Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cß on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Txp40, an insecticidal toxin protein from Xenorhabdus nematophila: Purification, toxicity assessment and biophysical characterization.

Txp40 is a ubiquitous toxin from Xenorhabdus and Photorhabdus bacteria, exhibits insecticidal activity against a wide range of insect pests belonging to Lepidoptera and Diptera orders. Initially, Txp40 affects midgut of the target insect and further damages some other tissues like fat bodies but the detailed mode of action is not known. Txp40 shares no significant sequence match to any proteins with known structure or function, suggesting that it is a novel type of insecticidal toxin. Here, we report purification, toxicity and biophysical characterization of the Txp40b toxin from X. nematophila (ATCC, 19061). The recombinant Txp40b was found toxic to Galleria mellonella larvae with LD50 of 30.42 ng larva-1. Circular dichroism spectroscopy revealed that purified Txp40b is an α-helix rich protein with a relatively lower melting temperature of 45 °C. In-silico model generated suggests two domain structure of Txp40b toxin. Detailed structural analysis of Txp40b will provide new insights about the mode of action and possibly it would illustrate a new domain and/or motif in the area of insecticidal proteins.

Identification of Volatile Organic Compounds Produced by Xenorhabdus indica Strain AB and Investigation of Their Antifungal Activities.

Xenorhabdus spp. are symbiotic bacteria associated with entomopathogenic nematodes to form a model complex that is used for the biological control of insect pests. These bacteria also produce secondary metabolites that have commercial potential in the pharmaceutical and agroforestry industries. Volatile organic compounds (VOCs) produced by the Xenorhabdus indica "strain AB" have been shown to have significant antifungal activity against Fusarium oxysporum f. sp. cucumerinum. Using gas chromatography-mass spectrometry, we identified 61 volatiles in the mixture of VOCs emitted by strain AB compared to a control strain, 6 of which were investigated for their antifungal activities. Of these, methyl anthranilate exhibited the highest mycelial growth suppression toward F. oxysporum, with a minimum inhibitory volume (MIV) of 50 µL/plate. Fluorescence assays, scanning electron microscopy, and measurements of the leakage of intracellular components revealed that the use of methyl anthranilate changed cell wall and cell membrane integrity as well as the permeability of the plasma membrane. Furthermore, methyl anthranilate treatment upregulated the transcription level of target genes related to redox reactions and the cell wall integrity pathway. The results suggest a novel mechanism used by Xenorhabdus spp. to overcome competitors during its life cycle and open up a new approach to using these bacteria in biological control. IMPORTANCE Fungal phytopathogens, particularly Fusarium oxysporum, are a major problem worldwide, especially in the postharvest of vital economic crops. Concerns about negative effects on the environment and human health have led to increasing restrictions on the use of chemical fungicides, and therefore, biological control agents are now being considered alternatives. It is in this context that we investigated the antifungal activity of VOCs produced by X. indica strain AB against F. oxysporum. We found that AB VOCs have a strong effect on the growth of the fungal phytopathogen. In addition, 85% of the identified volatile compounds were determined to be new compounds, opening up new lines of research to discover their properties, effects, and potential for pharmaceutical and agricultural applications. Antifungal assays proved that four of the six compounds with a high concentration in the GC-MS profile had a significant inhibitory effect on pathogen growth. Accordingly, this study opens up a new approach for the use of these bacteria in biocontrol.

A study on Xenorhabdus and Photorhabdus isolates from Northeastern Thailand: Identification, antibacterial activity, and association with entomopathogenic nematode hosts.

Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5_TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates.

Structure and biosynthesis of deoxy-polyamine in Xenorhabdus bovienii.

Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.

Microbial Cationic Peptides as a Natural Defense Mechanism against Insect Antimicrobial Peptides.

Bacteria produce a plethora of specialized metabolites (SM), with the ecological function of most of them not known. A major group of SM are peptides derived from nonribosomal peptide synthetases (NRPS). In entomopathogenic bacteria of the genus Xenorhabdus, PAX (peptide-antimicrobial-Xenorhabdus) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in Xenorhabdus doucetiae and found the majority bound to the cells. We derivatized PAX with fluorophores and show binding to cells when added externally using super-resolution microscopy. Externally added PAX in X. doucetiae and E. coli as well as inducible PAX production in X. doucetiae showed a protective effect against various antimicrobial peptides (AMPs) from insects, where they are used as a defense mechanism against pathogens. Because AMPs are often positively charged, our results suggest a PAX-induced repulsive force due to positive charge at the bacterial cell wall.

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

Mode of entry of secondary metabolites of the bacteria Xenorhabdus szentirmaii and X. nematophila into Tetranychus urticae, and their toxicity to the predatory mites Phytoseiulus persimilis and Neoseiulus californicus.

The bacterial metabolites in supernatants of Xenorhabdus species have acaricidal activity, but this mode of entry into mites has not yet been elucidated. Herein, we report on the possible mode of entry of Xenorhabdus szentirmaii and Xenorhabdus nematophila supernatants into Tetranychus urticae (Acari: Tetranychidae) adult females. We also assessed the toxicity of the supernatants against the developmental stages of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae). Experiments were conducted at 25 ± 1 °C, 70 ± 5% relative humidity, and 16:8h light:dark conditions. Our data showed that the bioactive acaricidal compound is most effective (86.5 to 89% mortality) when the entire integument of T. urticae comes in contact with it compared to contact of the ventral side only (26.5-34%). Against P. persimilis and N. californicus at 6 days post-application (dpa), the eggs were not affected by the X. szentirmaii or X. nematophila supernatant, whereas mortality of the mobile stages (larva, protonymph, deutonymph, adult) was 18.5% to 39.2%. Overall, the predatory mites were less affected by the bacterial metabolites than T. urticae. We hypothesize that the differences in morphology such as longer legs and thicker cuticle, as well as the diet of the predatory mites, reduce the contact of the body parts to the supernatant-treated surfaces. We need to isolate, identify, and characterize the X. szentirmaii and X. nematophila metabolite(s) and demonstrate efficacy to pestiferous mites and safety to plants, non-target organisms and the environment before it can be used as an acaricide.

Biogenic larvicidal formulation of metabolites from Steinernema saimkayi symbiont Xenorhabdus stockiae KUT6 against dengue vector Aedes aegypti.

To characterize the production and larvicidal activity of Xenorhabdus stockiae KUT6 Petroleum ether extracts from Luria Broth and induced Quorum sensing medium containing N-3- oxododecanoyl Homoserine Lactone inducer against dengue vector Aedes aegypti. The Galleria mellonella larvae were reared for the isolation of Steinernema saimkayi symbiont Xenorhabdus stockiae KUT6 from Cucumber field soil sample in NBTA. Then for the extraction of compounds the KUT6 strains were cultured in Luria Broth and Quorum Sensing optimized media using N-3-oxododecanoyl homoserine lactone inducer. The larvicidal activity of Xenorhabdus stockiae KUT6 of petroleum ether extracts were bioassayed against 4th instar Aedes aegypti dengue vector. The maximum rate of mortality were recorded of the samples A-24h, B-48h, C-72h, A1-24h, B1-48h, C1-72h at different concentrations 50 µg/ml, 100 µg/ml and 150 µg/ml respectively for 24h to 72h of exposure treatment. The morphological characteristics of Xenorhabdus stockiae KUT6 in NBTA were red core colonies with blue background surrounded by zone of inhibition. After 24h exposure maximum rate of 100% mortality of Aedes aegypti 4th instar larvae was attained when treated with sample C1-72h 50 µg/ml of the petroleum ether extracts of quorum sensed medium whereas the sample C 72h petroleum ether extracts of KUT6 cultured in Luria broth recorded 100% mortality at 150 µg on 24h exposure indicates enhancement in the product yield. The study assures the use of Xenorhabdus stockiae KUT6 petroleum ether extracts as biocontrol agent could be beneficial for the control of dengue vectors.

SrfABC Toxin from Xenorhabdus stockiae Induces Cytotoxicity and Apoptosis in HeLa Cells.

Our previous study showed that the srfABC operon, which was originally identified in Salmonella enterica as an SsrB-regulated operon clustered with the flagellar class 2 operon, exhibited significant cytotoxicity against insect midgut CF-203 cells and injectable insecticidal activity against Helicoverpa armigera larvae. The srfABC operon was widely distributed among bacteria, which raises the question of their biological roles in different species. In this study, we investigated the cytotoxic effect of SrfABC toxin on mammalian cell lines. When simultaneously expressed in the Escherichia coli cytoplasm, SrfABC exhibited cytotoxicity against all tested mammalian cancer cell lines (B16, 4T-1, Hep-3B, and HeLa) in a dose-dependent manner. Intracellular expression of SrfA-FLAG, SrfB-FLAG, or SrfC-FLAG also resulted in inhibition of proliferation and apoptosis on HeLa cells. When incubated with HeLa cells separately, SrfA, SrfB, and SrfC proteins alone could enter HeLa cells, then induce apoptosis and cytotoxicity. SrfC protein shifts its localization from cytoplasm to nucleus with the aid of SrfA and/or SrfB protein. Although SrfA, SrfB, and SrfC proteins alone exhibited a cytotoxic effect against HeLa cells, all three components were essential for the full cytotoxicity. Native PAGE and co-immunoprecipitation assay demonstrated that SrfA, SrfB, and SrfC proteins could interact with each other and form a heteromeric complex.

Anti-Trypanosoma activity of bioactive metabolites from Photorhabdus luminescens and Xenorhabdus nematophila.

Only two drugs are currently available for the treatment of Chagas disease and their effectiveness are unsatisfactory. Photorhabdus luminescens and Xenorhabdus nematophila, two enteric bacteria highly pathogenic to a broad range of insects, have been studied as potential source for bioactive metabolites against protozoa causing neglected tropical diseases. Therefore, we tested the in vitro anti-Trypanosoma cruzi activity of secreted metabolites from these bacteria. The conditioned medium of X. nematophila and P. luminescens showed significant parasiticidal activity in a concentration-dependent manner (IC50XN = 0.34 mg/mL, IC50PL = 1.0 mg/mL). The parasiticidal compound was identified as a small molecule stable to heating and pH changes ranging from 2 to 12. Moreover, anti-Trypanosoma molecules secreted by both bacteria stimulate the trypanocidal activity of macrophages by a mechanism independent of nitric oxide. Summarizing, our studies reveal that P. luminescens and X. nematophila are potential sources of putative novel drugs against Chagas disease.

Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus budapestensis.

Two novel depsipeptides xenematides F and G (1, 2), were isolated from entomopathogenic Xenorhabdus budapestensis SN84 along with a known compound xenematide B. The structures of the two new molecules were elucidated using NMR, MS and Marfey's method. The xenematide G (2) contains α-aminoheptanoic acid, a non-protein amino acid that is rarely found in secondary metabolites from entomopathogenic bacteria. Xenematides F and G were tested for antibacterial activity. Xenematide G (2) exhibited moderate antibacterial activity.

Characteristics and Function of the Chitin Binding Protein from Xenorhabdus nematophila.

BACKGROUND: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). OBJECTIVE: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. METHODS: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. RESULTS: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. CONCLUSION: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

Bacteria: A novel source for potent mosquito feeding-deterrents.

Antibiotic and insecticidal bioactivities of the extracellular secondary metabolites produced by entomopathogenic bacteria belonging to genus Xenorhabdus have been identified; however, their novel applications such as mosquito feeding-deterrence have not been reported. Here, we show that a mixture of compounds isolated from Xenorhabdus budapestensis in vitro cultures exhibits potent feeding-deterrent activity against three deadly mosquito vectors: Aedes aegypti, Anopheles gambiae, and Culex pipiens. We demonstrate that the deterrent active fraction isolated from replicate bacterial cultures is highly enriched in two compounds consistent with the previously described fabclavines, strongly suggesting that these are the molecular species responsible for feeding-deterrence. The mosquito feeding-deterrent activity in the putative fabclavine-rich fraction is comparable to or better than that of N,N-diethyl-3-methylbenzamide (also known as DEET) or picaridin in side-by-side assays. These findings lay the groundwork for research into biologically derived, peptide-based, low-molecular weight compounds isolated from bacteria for exploitation as mosquito repellents and feeding-deterrents.

Rhabdopeptide/Xenortide-like Peptides from Xenorhabdus innexi with Terminal Amines Showing Potent Antiprotozoal Activity.

Seven new rhabdopeptide/xenortide-like peptides (RXPs) (1-7) with putrescine or ammonia as the C-terminal amines were isolated from Xenorhabdus innexi DSM 16336. Their chemical structures were elucidated by high-resoultion mass spectroscopy (HR-MS) and one-dimensional (1D) and two-dimensional (2D) NMR. They were evaluated for their activities against protozoan parasites and cytotoxicity against rat skeletal myoblasts (L6 cells). All tested compounds exhibited strong effects against Trypanosoma brucei rhodesiense and Plasmodium falciparum, with IC50 values of 0.07-6.25 and 0.091-3.16 µM, respectively, making them the most active RXP derivatives known to date.

Efficient syntheses and anti-cancer activity of xenortides A-D including ent/epi-stereoisomers.

A one-pot, two-step, total synthesis of naturally occurring xenortides A, B, C and D, (Xens A-D) isolated from the bacterium Xenorhabdus nematophila, and an entire complementary set of stereoisomers, has been achieved. Compounds were synthesized utilizing an isocyanide-based Ugi 4-CR followed by facile N-Boc deprotection. The reaction sequence took advantage of the chiral pool of N-Boc protected amino acids (l-Leu/Val and d-Leu/Val) with aryl isocyanides, phenyl acetaldehyde and methylamine giving the desired Xens A-D (A and B >98% ee) and all subsequent stereoisomers in reasonable yields upon deprotection followed by separation of diastereomers. Also, detailed mechanistic insights for diastereoselectivity of (-)-Xen A, as a model in the Ugi 4-CR, has been described. Moreover, for the first time, this focused library was screened for cytotoxicity against a panel of epithelial cancer cell lines as well as normal cell lines with an MTT proliferation assay. The structure-activity relationship (SAR) study demonstrated that tryptamides Xen B and D were more active than phenylethylamides Xen A and C. Furthermore, (-)-Xen B (IC50 = 19-25 µM) and ent-(+)-Xen D (IC50 = 21-26 µM) gave the highest cytotoxicity and they were also found to be non-toxic toward normal cells. Importantly, the SAR results indicate that the stereochemistry at C8 and C11 in (-)-Xen B and ent-(+)-Xen D play a critical role in cytotoxic activity.

Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

Isolation and characterization of toxins from Xenorhabdus nematophilus against Ferrisia virgata (Ckll.) on tuberose, Polianthes tuberosa.

This study was aimed to evaluate the toxins of Xenorhabdus nematophilus bacterial isolate MDUStBa15 isolated from the nematode Steinernema carpocapsae that can parasitize two tailed mealybug Ferrisia virgata which is a new pest on tuberose. Soluble protein and organic fractions were characterized from cell free extract of X. nematophilus. Using SDS PAGE, presence of low molecular weight toxic proteins (12, 42 and 60 kDa) was observed in cell free extracts of X. nematophilus. Among these three proteins, 12 kDa was newly found in this study which showed anti-feedant activity and the maximum of 87.50% and 82.50% mortality of crawlers and adults of F. virgata, respectively at 72 h after treatment. GC-MS analysis of culture filtrates revealed the presence of five major compounds, all are exhibiting insecticidal property. Among several organic fractions, 1, 4 - epoxynaphthalene - 1 (2H) - methanol, 4,5,7-tris (1,1 - dimethylethyl) - 3,4 - dihydro; Pentacosane and Hexacosane were found in this study. Pot culture study revealed that an optimum dose of 5 ml/l of crude toxin caused the maximum mortality in crawlers (100%) and in adults (96.8%) of F. virgata at 72 h after spraying. In a field study application of 5 ml/l crude toxin along with biocontrol agent (Ladybird beetle - Cryptolaemus montrouzieri) registered the 90.55% mortality in crawlers and 73.60% mortality in adults of F. virgata at 7 days after spraying. The present study provides the clear evidence for the toxicity of protein; organic fraction and crude toxin which was obtained from X. nematophilus isolate MDUStBa15 against F. virgata on tuberose both in lab and field conditions. Hence, it can be utilised to manage the F. virgata on tuberose.

Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their Nematicidal Activities against Meloidogyne incognita.

For decades, plant parasitic nematodes have caused serious damage to crop production. Most nematicides are banned because of their negative impacts on the environment and public health. The repeated application of the few commercially available nematicides has caused more incidences of nematicide resistance. To seek novel nematicides, seven linear peptides named rhabdopeptides I-O, 1-7, were isolated from culture broth of Xenorhabdus budapestensis SN84. The structures of the peptides were elucidated on the basis of extensive mass spectrometry (MS), and nuclear magnetic resonance analyses. 3, 4, and 7 were novel compounds. 1, 2, 5, and 6 were isolated and purified for the first time, despite being previously elucidated from an extract mixture based on labeling and MS experiments. All seven compounds were tested for their nematicidal activities against the second-stage juveniles (J2) of Meloidogyne incognita using 24-microwell plates. Rhabdopeptide J, 2, demonstrated strong inhibitory activity with an LC50 value of 27.8 µg/mL. Rhabdopeptide K, 3, and M, 5, showed moderate inhibitory activity with LC50 values of 46.3 and 42.4 µg/mL, respectively.

PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella.

PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila. The pirA, pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly)4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella. The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella. Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD50 of 2.718µg/larva or 1.566µg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin.