial population becomes increasingly low-Lrp expressing, based on expression of an Lrp-dependent fluorescent reporter, as IJs age. These data support a model that virulent X. nematophila have a selective advantage and accumulate in aging IJs in advance of exposure to insect hosts in which this phenotype is necessary.

Immune interactions between Drosophila and the pathogen Xenorhabdus.

Deciphering host innate immune function and bacterial pathogenic tactics require a system that facilitates both facets of host-pathogen interactions. In recent years, a model that becomes established in dissecting mechanisms of host antibacterial immune response through probing with a potent bacterial pathogen involves the fruit fly Drosophila melanogaster and the insect pathogenic bacteria Xenorhabdus spp. The elegance of this system involves not only the genetic tractability of D. melanogaster, but also the association of Xenorhabdus with parasitic nematodes of insects that supervise the release of the bacteria as well as influence their pathogenic properties during the infection process. These dynamic aspects have enabled us to start decoding the specific features of the D. melanogaster host defense that participate in confronting the activity of Xenorhabdus molecular components, which are designed to evade the immune system. Here we outline recent information on the cellular, humoral and phenoloxidase reactions that are induced in D. melanogaster larvae and adults to oppose the Xenorhabdus attack, and the bacterial factors responsible for triggering these effects. This knowledge is critical not only for understanding how invertebrate immunity operates, but also for devising novel approaches to exploit the virulence ability of certain bacteria with the ultimate goal to counteract harmful insect pests or vectors of infectious disease.

The great potential of entomopathogenic bacteria Xenorhabdus and Photorhabdus for mosquito control: a review.

The control of insects of medical importance, such as Aedes aegypti and Aedes albopictus are still the only effective way to prevent the transmission of diseases, such as dengue, chikungunya and Zika. Their control is performed mainly using chemical products; however, they often have low specificity to non-target organisms, including humans. Also, studies have reported resistance to the most commonly used insecticides, such as the organophosphate and pyrethroids. Biological control is an ecological and sustainable method since it has a slow rate of insect resistance development. Bacterial species of the genera Xenorhabdus and Photorhabdus have been the target of several research groups worldwide, aiming at their use in agricultural, pharmaceutical and industrial products. This review highlights articles referring to the use of Xenorhabdus and Photorhabdus for insects and especially for mosquito control proposing future ways for their biotechnological applicability. Approximately 24 species of Xenorhabdus and five species of Photorhabdus have been described to have insecticidal properties. These studies have shown genes that are capable of encoding low molecular weight proteins, secondary toxin complexes and metabolites with insecticide activities, as well as antibiotic, fungicidal and antiparasitic molecules. In addition, several species of Xenorhabdus and Photorhabdus showed insecticidal properties against mosquitoes. Therefore, these biological agents can be used in new control methods, and must be, urgently considered in short term, in studies and applications, especially in mosquito control.

Glycan-dependent cell adhesion mechanism of Tc toxins.

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.

Biological control of Phlebotomus papatasi larvae by using entomopathogenic nematodes and its symbiotic bacterial toxins.

The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) - Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 - were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.

Screening a fosmid library of Xenorhabdus stockiae HN_xs01 reveals SrfABC toxin that exhibits both cytotoxicity and injectable insecticidal activity.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with the nematodes of the Steinernematid family, are known to produce several toxic proteins that interfere with the cellular immune responses of insects. In order to identify novel cytotoxins from Xenorhabdus spp., a fosmid library of X. stockiae HN_xs01 strain was constructed and the cytotoxicity of fosmid clones was tested against insect midgut CF-203 cells. An FS2 clone bearing the srfABC operon, originally identified in Salmonella enterica, exhibited excellent cytotoxicity against CF-203 cells. The srfABC operon alone exhibited cytotoxic effects and all three components of SrfABC toxin were essential for full cytotoxicity. Immunofluorescence studies showed that SrfABC toxin could depolymerize microtubules and disrupt mitochrondria. Flow cytometer analysis demonstrated that SrfABC toxin significantly induced G2/M phase arrest and apoptosis in CF-203 cells. Furthermore, SrfABC toxin exhibits highly injectable insecticidal activity against Helicoverpa armigera larvae. As is often found in host-associated microorganisms, SrfABC toxin is thought to play an important role in host colonization.

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.

ngrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Sand crickets (Gryllus firmus) have low susceptibility to entomopathogenic nematodes and their pathogenic bacteria.

The entomopathogenic nematode, Steinernema scapterisci, a specialist parasite of crickets, has been successfully used to combat the southern mole cricket, Neoscapteriscus borellii, which is an invasive pest of turf grass. As an entomopathogenic nematode, S. scapterisci causes rapid death of the insects it infects and uses bacteria to facilitate its parasitism. However, our understanding of the relative contributions of the nematode, S. scapterisci, and its bacterial symbiont, Xenorhabdus innexi, to parasitism remains limited. Here we utilized the sand cricket, Gryllus firmus, as a model host to evaluate the contributions of the EPNs S. scapterisci and S. carpocapsae, as well as their symbiotic bacteria, X. innexi and X. nematophila, respectively, to the virulence of the nematode-bacterial complex. We found that G. firmus has reduced susceptibility to infection from both S. scapterisci and the closely related generalist parasite S. carpocapsae, but that S. scapterisci is much more virulent than S. carpocapsae. Further, we found that N. borellii has reduced susceptibility to X. nematophila, and that G. firmus has reduced susceptibility to X. nematophila, X. innexi, and Serratia marcescens, much more so than other insects that have been studied. We found that the reduced susceptibility of G. firmus to bacterial infection is dependent on development, with adults being less susceptible to infection than nymphs. Our data provide evidence that unlike other EPNs, the virulence of S. scapterisci to crickets is dependent on the nematode rather than the bacterial symbiont that it carries and we speculate that S. scapterisci may be evolving independence from X. innexi.

Refining the Natural Product Repertoire in Entomopathogenic Bacteria.

Members of the genera Xenorhabdus and Photorhabdus are capable of producing a huge repertoire of different natural products to support a complex life cycle involving insect pathogenesis and nematode symbiosis. Many of the natural products have direct functions, specifically targeting different facets of nematode development or the insect immune system. These adaptations have allowed the bacteria to thrive in a unique environment and become highly efficient, versatile insect pathogens. Here, we discuss the ecological advantages afforded to the bacteria by the acquisition of the gene clusters responsible for producing this repertoire of chemical compounds.

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.

The insect pathogenic bacterium Xenorhabdus innexi has attenuated virulence in multiple insect model hosts yet encodes a potent mosquitocidal toxin.

BACKGROUND: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS: Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS: The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Identification and occurrence of the hydroxamate siderophores aerobactin, putrebactin, avaroferrin and ochrobactin C as virulence factors from entomopathogenic bacteria.

Effective iron acquisition and fine-tuned intracellular iron storage systems are the main prerequisites for a successful host invasion by a pathogen. Bacteria have developed several different strategies to sequester this essential element from their environment, one relies on the secretion of low molecular weight compounds with high affinity for ferric iron, the so-called siderophores. Here, we report hydroxamate siderophore structures produced by entomopathogenic bacteria of the species Xenorhabdus and Photorhabdus, which are known for their potential to produce bioactive natural products, required for their role as nematode symbiont and insect pathogen. Four siderophores could be identified, namely aerobactin, putrebactin, avaroferrin and ochrobactin C, which was found previously only in marine bacteria. While the putrebactin and avaroferrin producing biosynthesis gene cluster (BGC) is more widespread and most likely was present in a common ancestor of these bacteria, the aerobactin and ochrobactin producing BGC was probably taken up by a few strains individually. For aerobactin a role in virulence towards Galleria mellonella larvae is shown.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

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.

High Levels of the Xenorhabdus nematophila Transcription Factor Lrp Promote Mutualism with the Steinernema carpocapsae Nematode Host.

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states.IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, produces oxindole and suppresses host insect immune response by inhibiting eicosanoid biosynthesis.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, was isolated from an entomopathogenic nematode, Steinernema monticolum. X. hominickii exhibited significant insecticidal activities at ≥6.6×102 colony-forming units per larva against a lepidopteran insect, Spodoptera exigua with hemocoelic injection. The insecticidal activity of X. hominickii was reduced by an addition of arachidonic acid (AA, a catalytic product of PLA2), but enhanced by an addition by dexamethasone (DEX, a specific inhibitor of PLA2). S. exigua could defend the bacterial infection by forming hemocyte nodules. However, live X. hominickii significantly reduced the hemocytic nodulation compared to similar treatment with heat-killed X. hominickii. An addition of AA to live X. hominickii significantly rescued the immunosuppression. X. hominickii also inhibited phenoloxidase activity in hemolymph of S. exigua larvae. Furthermore, the bacteria suppressed gene expressions of antimicrobial peptides, such as attacin-1, attacin-2, defensin, gallerimycin and transferrin-1 of S. exigua. An organic extract of X. hominickii-cultured broth with ethyl acetate possessed oxindole and significantly suppressed hemocyte nodulation. Again, an addition of AA diminished the inhibitory activity of the organic extract against hemocyte nodulation. Oxindole alone inhibited hemocyte nodulation and PLA2 enzyme activity. These results suggest that the entomopathogenicity of X. hominickii comes from its inhibitory activity against eicosanoid biosynthesis of target insects.

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity.