The transcriptionally active bacterial communities of grapevine rhizosphere in dependence on rootstock and scion variety

The rhizosphere is where crucial processes for the productivity of viticultural systems occur. The composition of the bacterial communities associated with the rhizosphere of grapevines is known to depend on plant genotype. However, the genotype of grafted grapevines differs between scion and rootstock; the role of each genotype is unclear. To untangle the effect of scion and rootstock, the rRNA (V4–V5 region of 16S rRNA) extracted from the rhizosphere of the grape varieties Riesling and Mueller-Thurgau ungrafted vs grafted on different rootstocks was sequenced. The bioinformatic analysis with tools designed to be robust for compositional data showed that the investigated rootstocks or scions or combinations, respectively, recruited bacterial communities with distinguishable traits. Statistical differences were revealed between ungrafted Riesling vs Mueller-Thurgau, between grafted Riesling vs ungrafted Riesling, and between ungrafted Mueller-Thurgau vs grafted Mueller-Thurgau. Thus, confirming the role of scion and rootstock genotype as a driver of the structure and composition of bacterial communities in the rhizosphere of grapevines

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

International audienceVarious pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins

Semicarbazone EGA inhibits uptake of diphtheria toxin into human cells and protects cells article from intoxication

Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA) from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA) has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT). Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria

Rootstocks Shape Their Microbiome—Bacterial Communities in the Rhizosphere of Different Grapevine Rootstocks

The microbiota associated with the rhizosphere is responsible for crucial processes. Understanding how the plant and its bacterial community interact is of great importance to face the upcoming agricultural and viticultural challenges. The composition of the bacterial communities associated with the rhizosphere of grapevines is the result of the interaction between many drivers: biogeography, edaphic factors, soil management and plant genotype. The experimental design of this study aimed to reduce the variability resulting from all factors except the genotype of the rootstock. This was made possible by investigating four ungrafted grapevine rootstock varieties of the same age, grown on the same soil under the same climatic conditions and managed identically. The bacterial communities associated with the rhizosphere of the rootstocks 1103 Paulsen, 140 Ruggeri, 161-49 Couderc and Kober 5BB were characterized with the amplicon based sequencing technique, targeting regions V4–V5 of 16S rRNA gene. Linear discriminant analysis effect Size (LEfSe) analysis was performed to determine differential abundant taxa. The four rootstocks showed similarities concerning the structure of the bacteria assemblage (richness and evenness). Nonetheless, differences were detected in the composition of the bacterial communities. Indeed, all investigated rootstocks recruited communities with distinguishable traits, thus confirming the role of rootstock genotype as driver of the bacteria composition

EGA protects mammalian cells from clostridiumdifficile CDT, clostridium perfringens iota toxin clostridium botulinum C2 toxin

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxin

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

International audienceThe antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

CD44⁻ cells are resistant to iota and iota-like toxins versus CD44⁺ cells.

<p>(<b>A</b>) Dose-response of iota toxin on cells with controls consisting of cells in media only. The Y-axis represents the “% control” of F-actin content (Alexa-488 phalloidin stained after 5 h) in intoxicated cells versus controls in media only. (<b>B</b>) Like iota toxin, CD44⁺ RPM cells are also susceptible to <i>C. difficile</i> (CDT) and <i>C. spiroforme</i> (CST) binary toxins. Each assay was done in duplicate and values represent mean +/− standard deviation from three separate experiments.</p

Binding of iota-family B components to purified CD44 in solution.

<p>(<b>A and B</b>) The B component (10 µg) of each toxin was added to CD44-IgG (10 µg in 50 µl) at room temperature for 60 min. Protein A - agarose beads were then added for 5 min at room temperature, gently centrifuged, and washed with buffer. SDS-PAGE sample buffer containing reducing agent was added to the beads, the mixture heated, and protein separated from the beads by centrifugation. Supernatant proteins were then resolved on a 10% gel, transferred onto nitrocellulose, and clostridial B component detected with either 1∶1000 dilutions of rabbit anti-Ib (Panel A) or anti-C2II sera (Panel B). Protein A - peroxidase conjugate was used to detect bound antibody, and following washes, specific bands were visualized with chemiluminescent substrate. <b>(C)</b> Like the CD44-IgG construct, Ib also binds specifically to a CD44-GST construct. A GST version of <i>C. botulinum</i> C3 exoenzyme, used as a negative control, does not bind to Ib in pull-down experiments done similarly for panels A and B, with an exception being the use of glutathione-sepharose (instead of Protein A-agarose) beads.</p

Comparative binding of Ib to CD44⁺, versus CD44⁻, cells.

<p>Confocal microscopy was done with RPM (CD44⁻ and CD44⁺) cells incubated with Cy3-labeled Ib (10⁻⁷ M in DMEM +0.1% BSA) for 3 min at 37°C, washed with PBS, and mounted in mowiol. Blue represents Dapi-stained nuclei and red indicates cell-bound Ib. A representative field of cells is shown.</p