Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos

Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18G into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18G depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18G inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes.


Supplementary Figure 2. Afp18 G NxN colocalizes with RHOA at the cell membrane.
(a) Confocal images (single plane) of double immunofluorescence against EGFP-Afp18 G NxN and RHOA at 60% epiboly. EGFP-Afp18 G NxN and RHOA vector DNA were injected in single blastomeres at 16-cell stage. Upper row: single channel images and merged image of representative mosaic labeled blastomeres. Lower row: Magnification of region depicted as white rectangle of the upper row. EGFP-Afp18 G NxN is targeted to the membrane and colocalizes with RHOA. Scale bar 2 µm.
(b) Corresponding graph shows evaluation of co-localization. Line scans were recorded along the same regions of blastomere membranes (representatively depicted by white line). Average fluorescent profiles of the line scans illustrate co-localization of Afp18 G NxN with RHOA at the membrane (n = 8 line scans of different blastomeres and embryos). Zero represents the manually defined center point of the membrane, (-) values correspond to distance to the outside, (+) values to the distance to the inside of the labelled blastomere.

Supplementary Figure 3. Consequences of Afp18 G catalyzed tyrosine GlcNAcylation.
(a) HeLa cells were intoxicated with 6xHis-tagged Afp18 G and Afp18 G NxN in combination with anthrax protective antigen (PA) as translocation system or PA alone as control. Actin cytoskeleton was stained with TRITC-phalloidin and the nucleus was stained with DAPI. Scale bar, 10 µm.
(b) Autoradiogram of SDS-PAGE to assess substrate specificity of Afp18. Rho, Ras, and Rab family proteins were in vitro glycosylated by Afp18 G with UDP-[ 14 C]GlcNAc.
(c) Stereo representation of the α-GlcNAc modified switch I tyrosine-34 in the crystal structure of RhoA-GlcNAc. Electron density maps for the N-acteylglucosaminyl moiety (Fo-Fc omit map, green mesh) was contoured at 2.5 σ and for the protein (2Fo-Fc, grey mesh) at 1 σ. Protein is depicted with atom colors and grey carbons, the GlcNAc moiety with yellow carbons.
(d) Tandem mass spectrometric analysis of Afp18-catalyzed GlcNAcylation of Cdc42. MS-MS spectrum of the GlcNAc-modified peptide 24TTNKFPSEYVPT35 of Afp18 G -modified Cdc42. Sequence-specific fragment b-type and y-type ions are annotated. Switch I Y32 residue in Cdc42 was identified as acceptor amino acid for GlcNAc. m/z, mass-to-charge.
(e) Autoradiograms and Coomassie stainings of Afp18 G -catalyzed in vitro 14 C-GlcNAcylation of wild-type (WT) Rac1 and Cdc42 and the indicated mutants. (c) Afp18 G -mediated GlcNAcylation of RhoA inhibits the function of RhoGAP. Time course of [γ-32 P]GTP hydrolysis by wild-type RhoA and GlcNAcylated RhoA in the presence and absence of p50Rho-GAP. Non-hydrolyzed [γ-32 P]GTP bound to RhoA was determined. Data are relative to initial loading (mean ± s.d., n = 3 technical replicates).

Supplementary
(b) Western blot analysis of proteins in vitro translated from mRNAs as indicated. We used Afp18 G , Afp18 G NxN, RHOA, RHOA Y34F, and Lifeact-GFP expression constructs to synthesize mRNAs for in vitro translation. Luciferase mRNA, provided by Promega, served as loading control according the manufacturers manual of Transcend TM Non-radioactive Translation Detection System (Promega). The electrophoretic mobility of proteins correlates with predicted molecular masses (Afp18 G 40 kDa, RhoA 22 kDa, Lifeact-GFP 33 kDa, Luciferase 63 kDa); the minor non-specific bands (200,80,32, and 17 kDa) also occur in control reaction and thus derive from mRNAs still active in the translation mix. Figure 6. Full length images of SDS-PAGE gels, immunoblots and autoradiographs from the corresponding figures of the main text including molecular weight markers in [kDa]. Boxes represent areas used in the figures.