-
Frontiers in Veterinary Science 2022The light emitting module operon () of can be integrated into a "dark" bacterium for expression under a suitable promoter. The technique has been used to monitor...
The light emitting module operon () of can be integrated into a "dark" bacterium for expression under a suitable promoter. The technique has been used to monitor kinetics of infection, e.g., by studying gene expression in using mouse models and . Here, we applied the bioluminescence imaging (BLI) technique to track Enteritidis (SEn) strains carrying the operon expressed under a constitutive promoter sequence (sigma 70) in chicken after oral challenge. Detectable photon signals were localized in the crop, small intestine, cecum, and yolk sac in orally gavaged birds. The level of colonization was determined by quantification of signal intensity and SEn prevalence in the cecum and yolk sac. Furthermore, an isogenic SEn mutant strain tagged with the lux operon allowed for us to assess virulence determinants regarding their role in colonization of the cecum and yolk sac. Interestingly, mutations of (Salmonella Pathogenicity Island 1) and (ferric uptake regulator) showed significantly decreased colonization in yolk sac that was correlated with the BLI data. A similar trend was detected in a Δ strain by analyzing enrichment culture data. The inherently low quantum yield, light scattering, and absorption by tissues did not facilitate detection of signals from live birds. However, the detection limit of operon has the potential to be improved by resonance energy transfer to a secondary molecule. As a proof-of-concept, we were able to show that sensitization of a fluorescent-bound molecule known as the lumazine protein (LumP) improved the limit of detection to a certain extent.
PubMed: 35982923
DOI: 10.3389/fvets.2022.948448 -
International Journal of Molecular... Jun 2022Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by...
Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-β4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.
Topics: ADP Ribose Transferases; Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Adenosine Diphosphate; Photorhabdus
PubMed: 35806028
DOI: 10.3390/ijms23137026 -
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 -
Applied and Environmental Microbiology Jun 2022Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable...
Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of as a plant-growth-promoting and plant-protecting organism in agriculture. The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of as a plant-growth-promoting organism and biopesticide.
Topics: Animals; Chitin; Fusarium; Insecta; Nematoda; Photorhabdus; Soil; Symbiosis
PubMed: 35604230
DOI: 10.1128/aem.00645-22 -
MBio Jun 2022With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups...
With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured , a betaproteobacterium; odilorhabdins, from , are broad-spectrum inhibitors of protein synthesis, and darobactins from target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. and are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to and , kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express . To gain access to silent operons, we first fractionated supernatant from and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Biological Products; Deoxyguanosine; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Guanosine; Guanosine Triphosphate; Nematoda; Operon; Photorhabdus; Prodrugs; Xenorhabdus
PubMed: 35575547
DOI: 10.1128/mbio.00700-22 -
Inorganic Chemistry May 2022Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable...
Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active AA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu -tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of -tensors, giving confidence in our evaluation of AA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of AA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in AA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.
Topics: Copper; Electron Spin Resonance Spectroscopy; Mixed Function Oxygenases; Photorhabdus; Polysaccharides
PubMed: 35549254
DOI: 10.1021/acs.inorgchem.2c00766 -
Frontiers in Immunology 2022Nematodes and bacteria are prevalent in soil ecosystems, and some have evolved symbiotic relationships. In some cases, symbionts carry out highly specialized functions:...
Nematodes and bacteria are prevalent in soil ecosystems, and some have evolved symbiotic relationships. In some cases, symbionts carry out highly specialized functions: a prime example being entomopathogenic nematodes (EPNs), which vector bacteria ( or ) into insect hosts, killing them to provide a food source for the nematodes. It is thought that the commercially available malacopathogenic (kills slugs and snails) biocontrol nematode vectors a bacterium () into slugs to kill them. To investigate this further we used a metagenomic approach to profile the bacteria present in the commercial strain of , a wild strain of and two other species ( and ), after they had killed their slug host (). We show that these nematodes do not exclusively associate with one bacterium but a range of species, with members of the phyla Pseudomonadota, Bacillota, Actinobacteriota and Bacteroidota the most prevalent. The commercial strain of had the least diverse bacterial community. Furthermore, we found that the bacterium has been cultured on for 25 years is not the expected species but is spp. and the only strain of the species to associate with spp. was the commercial strain of . In summary, we found no evidence to show that rely exclusively on one bacterium to cause host mortality but found variable and diverse bacterial communities associated with these nematodes in their slug hosts.
Topics: Animals; Microbiota; Nematoda; Rhabditoidea; Snails; Soil
PubMed: 35515005
DOI: 10.3389/fimmu.2022.878783 -
Science Advances Apr 2022Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was...
Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo-electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.
Topics: Animals; Cryoelectron Microscopy; Photorhabdus; Polyvinyl Chloride; Protein Sorting Signals; Virulence
PubMed: 35486720
DOI: 10.1126/sciadv.abm2343 -
Nature Chemistry Jun 2022Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living...
Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.
Topics: Animals; Biological Products; Humans; Insecta; Multigene Family; Nematoda; Photorhabdus; Symbiosis; Xenorhabdus
PubMed: 35469007
DOI: 10.1038/s41557-022-00923-2 -
Journal of Fungi (Basel, Switzerland) Apr 2022is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus exhibit antimicrobial capability and...
is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus exhibit antimicrobial capability and have been found to have the potential for use as biocontrol agents against . species exhibit two phase variations with a differentiated composition of secondary metabolites designated to each phase. In this study, sp. nov. 0813-124 exhibited phase I (PL1) and phase II (PL2); however, only PL1 displayed distinct inhibition of in the confrontation assay. We identified the bioactive ingredients of sp. nov. 0813-124 to be glidobactin A and cepafungin I, with MIC values lower than 1.5 and 2.0 µg/mL, respectively. Furthermore, we revealed the biosynthetic gene cluster (BGC) of corresponding bioactive molecules through genomics analysis and determined its expression level in PL1 and PL2. The expression of glidobactin BGC in PL1 increased rapidly within 24 h, while PL2 was eventually stimulated after 60 h. In summary, we demonstrated that sp. nov. 0813-124 could potentially be used as a biocontrol agent or part of a natural product repertoire for combating .
PubMed: 35448634
DOI: 10.3390/jof8040403