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MicroPublication Biology 2023The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The...
The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode . Here we find that oral infection of larvae with moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.
PubMed: 37711508
DOI: 10.17912/micropub.biology.000938 -
Nature Communications Dec 2023The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1...
The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly understood. Here, we present the cryo-EM structure of Mcf1 from Photorhabdus luminescens, revealing a seahorse-like shape with a head and tail. While the three head domains contain two effectors, as well as an activator-binding domain (ABD) and an autoprotease, the tail consists of two putative translocation and three putative receptor-binding domains. Rearrangement of the tail moves the C-terminus away from the ABD and allows binding of the host cell ADP-ribosylation factor 3, inducing conformational changes that position the cleavage site closer to the protease. This distinct activation mechanism that is based on a hook-loop interaction results in three autocleavage reactions and the release of two toxic effectors. Unexpectedly, the BH3-like domain containing ABD is not an active effector. Our findings allow us to understand key steps of Mcf1 intoxication at the molecular level.
Topics: Animals; Bacterial Toxins; Lepidoptera; Apoptosis; Peptide Hydrolases
PubMed: 38086871
DOI: 10.1038/s41467-023-44069-2 -
Archives of Biochemistry and Biophysics May 2024Recent research into membrane interactions has uncovered a diverse range of therapeutic opportunities through the bioengineering of human and non-human macromolecules....
Recent research into membrane interactions has uncovered a diverse range of therapeutic opportunities through the bioengineering of human and non-human macromolecules. Although the majority of this research is focussed on fundamental developments, emerging studies are showcasing promising new technologies to combat conditions such as cancer, Alzheimer's and inflammatory and immune-based disease, utilising the alteration of bacteriophage, adenovirus, bacterial toxins, type 6 secretion systems, annexins, mitochondrial antiviral signalling proteins and bacterial nano-syringes. To advance the field further, each of these opportunities need to be better understood, and the therapeutic models need to be further optimised. Here, we summarise the knowledge and insights into several membrane interactions and detail their current and potential uses therapeutically.
PubMed: 38387829
DOI: 10.1016/j.abb.2024.109939 -
Toxins Jan 2024and bacterial symbionts of entomopathogenic nematodes and , respectively, have several biological activities including insecticidal and antimicrobial activities.... (Comparative Study)
Comparative Study
and bacterial symbionts of entomopathogenic nematodes and , respectively, have several biological activities including insecticidal and antimicrobial activities. Thus, XnChi, XhChi, and PtChi, chitinases of , , and isolated from Korean indigenous EPNs GJ1-2, GJ11-1, and GJ1-2 were cloned and expressed in BL21 to compare their biological activities. Chitinase proteins of these bacterial symbionts purified using the Ni-NTA system showed different chitobiosidase and endochitinase activities, but N-acetylglucosamidinase activities were not shown in the measuring of chitinolytic activity through N-acetyl-D-glucosarmine oligomers. In addition, the proteins showed different insecticidal and antifungal activities. XnChi showed the highest insecticidal activity against , followed by PtChi and XhChi. In antifungal activity, XhChi showed the highest half-maximal inhibitory concentration (IC) against with 0.031 mg/mL, followed by PtChi with 0.046 mg/mL, and XnChi with 0.072 mg/mL. XhChi also showed the highest IC against with 0.040 mg/mL, but XnChi was more toxic than PtChi with 0.055 mg/mL and 0.133 mg/mL, respectively. This study provides an innovative approach to the biological control of insect pests and fungal diseases of plants with the biological activity of symbiotic bacterial chitinases of entomopathogenic nematodes.
Topics: Animals; Antifungal Agents; Bacteria; Chitinases; Escherichia coli; Insecticides; Nematoda; Symbiosis; Republic of Korea
PubMed: 38251242
DOI: 10.3390/toxins16010026 -
Antibiotics (Basel, Switzerland) Sep 2023Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug...
Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen and the gallinaceous bird pathogen ) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that and are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.
PubMed: 37760758
DOI: 10.3390/antibiotics12091462 -
Parasites & Vectors Oct 2023Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural...
Taxonomic and molecular characterization of a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and whole genome sequencing of its associated bacterial symbiont.
BACKGROUND
Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis.
METHODS
Six Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria.
RESULTS
The newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77-100 vs. 121-130 μm), V% [(anterior end of vulva/body length) × 100] (46-57 vs. 41-47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13-2.0 vs. 0.32-0.39 mm), body diameter (73-150 vs. 160-220 μm), anterior end to the excretory pore distance (135-157 vs. 174-214 μm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with Photorhabdus luminescence subsp. clarkei symbiotic bacteria.
CONCLUSIONS
The discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India.
Topics: Female; Animals; Rhabditoidea; Phylogeny; Nematoda; Photorhabdus; Whole Genome Sequencing
PubMed: 37880744
DOI: 10.1186/s13071-023-05990-z -
The Journal of General and Applied... May 2024There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase...
There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and Km marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.
Topics: Plasmids; Genes, Reporter; Luciferases; Photorhabdus; Conjugation, Genetic; Escherichia coli; Promoter Regions, Genetic; Operon; Salmonella enterica
PubMed: 37940551
DOI: 10.2323/jgam.2023.10.001 -
Bio-protocol Jul 2023The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs)...
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
PubMed: 37449040
DOI: 10.21769/BioProtoc.4709 -
Journal of Fungi (Basel, Switzerland) Feb 2024Fungal diseases such as Fusarium head blight (FHB) are significant biotic stressors, negatively affecting wheat production and quality. This study explored the...
Fungal diseases such as Fusarium head blight (FHB) are significant biotic stressors, negatively affecting wheat production and quality. This study explored the antifungal activity of the metabolites produced by the bacterial symbionts of entomopathogenic nematodes (EPNs) against FHB-causing sp. . To achieve this, the symbiotic bacteria of nine EPN isolates from the EPN collection at the Agricultural Research Council-Small Grains (ARC-SG) were isolated from the cadavers of (Lepidoptera: ) larvae after infection with EPNs. Broth cultures (crude) and their supernatants (filtered and autoclaved) of each bacterial isolate were used as bacterial metabolite treatments to test their inhibitory effect on the mycelial growth and spore germination of . Mycelial growth inhibition rates varied among both bacterial isolates and treatments. Crude metabolite treatments proved to be more effective than filtered and autoclaved metabolite treatments, with an overall inhibition rate of 75.25% compared to 23.93% and 13.32%, respectively. From the crude metabolite treatments, the SGI 197 bacterial isolate from SGI 197 had the highest mean inhibition rate of 96.25%, followed by SGI 170 bacteria isolated from SGI 170 with a 95.79% mean inhibition rate. The filtered metabolite treatments of all bacterial isolates were tested for their inhibitory activity against spore germination. Mean spore germination inhibition rates from spp. bacterial isolates were higher (83.91 to 96.29%) than those from spp. (6.05 to 14.74%). The results obtained from this study suggest that EPN symbiotic bacterial metabolites have potential use as biological control agents of FHB. Although field efficacy against FHB was not studied, the significant inhibition of mycelial growth and spore germination suggest that the application of these metabolites at the flowering stage may provide protection to plants against infection with or spread of . These metabolites have the potential to be employed as part of integrated pest management (IPM) to inhibit/delay conidia germination until the anthesis (flowering stage) of wheat seedlings has passed.
PubMed: 38392820
DOI: 10.3390/jof10020148 -
Animals : An Open Access Journal From... May 2024Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the...
Acute hepatopancreatic necrosis disease (AHPND) poses a significant threat to shrimp aquaculture worldwide, necessitating the accurate and rapid detection of the pathogens. However, the increasing number of species that cause the disease makes diagnosis and control more difficult. This study focuses on developing a monoclonal antibody against the insect-related (Pir) toxin B (PirB), a pivotal virulence factor in AHPND-causing , and establishing a colloidal gold immunochromatographic assay for the enhanced early diagnosis and monitoring of AHPND. Monoclonal antibodies targeting PirB were developed and utilized in the preparation of colloidal-gold-labeled antibodies for the immunochromatographic assay. The specificity and sensitivity of the assay were evaluated through various tests, including antibody subclass detection, affinity detection, and optimal labeling efficiency assessment. The developed PirB immunochromatographic test strips exhibited a good specificity, as demonstrated by the positive detection of AHPND-causing and negative results for non-AHPND-causing . The study highlights the potential of the developed monoclonal antibody and immunochromatographic assay for the effective detection of AHPND-causing . Further optimization is needed to enhance the sensitivity of the test strips for improved practical applications in disease prevention and control in shrimp aquaculture.
PubMed: 38891648
DOI: 10.3390/ani14111600