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Nature Apr 2023Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile...
Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells, raising the possibility that these systems could be harnessed for therapeutic protein delivery. However, whether eCISs can function in human cells remains unknown, and the mechanism by which these systems recognize target cells is poorly understood. Here we show that target selection by the Photorhabdus virulence cassette (PVC)-an eCIS from the entomopathogenic bacterium Photorhabdus asymbiotica-is mediated by specific recognition of a target receptor by a distal binding element of the PVC tail fibre. Furthermore, using in silico structure-guided engineering of the tail fibre, we show that PVCs can be reprogrammed to target organisms not natively targeted by these systems-including human cells and mice-with efficiencies approaching 100%. Finally, we show that PVCs can load diverse protein payloads, including Cas9, base editors and toxins, and can functionally deliver them into human cells. Our results demonstrate that PVCs are programmable protein delivery devices with possible applications in gene therapy, cancer therapy and biocontrol.
Topics: Animals; Humans; Mice; Cell Membrane; Eukaryotic Cells; Photorhabdus; CRISPR-Associated Protein 9; Toxins, Biological; Proteins; Drug Delivery Systems; Protein Transport
PubMed: 36991127
DOI: 10.1038/s41586-023-05870-7 -
Nature Dec 2019The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens. These microorganisms have a highly restrictive permeability...
The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cell Line; Disease Models, Animal; Drug Discovery; Drug Resistance, Microbial; Escherichia coli Proteins; Female; Gastrointestinal Microbiome; Gram-Negative Bacteria; Humans; Mice; Microbial Sensitivity Tests; Microbial Viability; Mutation; Nematoda; Operon; Phenylpropionates; Photorhabdus; Substrate Specificity; Symbiosis
PubMed: 31747680
DOI: 10.1038/s41586-019-1791-1 -
Ecology and Evolution Feb 2024Understanding how parasites evolved is crucial to understand the host and parasite interaction. The evolution of entomopathogenesis in rhabditid nematodes has... (Review)
Review
Understanding how parasites evolved is crucial to understand the host and parasite interaction. The evolution of entomopathogenesis in rhabditid nematodes has traditionally been thought to have occurred twice within the phylum Nematoda: in Steinernematidae and Heterorhabditidae families, which are associated with the entomopathogenic bacteria and , respectively. However, nematodes from other families that are associated with entomopathogenic bacteria have not been considered to meet the criteria for "entomopathogenic nematodes." The evolution of parasitism in nematodes suggests that ecological and evolutionary properties shared by families in the order Rhabditida favor the convergent evolution of the entomopathogenic trait in lineages with diverse lifestyles, such as saprotrophs, phoretic, and necromenic nematodes. For this reason, this paper proposes expanding the term "entomopathogenic nematode" considering the diverse modes of this attribute within Rhabditida. Despite studies are required to test the authenticity of the entomopathogenic trait in the reported species, they are valuable links that represent the early stages of specialized lineages to entomopathogenic lifestyle. An ecological and evolutionary exploration of these nematodes has the potential to deepen our comprehension of the evolution of entomopathogenesis as a convergent trait spanning across the Nematoda.
PubMed: 38352205
DOI: 10.1002/ece3.10966 -
Microbiology (Reading, England) Apr 2020Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between... (Review)
Review
Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family . The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore has three distinct and obligate roles to play during this life-cycle: (1) must kill the insect host; (2) must be capable of supporting nematode growth and development; and (3) must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.
Topics: Animals; Bacterial Toxins; Gastrointestinal Tract; Host Microbial Interactions; Insecta; Life Cycle Stages; Photorhabdus; Rhabditoidea; Signal Transduction; Symbiosis
PubMed: 32209172
DOI: 10.1099/mic.0.000907 -
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 -
Microbiological Research Mar 2021In last years, the main studied microbial sources of natural blue pigments have been the eukaryotic algae, Rhodophytes and Cryptophytes, and the cyanobacterium... (Review)
Review
In last years, the main studied microbial sources of natural blue pigments have been the eukaryotic algae, Rhodophytes and Cryptophytes, and the cyanobacterium Arthrospira (Spirulina) platensis, responsible for the production of phycocyanin, one of the most important blue compounds approved for food and cosmetic use. Recent research also includes the indigoidine pigment from the bacteria Erwinia, Streptomyces and Photorhabdus. Despite these advances, there are still few options of microbial blue pigments reported so far, but the interest in these products is high due to the lack of stable natural blue pigments in nature. Filamentous fungi are particularly attractive for their ability to produce pigments with a wide range of colors. Bikaverin is a red metabolite present mainly in species of the genus Fusarium. Although originally red, the biomass containing bikaverin changes its color to blue after heat treatment, through a mechanism still unknown. In addition to the special behavior of color change by thermal treatment, bikaverin has beneficial biological properties, such as antimicrobial and antiproliferative activities, which can expand its use for the pharmaceutical and medical sectors. The present review addresses the production natural blue pigments and focuses on the properties of bikaverin, which can be an important source of blue pigment with potential applications in the food industry and in other industrial sectors.
Topics: Color; Fusarium; Pigments, Biological; Xanthones
PubMed: 33302226
DOI: 10.1016/j.micres.2020.126653 -
Nature Oct 2022Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces...
Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Topics: Animals; Bacterial Toxins; Biological Control Agents; CRISPR-Cas Systems; Culicidae; Drosophila Proteins; Drosophila melanogaster; Fat Body; Gene Editing; Gene Knockdown Techniques; Hemocytes; Humans; Moths; Mucins; Pest Control, Biological; Phagocytosis; Photorhabdus; Repetitive Sequences, Amino Acid; Transgenes; Virulence Factors
PubMed: 36171290
DOI: 10.1038/s41586-022-05250-7 -
Applied and Environmental Microbiology Aug 2020The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease...
The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The -EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of in the rhizosphere. We performed transcriptome analysis to show how responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions. Insect-pathogenic bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that can specifically react to and interact with plant roots. Understanding the adaptation of in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.
Topics: Biological Control Agents; Chemotaxis; Exudates and Transudates; Fungi; Gene Expression Profiling; Genes, Bacterial; Photorhabdus; Plant Roots; RNA-Seq; Rhizosphere
PubMed: 32591378
DOI: 10.1128/AEM.00891-20 -
Heliyon Mar 2023For decades, transcription of -operon was considered being constitutive. Therefore, this -operon has been used for measurements in non-specific bacterial luminescent...
For decades, transcription of -operon was considered being constitutive. Therefore, this -operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of -operon under high temperature was studied. The expression was researched in the natural strain and in the heterologous system of . FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of genes, which was confirmed by RT-PCR. The promoter of the -operon of the related bacterium ZM1 from the forests of Moldova, being cloned in the heterologous system of , is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of -operon increases only in the natural strain of , but not in the heterologous system of cells. In addition, the activation of the -operon of persists in strains deficient in both the and genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the -operon of in the heterologous system.
PubMed: 36950606
DOI: 10.1016/j.heliyon.2023.e14527 -
PLoS Biology Nov 2020Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy....
Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.
Topics: Actins; Animals; Bacterial Toxins; Binding Sites; Binding, Competitive; Cofilin 1; Cryoelectron Microscopy; Fluorescent Dyes; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; In Vitro Techniques; Microfilament Proteins; Microscopy, Confocal; Models, Molecular; Myosins; Peptide Fragments; Protein Engineering; Protein Interaction Domains and Motifs; Rabbits; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins
PubMed: 33216759
DOI: 10.1371/journal.pbio.3000925