Identification of natural products regulating the symbiosis between entomopathogenic nematodes and their bacterial symbionts

The associations between bacteria and higher eukaryotes have been the subject of analysis in several academic disciplines. The study of the regulation of these interactions in a simple and easily manipulated symbiotic system, has the potential to enhance our understanding of such relationships in complex organisms. A suitable and promising model that meets these criteria is entomopathogenic nematodes (ENPs). The soil-dwelling EPNs from the genera Heterorhabditis and Steinernema are symbiotically associated with the bacteria Photorhabdus and Xenorhabdus, respectively. This mutualistic consortium is capable of causing the death of the host insect within 48 hours. EPNs exhibit a complex life cycle, which includes a single free-living stage, referred to as the third infective juvenile (IJ) or dauer stage. The IJ shows morphological and metabolic characteristics that confer resistance to adverse conditions for locating and infecting a new host, retaining the bacterial symbiont in its intestinal tract. Once the IJ gains access through the insects' natural openings into the hemocoel, the held endosymbiont is released. Upon contact with the hemolymph, the bacterium proliferates and produces a wide range of compounds, including toxins and hydrolytic enzymes. Some of these compounds facilitate the conversion of insect tissues into a nutritional source for optimal nematode development and reproduction. In addition, the bacteria synthesize a diverse array of small molecules through the non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways, which are involved in multiple stages of the nematode-bacterial life cycle. These stages include those related to the regulation of the tripartite association such as overcoming the host immune system, insect pathogenicity and protection of the consortium against opportunistic organisms. After the optimal conditions for nematode growth have been established by the bacterial endosymbiont within the insect, the IJs proceed to develop into a juvenile stage 4. This process is initially characterized by a recovery phase, whereby the IJ detects specific signals that induce it to exit from the arrested metabolism and continue the nematode reproduction and life cycle, resulting in the development into the adult stage. The signals required to activate this developmental change vary between nematode species. In the case of Heterorhabditis, the influence of isopropyl stilbene (IPS) produced by Photorhabdus sp. has been identified as a compound required for the nematode recovery. However, no signals for Steinernema have been described yet. The objective of this study was to identify natural products (NPs) synthesized by bacteria X. doucetiae that may be required for optimal development of its host, the nematode S. diaprepesi. To ascertain the role of NPs as potential signals for the transition from IJ to 1st generation adult stage, the recovery and maturation of the EPN S. diaprepesi was analyzed after exposure to several mutant strains of X. doucetiae, which were deficient in NP synthesis according to deletion or/and promoter exchange (induced and non-induced) performed in their respective biosynthetic gene clusters (BGCs). The compounds subjected to analysis included xenocoumacin (XCN), GameXPeptide (GXP), xenoamicin (XAB), xenorhabdin (XRD), protegomycin (PRT), and phenylethylamides (Phe-D). X. doucetiaehfq and ngrA mutants were also evaluated, which do not produce most of the mentioned NP classes. The results demonstrated a reduction in nematode development following exposure to X. doucetiae hfq and ngrA, with a pronounced decline observed in the latter. Subsequently, when the requirement of the compounds was evaluated separately, it was determined that XCN, GXP, XAB, and XRD did not affect recovery to the first generation of adults. Conversely, the suppression of phenylethylamine (Phe-N) and Phe-D resulted in the complete inhibition of development. It is noteworthy that a slight decline in growth was observed in nematodes exposed to strains deficient in PRT production. Upon examination of the adult stages, it was determined that the number of females exhibiting endotokia matricida (EM), a defect in the egg-laying process, increased. This anomaly is caused by the detection of stress signals, such as the depletion of a nutritional source. The aforementioned effects of Phe-N, Phe-D and PRT were validated by induction of the respective exchanged promoter mutants and supplementation of the medium with the corresponding compounds, in accordance with the genetic modifications made in the mutant strains. The results demonstrated the restoration of nematode growth and a reduction in developmental defects, thereby confirming the requirement of these NPs. It was determined that GXP is not a compound essential for the nematode development. However, the overproduction of this NP caused an inhibition of the nematode growth, exhibiting a negative effect. Therefore, a chemotaxis analysis testing GXP against the free-living nematode Caenorhabditis elegans was performed, which demonstrated a repellence effect caused by this compound. This result suggests a new potential biological function of GXP against soil-living nematodes. The production of Phe-D, PRT and GXP was analyzed during EPN infection, in order to gain insight into their association with insect infection progression and nematode development. To this end, instar larvae of Galleria mellonella were infected with the nematode S. diaprepesi containing its cognate symbiont bacterium X. doucetiae WT. At designated time points following infection, hemolymph samples were collected and subjected to analysis via high-performance liquid chromatography with mass spectrometry (HPLC-MS). Moreover, evidence of infection was recorded, and the nematode development was assessed through the dissection of insects and observation of the different nematode stages. It was demonstrated that the aforementioned compounds were detected 48 hours following infection. The maximum production of Phe-D was observed after 48 hours, which coincided with the development of the nematodes into adults. At this time point, all insects exhibited symptoms indicative of infection. In contrast, it was noted that PRT was synthesized predominantly between 72 and 96 hours. During this period, females containing unhatched eggs were observed. In the case of GXP, it was determined that GameXPeptide C was produced from the beginning of the infection between 26 and 36 hours, maintaining a constant concentration after 48 hours. In conclusion, our findings present the first report of NPs synthesized by X. doucetiae that are required for the recovery and development of the nematode S. diaprepesi at different stages. The results demonstrated that Phe-N and Phe-D are essential for the maturation from IJs to the first generation of adults. PRT has been identified as a signal related to the optimal conditions for egg deposition, while GXP has been shown to act as a repellent compound towards other soil-dwelling nematodes, thereby serving as a defense mechanism.