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A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins

Nature Structural & Molecular Biology volume 20, pages 1273–1280 (2013)Cite this article

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Abstract

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP–N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. 1H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

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Figure 1: PaTox and toxic effects.
Figure 2: Photorhabdus glycosyltransferase GlcNAcylates Rho GTPases at a tyrosine residue.
Figure 3: Crystal structure of PaToxG.
Figure 4: Functional consequences of RhoA GlcNAcylation at Y34.
Figure 5: 1H-NMR analysis of GlcNAc-modified RhoA.
Figure 6: The SseI-like domain of PaTox deamidates heterotrimeric Gα proteins.

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Acknowledgements

We thank P. Gebhardt for technical assistance, P. Papatheodorou for discussions and U. Lanner for supporting MS analyses. We thank S. Offermanns (Max-Planck-Institute) for providing Gαq/11- and Gα12/13-deficient MEFs, Y. Horiguchi (Osaka University) for the deamidation-specific antibody anti-Gα QE (3G3) and A.E. Lang (University of Freiburg) for providing anthrax protective antigen. The plasmids pGEX4T1-LARG (residues 766–1138) and pGEX4T1-PDZ-RhoGEF (residues 712–1081) were kindly provided by M. Reza Ahmadian (University Düsseldorf). We thank the BM14 (European Synchrotron Radiation Facility) and PXI (Swiss Light Source) beamline staff for their support. This work was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center 746 (K.A., J.H.C.O. and C.H.), Deutsche Forschungsgemeinschaft AK6/22-1 (K.A.) and the Excellence Initiative of the German Federal and State Governments EXC 294 BIOSS (K.A., C.H. and B.W.).

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Authors and Affiliations

  1. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Thomas Jank, Kira E Böhmer, Marcus Steinemann, Joachim H C Orth & Klaus Aktories

  2. Institut für Biochemie und Molekularbiologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Xenia Bogdanović, Christophe Wirth & Carola Hunte

  3. Zentrum für Biosystemanalyse, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Erik Haaf & Bettina Warscheid

  4. Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Regensburg, Germany

    Michael Spoerner & Hans Robert Kalbitzer

  5. Institut für Biologie II, Fakultät für Biologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Bettina Warscheid

  6. Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Bettina Warscheid, Carola Hunte & Klaus Aktories

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  1. Thomas Jank
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Contributions

T.J. designed the study, performed experiments, analyzed the data and wrote the paper; K.A. designed the study, analyzed data and wrote the paper; K.E.B. and M. Steinemann collected data; J.H.C.O. designed the study; E.H. and B.W. performed MS analyses; X.B., C.W. and C.H. performed X-ray analysis; M. Spoerner and H.R.K. performed NMR analysis; and all authors discussed the results and commented on the manuscript.

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Correspondence to Klaus Aktories.

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Integrated supplementary information

Supplementary Figure 1 Sequence similarity of PaTox with glycosyltransferases and deamidases, sugar-donor substrate specificity and enzyme activity of PaTox glycosyltransferase.

(a) Amino acid sequence alignment of the region surrounding the DxD-motif (marked) of P. asymbiotica PaTox (accession number C7BKP9) with different glycosyltransferases: Toxin B (C. difficile toxin B, accession number P18177), Lethal toxin (C. sordellii lethal toxin, accession number Q46342), α-Toxin (C. novyi alpha toxin, accession number Q46149), Lgt1 (Legionella pneumophila glucosyltransferase 1, accession number Q5ZVS2). (b) Amino acid sequence alignment of a region of the SseI-like domain of PaTox with Salmonella enterica virulence factor SseI(SrfH) (accession number Q8ZQ79), and Pasteurella multocida toxin (PMT, accession number P17452). The catalytic Cys-His-Asp triad of papain-like proteases and deamidases is highlighted. The alignments were prepared using Clustal W. Identical residues are boxed and shown in red, similar residues are shown in blue. The accession numbers of amino acid sequences were obtained from Uniprot. (c) In vitro glycohydrolase activity of PaTox to assess sugar donor specificity. PaToxG-catalyzed hydrolysis of the indicated radiolabeled UDP-sugars was analyzed by thin-layer chromatography and autoradiography. Data are means ± s.d. of three independent experiments. (d) Mutation of the DxD motif abrogates glycosyltransferase activity. Autoradiogram and Coomassie staining of in vitro glycosylation reactions with PaToxG and the mutant PaToxG(NxN) in combination with recombinant RhoA and Rac1.

Supplementary Figure 2 MS analyses of tyrosine GlcNAcylation of RhoA, Rac1, and Cdc42 by PaTox and glycosylation during P. asymbiotica infection.

(a-c) Extracted ion chromatograms of LC-MS analyses of modified (upper panel) and wild type GTPases (lower panel) digested with thermolysin. The indicated peptides of wild type RhoA (a), Rac1 (b), and Cdc42 (c) were partially shifted by 203.1 Da indicating a modification by N-acetylhexosamine (HexNAc). Chromatograms were scanned for unmodified (blue trace) and modified peptides (red trace). The HexNAc-modified forms of the peptides were exclusively detected in PaTox-treated GTPases. (d) MS-MS spectra of the GlcNAc-modified peptides 27-AFPGEYIPTVFDNYSAN-43 and 20-LISYTTNKFPSEYVPT-35 of PaTox-modified Rac1 and Cdc42, respectively. Sequence-specific fragment ions are annotated in blue (b-type ions) and green (y-type ions). In all GTPases studied here, the conserved switch I Y32 residue was identified as acceptor amino acid for GlcNAc. m/z, mass-to-charge. (e) Glycosylation of Rho in Sf9 insect cells was assessed after infection with P. asymbiotica and P. luminescens (multiplicity of infection of 10) for 14 h. Non-infected cells were controls. After extensive washing, cells were lysed and GlcNAcylation was analyzed by a consecutive glycosylation reaction with UDP-[14C]GlcNAc and PaToxG (top panel) and immunoblotting with an anti-Rac1 (Mab102) antibody which only recognizes non-glycosylated Rac1 (second panel). Infection of Sf9 cells by P. asymbiotica but not by P. luminescens, which does not present the PaTox-gene, resulted in a reduced signal indicating an infection-mediated GlcNAcylation of the switch I tyrosine; anti-Rac1 (23A8) illustrates total Rac1 content (third panel); anti-GAPDH antibody was used as loading control (bottom panel). (f) Proof that anti-Rac1 (Mab102) does not recognize glycosylated Rac protein. J774 macrophages were treated with PaToxG (100 ng/ml) and anthrax protective antigen (PA, 700 ng/ml), PA alone and C. difficile toxin B (100 ng/ml) for 14 h. Cells were washed, lysed and subjected to in vitro glycosylation with PaToxG as performed in (e) (top panel). Western blots were decorated with anti-Rac1 (Mab 102) (second panel), anti-Rac1 (23A8) (third panel), and anti-GAPDH (bottom panel) antibodies. Data in (e) and (f) are representative of two independent experiments.

Supplementary Figure 3 Description of secondary-structural elements of PaToxG and important amino acid residues.

(a) Sequence of P. asymbiotica PaToxG and assignment of secondary structural elements. Amino acids analyzed by site-directed mutagenesis are marked with an arrow (Supplementary Table 1). (b) Topology diagram of PaToxG with secondary structural elements (colored as in Fig. 3). The catalytic domain features a typical GT-A fold with nucleotide binding fold (blue) and acceptor binding fold (red), which are also referred to as individual domains. The adjacent 3-helix bundle is indicated in yellow. The first 17 N-terminal residues (monomer A; 18 residues monomer B), two short regions (A: residues 2300-2306, B: 2302-2305; A: 2386-2393, B: 2384-2392) and the C-terminal residues (A: 2421-2448; B: 2420-2448) are not resolved in the structure and likely to be disordered and shown as dotted lines. (c) Cellular effects of PaToxG-mutants with exchanged catalytic core residues. Micrographs of HeLa cells treated for 4 h with PaToxG wild type and the indicated catalytic core mutants (each 11 nM) in combination with PA (0.5 μg/ml) as delivery system. With the mutants PaToxG-D2260A and PaToxG-R2263A no cellular effects were observed. PA alone showed no effect (control). Scale bar, 50 μm. The experiment was repeated four times.

Supplementary Figure 4 Functional consequences of tyrosine GlcNAcylation of Rho proteins.

(a, b) Position of Y34 in RhoA in the inactive (GDP) and active (GTPγS) conformation. Comparison of the crystal stuctures of inactive RhoA·GDP (a, PDB 1FTN) and the constitutive active from RhoA-G14V·GTPγS (b, PDB 1A2B). Y34 is marked and shown in red, switch I (swI) region is shown in blue and switch II (swII) region in green, nucleotides are shown as sticks in black. (c, d) GlcNAcylation of Y34 does not alter nucleotide binding to RhoA. Fluorimetric analysis of mant-GDP (c) or mant-GppNHp (d) binding to wild type RhoA (closed circles) or glycosylated RhoA (open circles) bound to GDP. Nucleotide exchange was monitored by the increase in fluorescence upon mant-GDP or mant-GppNHp binding to RhoA. The curves were fitted by a single-exponential function (lines). Error bars are ± s.d. from three technical replicates. (e, f) Tyrosine GlcNAcylation of RhoA prevents PDZ- and LBC-stimulated nucleotide exchange. Fluorimetric analysis of mant-GDP exchange with wild type RhoA (circles) and GlcNAcylated RhoA (triangles). After 5 min, guanine nucleotide exchange factor PDZ-RhoGEF (e) and p47-LBC (f) was added (open signs). Data in (e) and (f) are representative of two independent experiments. (g) Tyrosine GlcNAcylation of RhoA, Rac1 and Cdc42 impairs effector interaction in a cell free system. Western blots of GST-effector pulldown experiments with recombinant RhoA, Rac1 and Cdc42 preglycosylated with PaToxG or the catalytic fragment of C. difficile toxin B with UDP-GlcNAc and UDP-glucose, respectively. Before co-precipitation by the effectors GST-Rhotekin (RhoA) and GST-PAK (Rac1, Cdc42), GTPases were loaded with GTPγS or GDPβS. (h, i) RhoA in the active conformation is the preferred substrate for GlcNAcylation by PaTox. (h) Autoradiogram and Coomassie staining of in vitro 14C-GlcNAcylation of RhoA preloaded with GTPγS or GDPβS by PaToxG (top panels). The catalytic domain of C. difficile toxin B (bottom panels), which uses the inactive GDP-conformation of RhoA for the glucosylation of T37, was used as a control with UDP-[14C]glucose. (i) Examples of time-dependent glycosylation of 2.5 μM RhoA–GppNHp (filled circles) and RhoA–GDP (open circles) by 0.5 nM PaToxG in the presence of 50 μM radiolabeled UDP-[14C]GlcNAc at 30°C. Quantification of modified RhoA was done after separation with SDS-PAGE by autoradiography. Data were fitted to a single exponential rise (dotted lines) and the initial velocities of GlcNAcylation were determined from the calculated slope (straight lines) at the beginning of the reactions. For this concentration of RhoA the values were 3.28 ± 0.08 μmol(product)/[mg(enzyme)·min] for the GppNHp form and 0.32 ± 0.10 μmol(product)/[mg(enzyme)·min] for the GDP form. Data are means ± s.d. of three biological replicates.

Supplementary Figure 5 Substrate specificity of PaTox regarding heterotrimeric Gα proteins and cellular uptake.

(a) Western blot of in vitro deamidation of GST-Gα proteins by 100 nM PaToxG(NxN)-D for 1 h at 30°C using the deamidation specific antibody anti-Gα (QE). The amount of total GST-Gα was detected by immunoblotting with an anti-GST antibody and served as a loading control. (b) Actin staining of PaToxG(NxN)-D or PaToxG(NxN)-D(CS) treated mouse embryonic fibroblasts from wild type, Gα12/13- and Gαq/11-deficient mice. Proteins were applied in combination with PA for 8 h. Scale bar, 10 μm. (c) Translocation of PaTox from early endosomes. Western blot showing PaTox-induced deamidation of heterotrimeric G proteins in murine macrophages (RAW 264.7-cells) preincubated without or with bafilomycin A1 (Baf, 100 nM) or brefeldin A (BFA, 100 nM) for 30 min. Cells were treated with recombinant full length PaTox (300 pM) or the fragment PaToxG-D(1.4 nM) without or with protective antigen (PA) as delivery system. After 18 h incubation, cells were washed and lysed. Toxin uptake and intracellular activity was analyzed by Western blotting with deamidation-specific antibody Gα QE. Anti-Gαq and anti-GAPDH were used as loading controls. Results in (a-c) were obtained in three independent experiments.

Supplementary Figure 6 Full-length images showing molecular-weight markers for SDS-PAGE gels, immunoblots and autoradiographs included in Figure 2.

(a) Full length radioimage of Fig. 2a. (b) Uncropped Western blots of Fig. 2a. (c) Uncropped autoradiograms of Fig. 2b. (d) Uncropped Coomassie stained SDS-PAGE gels of Fig. 2b. (e) Uncropped radioimage (upper panel) and Coomassie stained gel (lower panel) of Fig. 2d. (f) Uncropped radioimage (upper panel) and Coomassie stained gel (lower panel) of Fig. 2e. (g) Uncropped radioimage (upper panel) and Coomassie stained gel (lower panel) of Fig. 2f. See corresponding figure legends for details.

Supplementary Figure 7 Full-length images showing molecular-weight markers for immunoblots included in Figures 4 and 6.

(a) Full length Western blot analyses of the blots shown in Fig. 4c. Unspecific labeling of proteins by the anti-RhoA antibody is marked. (b) Full length Western blots of Fig. 4e. Unspecific labeling of proteins by the anti-GST antibody is marked. (c) Uncropped Western blots of Fig. 6b. Unspecific labeling of proteins is marked. (d) Uncropped Western blots of Fig. 6c. Unspecific bands are marked. See corresponding figure legends for details.

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Jank, T., Bogdanović, X., Wirth, C. et al. A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins. Nat Struct Mol Biol 20, 1273–1280 (2013). https://doi.org/10.1038/nsmb.2688

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