Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells

The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged as vital catalysts of cellular function. In order to unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, and developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed.

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Cell-penetrating and cargo-delivery ability of a spider toxin-derived peptide in mammalian cells

European Journal of Pharmaceutics and Biopharmaceutics ◽

10.1016/j.ejpb.2017.01.012 ◽

2017 ◽

Vol 114 ◽

pp. 145-153 ◽

Cited By ~ 24

Author(s):

Nisha Ponnappan ◽

Archana Chugh

Keyword(s):

Mammalian Cells ◽

Spider Toxin ◽

Cargo Delivery ◽

Cell Penetrating

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Glycosaminoglycans are specific endosomal receptors for Yersinia pseudotuberculosis Cytotoxic Necrotizing Factor

10.1101/2020.05.18.101790 ◽

2020 ◽

Author(s):

Stefanie Kowarschik ◽

Julian Schöllkopf ◽

Thomas Müller ◽

Songhai Tian ◽

Julian Knerr ◽

...

Keyword(s):

Cell Surface ◽

Conformational Change ◽

Mammalian Cells ◽

Yersinia Pseudotuberculosis ◽

Double Resonance ◽

Cytotoxic Necrotizing Factor ◽

Endosomal Membrane ◽

Terminal Domain ◽

Successful Infection ◽

Heparan Sulfates

AbstractThe Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author SummaryThe RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.

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Photothermal nanoblade for large cargo delivery into mammalian cells

2011 16th International Solid-State Sensors, Actuators and Microsystems Conference ◽

10.1109/transducers.2011.5969731 ◽

2011 ◽

Cited By ~ 5

Author(s):

T.-H. Wu ◽

T. Teslaa ◽

S. Kalim ◽

C.T. French ◽

S. Moghadam ◽

...

Keyword(s):

Mammalian Cells ◽

Cargo Delivery

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Cleavage of Escherichia coli Cytotoxic Necrotizing Factor 1 Is Required for Full Biologic Activity

Infection and Immunity ◽

10.1128/iai.01145-08 ◽

2009 ◽

Vol 77 (5) ◽

pp. 1835-1841 ◽

Cited By ~ 26

Author(s):

Zeynep Knust ◽

Britta Blumenthal ◽

Klaus Aktories ◽

Gudula Schmidt

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Catalytic Domain ◽

Small Gtpases ◽

Gtp Hydrolysis ◽

Cytotoxic Necrotizing Factor ◽

Endosomal Membrane ◽

Protein Toxin ◽

Late Endosomes ◽

Cytotoxic Necrotizing Factor 1

ABSTRACT Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by pathogenic Escherichia coli strains. CNF1 constitutively activates small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. The toxin is taken up into mammalian cells by receptor-mediated endocytosis and is delivered from late endosomes into the cytosol. Here, we show that an approximately 55-kDa fragment of CNF1, which contains the catalytic domain and an additional part of the toxin, is present in the cytosol. The processing of this fragment requires an acidic pH and insertion of the toxin into the endosomal membrane. We define the cleavage site region as the region located between amino acids 532 and 544 of CNF1. The data provide insight into the complex mechanism of uptake of bacterial toxins into mammalian cells.

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Ultrastructural Alterations in Hela Cells Induced by Spider Venom

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060118 ◽

1967 ◽

Vol 25 ◽

pp. 20-21

Author(s):

Dale E. McClendon ◽

Paul N. Morgan ◽

Bernard L. Soloff

Keyword(s):

Growth Medium ◽

Mammalian Cells ◽

Carcinoma Cells ◽

Venom Protein ◽

Sterile Saline ◽

Carbon Coated ◽

Available Information ◽

Loxosceles Reclusa ◽

Essential Growth ◽

Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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