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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 -
Cell Apr 2019Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC)...
Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.
Topics: Bacteriophage T4; Cell Membrane; Cryoelectron Microscopy; Models, Molecular; Photorhabdus; Protein Conformation; Type VI Secretion Systems
PubMed: 30905475
DOI: 10.1016/j.cell.2019.02.020 -
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 -
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 -
Frontiers in Microbiology 2022The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity... (Review)
Review
The importance of and symbionts to their respective and nematode hosts is that they not only contribute to their entomopathogenicity but also to their fecundity through the production of small molecules. Thus, this mini-review gives a brief introductory overview of these nematophilic bacteria. Specifically, their type species, nematode hosts, and geographic region of isolations are tabulated. The use of nucleotide sequence-based techniques for their species delineation and how pangenomes can improve this are highlighted. Using the - association as an example, the bacterium-nematode lifecycle is visualized with an emphasis on the role of bacterial biomolecules. Those currently in drug development are discussed, and two potential antimalarial lead compounds are highlighted. Thus, this mini-review tabulates forty-eight significant nematophilic bacteria and visualizes the ecological importance of their biomolecules. It further discusses three of these biomolecules that are currently in drug development. Through it, one is introduced to and bacteria, their natural production of biomolecules in the nematode-bacterium lifecycle, and how these molecules are useful in developing novel therapies.
PubMed: 36187939
DOI: 10.3389/fmicb.2022.993688 -
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 -
Toxins Jun 2010Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae. Recent studies show the... (Review)
Review
Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae. Recent studies show the importance of this bacterium as an alternative source of insecticides, as well as an emerging human pathogen. Various toxins have been identified and characterized in this bacterium. These toxins are classified into four major groups: the toxin complexes (Tcs), the Photorhabdus insect related (Pir) proteins, the "makes caterpillars floppy" (Mcf) toxins and the Photorhabdus virulence cassettes (PVC); the mechanisms however of toxin secretion are not fully elucidated. Using bioinformatics analysis and comparison against the components of known secretion systems, multiple copies of components of all known secretion systems, except the ones composing a type IV secretion system, were identified throughout the entire genome of the bacterium. This indicates that Photorhabdus luminescens has all the necessary means for the secretion of virulence factors, thus it is capable of establishing a microbial infection.
Topics: Bacterial Proteins; Bacterial Toxins; Humans; Photorhabdus
PubMed: 22069636
DOI: 10.3390/toxins2061250