Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins.

Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.

Corrigendum: The Photorhabdus virulence cassettes RRSP-like effector interacts with cyclin-dependent kinase 1 and causes mitotic defects in mammalian cells.

[This corrects the article DOI: 10.3389/fmicb.2020.00366.].

Corrigendum: The Photorhabdus virulence cassettes RRSP-like effector interacts with cyclin-dependent kinase 1 and causes mitotic defects in mammalian cells.

[This corrects the article DOI: 10.3389/fmicb.2020.00366.].

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica.

The extracellular contractile injection systems (eCISs) are encoded in the genomes of a large number of bacteria and archaea. We have previously characterized the overall structure of Photorhabdus Virulence Cassette (PVC), a typical member of the eCIS family. PVC resembles the contractile tail of bacteriophages and exerts its action by the contraction of outer sheath and injection of inner tube plus central spike. Nevertheless, the biological function of PVC effectors and the mechanism of effector translocation are still lacking. By combining cryo-electron microscopy and functional experiments, here we show that the PVC effectors Pdp1 (a new family of widespread dNTP pyrophosphatase effector in eCIS) and Pnf (a deamidase effector) are loaded inside the inner tube lumen in a "Peas in the Pod" mode. Moreover, we observe that Pdp1 and Pnf can be directly injected into J774A.1 murine macrophage and kill the target cells by disrupting the dNTP pools and actin cytoskeleton formation, respectively. Our results provide direct evidence of how PVC cargoes are loaded and delivered directly into mammalian macrophages.

[Photorhabdus virulence cassette promotes bacterial invasion into macrophages by activating NF-κB and MAPK signaling pathway].

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.

Photorhabdus virulence cassette promotes bacterial invasion into macrophages by activating NF-κB and MAPK signaling pathway / 生物工程学报

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.

The Photorhabdus Virulence Cassettes RRSP-Like Effector Interacts With Cyclin-Dependent Kinase 1 and Causes Mitotic Defects in Mammalian Cells.

The "Photorhabdus virulence cassettes" (PVCs) secreted by Photorhabdus are defined as "extracellular contractile injection systems" (eCISs) and can deliver effectors to eukaryotic hosts for cytotoxicity. Previously, we demonstrated the cryogenic electron microscopy (cryo-EM) structure and assembly process of an intact PVC particle from Photorhabdus asymbiotica. In this work, we characterized the biological functions of a PVC effector, which is defined as a homologous protein of Ras/Rap1-specific endopeptidase domain (RRSP) in the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin from Vibrio vulnificus. In this work, we found that the RRSP homologous protein (RRSPPa) was associated with inhibition of cell proliferation and increased cell apoptosis and death of HeLa cells. Furthermore, we discovered that RRSPPa disturbed mitotic progression, including the induction of cell cycle alteration, retardation of cell abscission time, and regression of the cleavage furrow. In addition, we revealed that RRSPPa could target the cyclin-dependent kinase 1 (CDK1) protein and block activation of CDK1 through inhibition of Thr161 phosphorylation, which partially explained the crucial role of this effector in cell mitosis.

Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System.

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.

Synthesis of α-l-Fucopyranoside-Presenting Glycoclusters and Investigation of Their Interaction with Photorhabdus asymbiotica Lectin (PHL).

Photorhabdus asymbiotica is a gram-negative bacterium that is not only as effective an insect pathogen as other members of the genus, but it also causes serious diseases in humans. The recently identified lectin PHL from P. asymbiotica verifiably modulates an immune response of humans and insects, which supports the idea that the lectin might play an important role in the host-pathogen interaction. Dimeric PHL contains up to seven l-fucose-specific binding sites per monomer, and in order to target multiple binding sites of PHL, α-l-fucoside-containing di-, tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol were chosen as multivalent scaffolds, and the fucoclusters were built from the above-mentioned cores by coupling with different oligoethylene bridges and propargyl α-l-fucosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between fucoside derivates and PHL was investigated by several biophysical and biological methods, ITC and SPR measurements, hemagglutination inhibition assay, and an investigation of bacterial aggregation properties were carried out. Moreover, details of the interaction between PHL and propargyl α-l-fucoside as a monomer unit were revealed using X-ray crystallography. Besides this, the interaction with multivalent compounds was studied by NMR techniques. The newly synthesized multivalent fucoclusters proved to be up to several orders of magnitude better ligands than the natural ligand, l-fucose.