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Advanced Science (Weinheim,... Jun 2024Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii...
Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.
Topics: Anthraquinones; Polyketides; Melanins; Polyketide Synthases; Photorhabdus; Naphthols; Pigments, Biological
PubMed: 38491909
DOI: 10.1002/advs.202400184 -
Current Microbiology Jun 2024One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its...
One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its taxonomic position. The 16S rRNA gene sequences indicate that it belongs to the class Gammaproteobacteria, to the family Morganellaceae, to the genus Photorhabdus, and likely represents a novel bacterial species. This strain, designated here as CRI-LC, was therefore molecularly, biochemically, and morphologically characterized to describe the novel bacterial species. Phylogenetic reconstructions using 16S rRNA gene sequences show that CRI-LC is closely related to P. laumondii subsp. laumondii TT01 and to P. laumondii subsp. clarkei BOJ-47. The 16rRNA gene sequences between CRI-LC and P. laumondii subsp. laumondii TT01 are 99.1% identical, and between CRI-LC and P. laumondii subsp. clarkei BOJ-47 are 99.2% identical. Phylogenetic reconstructions using whole genome sequences show that CRI-LC is closely related to P. laumondii subsp. laumondii TT01 and to P. laumondii subsp. clarkei BOJ-47. Moreover, digital DNA-DNA hybridization (dDDH) values between CRI-LC and its two relative species P. laumondii subsp. laumondii TT01 and P. laumondii subsp. clarkei BOJ-47 are 65% and 63%, respectively. In addition, we observed that average nucleotide identity (ANI) values between CRI-LC and its two relative species P. laumondii subsp. laumondii TT01 and P. laumondii subsp. clarkei BOJ-47 are 95.8% and 95.5%, respectively. These values are below the 70% dDDH and the 95-96% ANI divergence thresholds that delimits prokaryotic species. Based on these genomic divergence values, and the phylogenomic separation, we conclude that CRI-LC represents a novel bacterial species, for which we propose the name Photorhabdus africana sp. nov. with CRI-LC (= CCM 9390 = CCOS 2112) as the type strain. The following biochemical tests allow to differentiate P. africana sp. nov. CRI-LC from other species of the genus, including its more closely related taxa: β-Galactosidase, citrate utilization, urease and tryptophan deaminase activities, indole and acetoin production, and glucose and inositol oxidation. Our study contributes to a better understanding of the taxonomy and biodiversity of this important bacterial group with great biotechnological and agricultural potential.
Topics: Phylogeny; Photorhabdus; Animals; RNA, Ribosomal, 16S; DNA, Bacterial; Rhabditoidea; Sequence Analysis, DNA; Bacterial Typing Techniques
PubMed: 38910178
DOI: 10.1007/s00284-024-03744-3 -
Biochemical Pharmacology Feb 2024Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic...
Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic nematodes. As part of our ongoing exploration to identify bioactive compounds from diverse natural resources, the chemical analysis of the cultures of P. luminescens KACC 12254 via LC/MS and TLC-based analyses enabled the isolation and identification of a major fluorescent compound. Its chemical structure was elucidated as 1,8-dihydroxy-3-methoxyanthraquinone (DMA) using HR-ESI-MS and NMR analysis. In this study, we conducted comprehensive investigations utilizing human colorectal cancer HCT116 cells, human umbilical cord vascular endothelial cells (HUVECs), and zebrafish embryos to assess the potential benefits of DMA in suppressing tumor angiogenesis. Our results convincingly demonstrate that DMA effectively suppresses the stability of hypoxia-inducible factor-1α (HIF-1α) protein and its target genes without inducing any cytotoxic effects. Furthermore, DMA demonstrates the ability to inhibit HIF-1α transcriptional activation and mitigate the production of reactive oxygen species (ROS). In our in vitro experiments, DMA exhibits notable inhibitory effects on VEGF-mediated tube formation, migration, and invasion in HUVECs. Additionally, in vivo investigations using zebrafish embryos confirm the antiangiogenic properties of DMA. Notably, DMA does not exhibit any adverse developmental or cardiotoxic effects in the in vivo setting. Moreover, we observe DMA's capability to restrain tumor growth through the downregulation of PI3K/AKT and c-RAF/ERK pathway. Collectively, these compelling findings underscore DMA's potential as a promising therapeutic candidate for targeted intervention against HIF-1α and angiogenesis in cancer treatment.
Topics: Animals; Humans; Angiogenesis; Anthraquinones; Cell Line, Tumor; Down-Regulation; Endothelial Cells; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Signal Transduction; Zebrafish
PubMed: 38072164
DOI: 10.1016/j.bcp.2023.115972 -
Plant Disease Nov 2023Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our...
Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from bacteria, -cinnamic acid (-CA), and (4)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN in a cowpea. Factorial combinations of five -CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 μg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the -CA + PPA mixture significantly reduced the penetration of infective juveniles (J2s) into the cowpea roots. The potential toxicity of -CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of -CA + PPA × nematode inoculation interactions and the -CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of -CA and PPA reduces J2's ability to infect the roots without impairing plant growth or chlorophyll content.
Topics: Animals; Tylenchoidea; Vigna; Photorhabdus; Antinematodal Agents; Chlorophyll
PubMed: 37330631
DOI: 10.1094/PDIS-11-22-2574-SC -
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 -
Biochemistry. Biokhimiia Sep 2023Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes,...
Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.
Topics: Animals; Photorhabdus; Protease Inhibitors; Insecta; Anti-Infective Agents; Antiviral Agents
PubMed: 37770402
DOI: 10.1134/S0006297923090158 -
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 -
Research in Microbiology 2023The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus...
The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus laumondii TT01 has emerged as a goldmine for novel anti-infective drug discovery. Outside plants, Photorhabdus is the only Gram-negative known to produce stilbene-derivatives including 3,5-dihydroxy-4-ethyl-trans-stilbene and 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS). IPS is a bioactive polyketide which received considerable attention, mainly because of its antimicrobial properties, and is currently in late-stage clinical development as a topical treatment for psoriasis and dermatitis. To date, little is known about how Photorhabdus survives in the presence of stilbenes. We combined genetic and biochemical approaches to assess whether AcrAB efflux pump exports stilbenes in P. laumondii. We demonstrated that the wild-type (WT) exerts an antagonistic activity against its derivative ΔacrA mutant, and that is able to outcompete it in a dual-strain co-culture assay. The ΔacrA mutant also showed high sensitivity to 3,5-dihydroxy-4-ethyl-trans-stilbene and IPS as well as decreased IPS concentrations in its supernatant comparing to the WT. We report here a mechanism of self-resistance against stilbene derivatives of P. laumondii TT01, which enables these bacteria to survive under high concentrations of stilbenes by extruding them out via the AcrAB efflux pump.
PubMed: 37196776
DOI: 10.1016/j.resmic.2023.104081 -
Applied Microbiology and Biotechnology Dec 2023The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect...
The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM HO; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.
Topics: Animals; Genotype; Hydrogen Peroxide; Nematoda; Insecta; Photorhabdus; Symbiosis
PubMed: 37733051
DOI: 10.1007/s00253-023-12775-y