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Nature Communications Jun 2020Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction...
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.
Topics: Animals; Bacterial Toxins; Binding Sites; Cell Adhesion; Cell Membrane; HEK293 Cells; Heparin; Humans; Insecta; Lewis X Antigen; Models, Molecular; Molecular Docking Simulation; Morganella morganii; Photorhabdus; Polysaccharides; Xenorhabdus
PubMed: 32483155
DOI: 10.1038/s41467-020-16536-7 -
The Journal of Biological Chemistry Apr 2017Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family...
Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl--stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.
Topics: Animals; Anti-Infective Agents; Biological Products; Catalysis; Chromatography, High Pressure Liquid; Crystallography, X-Ray; DNA Mutational Analysis; Epoxy Compounds; Gene Deletion; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Immunosuppressive Agents; Magnetic Resonance Spectroscopy; Molecular Conformation; Mutation; Photorhabdus; Protein Folding; Rhabditoidea; Stereoisomerism; Stilbenes; Symbiosis
PubMed: 28246174
DOI: 10.1074/jbc.M116.762542 -
Microbial Cell Factories Apr 2024Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life...
BACKGROUND
Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.
RESULTS
In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
CONCLUSIONS
The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
Topics: Xenorhabdus; Photorhabdus; Gene Editing; Biological Products; Clustered Regularly Interspaced Short Palindromic Repeats
PubMed: 38561780
DOI: 10.1186/s12934-024-02363-8 -
Scientific Reports Nov 2023The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study,...
The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.
Topics: Genome, Bacterial; Bacteria; Computational Biology; Multigene Family; Biosynthetic Pathways
PubMed: 38007490
DOI: 10.1038/s41598-023-47121-9 -
Scientific Reports Jun 2022Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce...
Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.
Topics: Antiprotozoal Agents; Entamoeba histolytica; Humans; Photorhabdus; Trypanosoma cruzi; Xenorhabdus
PubMed: 35750682
DOI: 10.1038/s41598-022-13722-z -
Journal of Invertebrate Pathology Jun 2023The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide....
The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had ∼ 55% and ∼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in ∼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.
Topics: Animals; Xenorhabdus; Photorhabdus; Moths; Larva; Vitis
PubMed: 36921888
DOI: 10.1016/j.jip.2023.107911 -
PloS One 2014Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological...
Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.
Topics: Antimicrobial Cationic Peptides; Bacterial Outer Membrane Proteins; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Genome, Bacterial; Humans; Magnesium Sulfate; Phenotype; Photorhabdus; Phylogeny
PubMed: 25333642
DOI: 10.1371/journal.pone.0110060 -
The FEBS Journal Feb 2021O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune...
O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two β-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.
Topics: Animals; Bacterial Proteins; Gram-Negative Bacterial Infections; Hemocytes; Hemolymph; Host-Pathogen Interactions; Humans; Immune System; Immunity; Lectins; Methylation; Moths; Photorhabdus; Sugars
PubMed: 32559333
DOI: 10.1111/febs.15457 -
Nature Microbiology Dec 2020Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage in their life cycles. In both stages, numerous...
Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence. Although regulators have been implicated in the regulation of these specialized metabolites, 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.
Topics: Animals; Biological Products; Gene Expression Regulation, Bacterial; Insecta; Nematoda; Photorhabdus; RNA, Bacterial; RNA, Small Untranslated; Symbiosis; Virulence; Xenorhabdus
PubMed: 33139881
DOI: 10.1038/s41564-020-00797-5 -
Nucleic Acids Research Mar 2015Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be...
Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed 'recombineering', in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5'-3' exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.
Topics: Amino Acid Sequence; Bacterial Proteins; Bacteriophage lambda; DNA, Bacterial; Escherichia coli; Exodeoxyribonuclease V; Genetic Engineering; Genome, Bacterial; Genomics; Molecular Sequence Data; Multigene Family; Operon; Photorhabdus; Plasmids; Recombination, Genetic; Sequence Homology, Amino Acid; Xenorhabdus
PubMed: 25539914
DOI: 10.1093/nar/gku1336