The Xenorhabdus nematophila LrhA transcriptional regulator modulates production of γ-keto-N-acyl amides with inhibitory activity against mutualistic host nematode egg hatching.

Xenorhabdus nematophila is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between X. nematophila secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new N-acyl amides, GKA-Arg (1) and GKA-Pro (2) which harbor a γ-keto acyl appendage. A lrhA null mutant produced elevated levels of compound 1 and reduced levels of compound 2 relative to wild type. N-acyl amides 1 and 2 were shown to be selective agonists for the human G-protein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist 2 deleteriously affected the hatch rate and length of Steinernema nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. IMPORTANCE: Xenorhabdus bacteria are of interest due to their symbiotic relationship with Steinernema nematodes and their ability to produce a variety of natural bioactive compounds. Despite their importance, the regulatory hierarchy connecting specific natural products and their regulators is poorly understood. In this study, comparative metabolomic profiling was utilized to identify the secondary metabolites modulated by the X. nematophila global regulator LrhA. This analysis led to the discovery of three metabolites, including an N-acyl amide that inhibited the egg hatching rate and length of Steinernema carpocapsae nematodes. These findings support the notion that X. nematophila LrhA influences the symbiosis between X. nematophila and S. carpocapsae through N-acyl amide signaling. A deeper understanding of the regulatory hierarchy of these natural products could contribute to a better comprehension of the symbiotic relationship between X. nematophila and S. carpocapsae.

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.

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.

The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila.

UNLABELLED: The bacterium Xenorhabdus nematophila engages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary for X. nematophila virulence and immunosuppression in insects, as well as colonization of the mutualistic host nematode Steinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp in X. nematophila host associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression of lrp varies within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels of lrp were attenuated in these processes relative to those with low to intermediate lrp expression. Furthermore, fixed expression of lrp at high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuating lrp expression levels are sufficient to drive phenotypic variation in X. nematophila. IMPORTANCE: Many bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogen Xenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation of X. nematophila phenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase.

Simple urea compounds ("phurealipids") have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic association with the nematode Steinernema carpocapsae. The nematode invades and traverses the gut of susceptible insects. X. nematophila is released in the insect blood (hemolymph), where it suppresses host immune responses and functions as a pathogen. X. nematophila produces diverse antimicrobials in laboratory cultures. The natural competitors that X. nematophila encounters in the hemolymph and the role of antimicrobials in interspecies competition in the host are poorly understood. We show that gut microbes translocate into the hemolymph when the nematode penetrates the insect intestine. During natural infection, Staphylococcus saprophyticus was initially present and subsequently disappeared from the hemolymph, while Enterococcus faecalis proliferated. S. saprophyticus was sensitive to X. nematophila antibiotics and was eliminated from the hemolymph when coinjected with X. nematophila. In contrast, E. faecalis was relatively resistant to X. nematophila antibiotics. When injected by itself, E. faecalis persisted (~10(3) CFU/ml), but when coinjected with X. nematophila, it proliferated to ~10(9) CFU/ml. Injection of E. faecalis into the insect caused the upregulation of an insect antimicrobial peptide, while the transcript levels were suppressed when E. faecalis was coinjected with X. nematophila. Its relative antibiotic resistance together with suppression of the host immune system by X. nematophila may account for the growth of E. faecalis. At higher injected levels (10(6) CFU/insect), E. faecalis could kill insects, suggesting that it may contribute to virulence in an X. nematophila infection. These findings provide new insights into the competitive events that occur early in infection after S. carpocapsae invades the host hemocoel.

Immune Signaling and Antimicrobial Peptide Expression in Lepidoptera.

Many lepidopteran insects are agricultural pests that affect stored grains, food and fiber crops. These insects have negative ecological and economic impacts since they lower crop yield, and pesticides are expensive and can have off-target effects on beneficial arthropods. A better understanding of lepidopteran immunity will aid in identifying new targets for the development of specific insect pest management compounds. A fundamental aspect of immunity, and therefore a logical target for control, is the induction of antimicrobial peptide (AMP) expression. These peptides insert into and disrupt microbial membranes, thereby promoting pathogen clearance and insect survival. Pathways leading to AMP expression have been extensively studied in the dipteran Drosophila melanogaster. However, Diptera are an important group of pollinators and pest management strategies that target their immune systems is not recommended. Recent advances have facilitated investigation of lepidopteran immunity, revealing both conserved and derived characteristics. Although the general pathways leading to AMP expression are conserved, specific components of these pathways, such as recognition proteins have diverged. In this review we highlight how such comparative immunology could aid in developing pest management strategies that are specific to agricultural insect pests.