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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 -
Life Science Alliance Oct 2019Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches....
Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.
Topics: Animals; Bacterial Proteins; Bacterial Toxins; Cloning, Molecular; Copepoda; Cysteine Endopeptidases; Drug Delivery Systems; HeLa Cells; Humans; Injections; Luciferases; Nanoparticles; Photorhabdus; Protein Engineering
PubMed: 31540947
DOI: 10.26508/lsa.201900485 -
International Journal of Systematic and... Sep 2021Two Gram-negative, rod-shaped bacteria, H1 and H3, isolated from the digestive tract of entomopathogenic nematodes were biochemically and molecularly characterized to...
Two Gram-negative, rod-shaped bacteria, H1 and H3, isolated from the digestive tract of entomopathogenic nematodes were biochemically and molecularly characterized to determine their taxonomic positions. The 16S rRNA gene sequences of these strains indicate that they belong to the Gammaproteobacteria, to the family , and to the genus. Deeper analyses using whole genome-based phylogenetic reconstructions show that strains H1 and H3 are closely related to DSM 15138, to DSM 22397, and to PB45.5. genomic comparisons confirm these observations and show that strain H1 shares 70.6, 66.8, and 63.5 % digital DNA-DNA hybridization (dDDH) with DSM 15138, DSM 22397, and PB45.5, respectively, and that strain H3 shares 76.6, 69.4, and 59.2 % dDDH with DSM 15138, DSM 22397, and PB45.5, respectively. Physiological and biochemical characterization reveals that these two strains differ from most of the validly described species and from their more closely related taxa. Given the clear phylogenetic separations, that the threshold to discriminate species and subspecies is 70 and 79% dDDH, respectively, and that strains H1 and H3 differ physiologically and biochemically from their more closely related taxa, we propose to classify H1 and H3 into new taxa as follows: H3 as a new subspecies within the species , and H1 as a new species within the genus, in spite that H1 shares 70.6 % dDDH with DSM 15138, score that is slightly higher than the 70 % threshold that delimits species boundaries. The reason for this is that H1 and DSM 15138 cluster apart in the phylogenetic trees and that dDDH scores between strain H1 and other strains are lower than 70 %. Hence, the following names are proposed: sp. nov. with the type strain H1 (=IARI-SGMG3,=KCTC 82683=CCM 9150=CCOS 1975) and subsp. subsp. nov. with the type strain H3 (=IARI-SGHR2=KCTC 82684=CCM 9149=CCOS 1976). These propositions automatically create subsp. subsp. nov. with DSM 15138 as the type strain (currently classified as ).
Topics: Animals; Bacterial Typing Techniques; Base Composition; DNA, Bacterial; Fatty Acids; Nematoda; Nucleic Acid Hybridization; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 34524954
DOI: 10.1099/ijsem.0.004998 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Nov 2021Photorhabdus is a Gram-negative bacterium from the family Enterobacteriaceae that lives in a symbiotic association with nematode or insects. In addition to the role of...
Photorhabdus is a Gram-negative bacterium from the family Enterobacteriaceae that lives in a symbiotic association with nematode or insects. In addition to the role of being insect pathogens, one species called Photorhabdus asymbiotica (Pa) causes human infection around the world. Nevertheless, how does this transkingdom infection occur remains elusive. Here we focus on one pathogenic determinant called Photorhabdus virulence cassette (PVC) that is founded in the Pa genome and many other pathogens. The RNA-seq and qPCR data showed that the NF-κB and MAPK pathways were drastically activated in the PVC-treated mammalian macrophages. Western blotting assays using samples treated with various inhibitors of the affected pathways confirmed the results we have observed for MAPK pathway previously. p65 translocation assays validated the NF-κB activation in the macrophages after PVC treatment. Moreover, the bacterial phagocytosis by macrophage was also promoted by PVC at the early stage, and this phagocytosis was inhibited by cytoskeleton inhibitors. Thus, the results indicated that PVC is involved in the bacterial invasion by activating NF-κB and MAPK signaling pathway, providing a new perspective for analyzing the pathogenicity of Pa in human infections.
Topics: Animals; Humans; Macrophages; NF-kappa B; Photorhabdus; Signal Transduction; Virulence
PubMed: 34841805
DOI: 10.13345/j.cjb.210148 -
Trends in Microbiology Dec 2010Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses... (Review)
Review
Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses entomopathogenic nematodes as vectors in a system that represents a useful tool for probing the molecular basis of immunity. During the course of infection, Photorhabdus multiplies rapidly within the insect, producing a range of toxins that inhibit phagocytosis of the invading bacteria and eventually kill the insect host. Photorhabdus bacteria have recently been established as a tool for investigating immune recognition and defense mechanisms in model hosts such as Manduca and Drosophila. Such studies pave the way for investigations of gene interactions between pathogen virulence factors and host immune genes, which ultimately could lead to an understanding of how some Photorhabdus species have made the leap to becoming human pathogens.
Topics: Animals; Humans; Immune Evasion; Insecta; Nematoda; Phagocytosis; Photorhabdus
PubMed: 21035345
DOI: 10.1016/j.tim.2010.09.006 -
International Journal of Systematic and... Jan 2021Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1, Q614 and PB68.1, isolated from the digestive system of entomopathogenic nematodes, were...
Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1, Q614 and PB68.1, isolated from the digestive system of entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family , and to the genus . Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1 is closely related to , that Q614 is closely related to and that PB68.1 is closely related to genomic comparisons confirm these observations: BA1 and 15138 share 68.8 % digital DNA-DNA hybridization (dDDH), Q614 and SF41 share 75.4 % dDDH, and PB68.1 and DSM 17609 share 76.6 % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1 as a new species within the genus , Q614 as a new subspecies within and PB68.1 as a new subspecies within . Hence, the following names are proposed for these strains: sp. nov. with the type strain BA1(=DSM 111180=CCOS 1943=LMG 31957), subsp subsp. nov. with the type strain Q614 (=DSM 111144=CCOS 1944=LMG 31959) and subsp subsp. nov. with the type strain PB68.1 (=DSM 111145=CCOS 1942). These propositions automatically create subsp subsp. nov. with SF41 as the type strain (currently classified as ) and subsp. subsp. nov. with DSM17609 as the type strain (currently classified as ).
Topics: Animals; Australia; Bacterial Typing Techniques; Base Composition; DNA, Bacterial; Digestive System; Egypt; Nematoda; Nucleic Acid Hybridization; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Thailand
PubMed: 33464198
DOI: 10.1099/ijsem.0.004610 -
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 -
Advances in Experimental Medicine and... 2012The outcome of any bacterial infection, whether it is clearance of the infecting pathogen, establishment of a persistent infection, or even death of the host, is as... (Review)
Review
The outcome of any bacterial infection, whether it is clearance of the infecting pathogen, establishment of a persistent infection, or even death of the host, is as dependent on the host as on the pathogen (Finlay and Falkow 1989). To infect a susceptible host bacterial pathogens express virulence factors, which alter host cell physiology and allow the pathogen to establish a nutrient-rich niche for growth and avoid clearance by the host immune response. However survival within the host often results in tissue damage, which to some cases accounts for the disease-specific pathology. For many bacterial pathogens the principal determinants of virulence and elicitors of host tissue damage are soluble exotoxins, which allow bacteria to penetrate into deeper tissue or pass through a host epithelial or endothelial barrier. Therefore, exploring the complex interplay between host tissue and bacterial toxins can help us to understand infectious disease and define the contributions of the host immune system to bacterial virulence. In this chapter, we describe a new model, the Drosophila embryo, for addressing a fundamental issue in bacterial pathogenesis, the elucidation of the in vivo targets of bacterial toxins and the monitoring of the first moments of the infection process in real-time. To develop this model, we used the insect and emerging human pathogen Photorhabdus asymbiotica and more specifically we characterised the initial cross-talk between the secreted cytotoxin Mcf1 and the embryonic hemocytes. Mcf1 is a potent cytotoxin which has been detected in all Photorhabdus strains isolated so far, which can rapidly kill insects upon injection. Despite several in vitro tissue culture studies, the biology of Mcf1 in vivo is not well understood. Furthermore, despite the identification of many Photorhabdus toxins using recombinant expression in E. coli (Waterfield et al. 2008), very few studies address the molecular mechanism of action of these toxins in relation to specific immune responses in vivo in the insect model.
Topics: Animals; Bacterial Toxins; Disease Models, Animal; Drosophila; Hemocytes; Humans; Photorhabdus; Virulence Factors
PubMed: 22127885
DOI: 10.1007/978-1-4419-5638-5_6 -
Current Topics in Microbiology and... 2017A general definition of secondary metabolism is that it consists of the metabolic pathways and the products of metabolism that are not absolutely required for the...
A general definition of secondary metabolism is that it consists of the metabolic pathways and the products of metabolism that are not absolutely required for the survival of the organism. Using this definition, it is now well established that Photorhabdus elaborate an extensive secondary metabolism during the post-exponential phase of bacterial growth. This secondary metabolism includes, but is not limited to, the production of light, a stilbene antibiotic and an anthraquinone pigment. In this chapter, the role of secondary metabolism during the life cycle of Photorhabdus will be discussed. Recent work has shown that secondary metabolism in Photorhabdus is required for the mutualistic association between the bacteria and its nematode partner, in particular bacterial secondary metabolism is required to support normal nematode growth and development. An isogenic population of Photorhabdus is phenotypically heterogenous and this facilitates functional partitioning within the population. The relationship between secondary metabolism and the various phenotypic and phase variants that exist in populations of Photorhabdus will also be discussed. Finally, this chapter will also describe the various regulatory nodes that have been identified as being part of the complex regulatory network that is used to control the temporal expression of secondary metabolism in Photorhabdus.
Topics: Anthraquinones; Photorhabdus; Secondary Metabolism; Symbiosis
PubMed: 27469305
DOI: 10.1007/82_2016_21 -
ELife Sep 2019is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small...
is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential.
Topics: Bacterial Toxins; Epithelial Cells; HeLa Cells; Humans; Photorhabdus; Protein Transport; Virulence; Virulence Factors
PubMed: 31526474
DOI: 10.7554/eLife.46259