The plasmids and strains acclimated in this abstraction are listed in Table 1. WT nleB (C. rodentium) and its acquired DAD221–223/AAA were cloned into pET42a. FADD was cloned into pET15a. WT gshB and its acquired gshB R256A, as able-bodied as gshA, were cloned in pET28a application ABC cloning34. The C. rodentium gshB abatement was generated application lambda red recombination35.
Proteins were bidding from E. coli BL21 (DE3) and induced with 0.5 mM IPTG back cultures able an OD600 of 0.4. Beef were developed for an added 4 h at 37 °C and afresh harvested application centrifugation. Corpuscle pellets were resuspended in 1/40th ability aggregate of 50 mM NaH2PO4 pH 8.0 supplemented with 0.5 mg/ml lysozyme and protease inhibitor affair (Thermo Scientific). Bacterial suspensions were incubated on ice for 30 min with casual shaking, at which time an according aggregate 50 mM NaH2PO4 pH 8.0, 2 M NaCl, 8 mM imidazole, 20% glycerol, 2% Triton X-100 was added for added evolution on ice for 30 min. Corpuscle lysates were sonicated, centrifuged, and accumulated with 2 ml Ni-NTA slurry (Qiagen) for 1 h at 4 °C with affable rotation. The admixture was loaded on a Poly-Prep Chromatography Cavalcade (Bio-Rad) and done in 50 mM NaH2PO4 pH 8.0, 600 mM NaCl, 60 mM imidazole, 10% glycerol). Proteins were eluted in 50 mM NaH2PO4 pH 8.0, 600 mM NaCl, 250 mM imidazole, 10% glycerol and afresh dialyzed into the aforementioned absorber defective imidazole17.
HEK293T beef were developed in DMEM to 80% confluency and afresh adulterated with EHEC O111:NM R82F236 at a complication of infection of 1.0 for 16 h. Adulterated beef were done three times in ice-cold PBS and lysed by abrading with ice-cold guanidinium chloride lysis absorber (6 M GdmCl, 100 mM Tris pH 8.5, 10 mM TCEP, 40 mM 2-Chloroacetamide) on a bed of ice. Lysates were calm and aloft at 95 °C for 10 minutes with afraid at 2,000 rpm to microburst DNA and inactivate protease activity. Lysates were afresh cooled for 10 minutes on ice and afresh aloft afresh at 95 °C for 10 minutes with afraid at 2,000 rpm. Protein samples (2 mg) were acetone precipitated and broiled protein pellets were resuspended in 6 M urea, 2 M thiourea, 40 mM NH4HCO3 and reduced/alkylated above-mentioned to assimilation with Lys-C (1/200 w/w) and afresh with trypsin (1/50 w/w) brief as ahead described37. Digested samples were acidified to a final absorption of 0.5% formic acerbic and desalted with 50 mg tC18 SEP-PAK (Waters Corporation). tC18 SEP-PAKs were conditioned with absorber B (80% ACN, 0.1% formic acid), done with 10 volumes of Absorber A* (0.1% TFA, 2% ACN), sample loaded, cavalcade done with 10 volumes of Absorber A* and apprenticed peptides eluted with absorber B afresh dried.
Arg-GlcNAc peptide affection ablution was performed as ahead described16. Protein A/G added Agarose chaplet (Santa Cruz, Santa Cruz CA) were done with immunoprecipitation absorber (IAP, 10 mM Na2HPO4, 50 mm NaCl, 50 mM MOPS, pH 7.2) and rotated brief with 10 μg of anti-Arg-GlcNAc antibiotic (ab195033, Abcam) at 4 °C. Coupled anti-Arg-GlcNAc chaplet were afresh done with 100 mM sodium borate (pH 9) to abolish non-bound proteins and cross-linked for 30 minutes of circling application 20 mM dimethyl pimelimidate in 100 mM HEPES, pH 8.0. Cross-linking was quenched by abrasion chaplet three times with 200 mM ethanolamine, pH 8.0 and afresh alternating the chaplet in an added 1 ml of 200 mM ethanolamine, pH 8.0 for 2 hours at 4 °C. Antiseptic peptides were resuspended in 1 ml IAP absorber and peptide lysates were afresh added to the able cross-linked anti-Arg-GlcNAc antibiotic chaplet and rotated for 3 hours at 4 °C. Antibiotic chaplet were centrifuged at 3,000 g for 2 minutes at 4 °C and the absolved peptide lysates collected. Antibiotic chaplet were afresh done with ice-cold IAP absorber and Arg-GlcNAc peptides eluted application two circuit of acerbic elution. For anniversary elution round, 100 μl of 0.2% TFA was added and antibiotic chaplet accustomed to angle at allowance temperature with affable afraid every minute for 10 minutes. Peptide supernatants were calm and desalted application C18 date tips38 afore actuality broiled bottomward and stored until LC-MS analysis.
Purified tagged GshB was bankrupt up application SP3 based ablution according to antecedent protocols39. Samples were aboriginal denatured and bargain application 1% SDS, 10 mM DTT, 100 mM HEPES by baking at 95 °C, 1,000 rpm for 10 minutes. Samples were afresh cooled and alkylated with 40 mM 2-chloroacetamide (CAA) for 1 hour at RT in the dark. The alkylation reactions were afresh quenched with 40 mM DTT for 10 minutes and afresh samples precipitated on to SeraMag Speed Chaplet (GE Healthcare, USA) with booze (final absorption 50% v/v). Samples were annoyed for 10 minutes to acquiesce complete precipitation assimilate chaplet and afresh done three times with 80% ethanol. The chaplet were resuspended in 100 mM ammonium bicarbonate absolute 1 μg lys-C 1/50 (w/w) and digested brief at 37 °C. Samples were centrifuged at 14,000 g for 5 minutes to pellet the chaplet and the afloat calm and desalted application C18 date tips afore actuality broiled for LC-MS analysis.
Purified peptides were resuspended in Absorber A* and afar application a two-column chromatography bureaucracy composed of a PepMap100 C18 20 mm × 75 μm allurement and a PepMap C18 500 mm × 75 μm analytic cavalcade (Thermo Fisher Scientific)16. Samples were concentrated assimilate the allurement cavalcade at 5 μl/min for 5 minutes and alloyed into either an Orbitrap Fusion Lumos Tribrid Accumulation Spectrometer (Thermo Fisher Scientific) for the appraisal of able Arg-GlcNAc peptides and PRM appraisal of Arg-GlcNAcylated GshB or an Orbitrap Elite (Thermo Fisher Scientific) for the allegory of Arg-GlcNAcylation levels aural antiseptic GshB. Gradients (120 minutes) were run by altering the absorber agreement from 1% absorber B to 28% B over 90 minutes, afresh from 28% B to 40% B over 10 minutes, afresh from 40% B to 100% B over 2 minutes, the agreement was captivated at 100% B for 3 minutes, and afresh alone to 3% B over 5 minutes and captivated at 3% B for accession 10 minutes for able Arg-GlcNAc adapted peptides and allegory of Arg-GlcNAcylation levels aural antiseptic GshB. For 120-minute gradients, the Lumos and Elite Accumulation Spectrometers were operated in a data-dependent approach automatically switching amid the accretion of a distinct Orbitrap MS browse HCD or CID fragmentation. For alongside acknowledgment ecology (PRM) experiments, the accepted Lys-C Arg-modified peptide of GshB (IARQIGPTLKEK) was monitored application EThcD breach targeting the predicted m/z for the 2 (m/z 650.2835) and 3 allegation (m/z 488.9248) states over a 95 minute gradient, altering the absorber agreement from 1% absorber B to 28% B over 60 minutes, afresh from 28% B to 40% B over 10 minutes, afresh from 40% B to 100% B over 2 minutes, captivated at 100% B for 3 minutes, and afresh alone to 3% B over 5 minutes and captivated at 3% B for accession 15 minutes.
Identification of Arg-glycosylated peptides was able application MaxQuant (v220.127.116.11)40. Searches were performed adjoin E. coli O127:H6 ache E2348/69 (Uniprot proteome id UP000008205- E. coli O127:H6 ache E2348/69/EPEC, downloaded 28-07-2014, 4,595 entries) or C. rodentium ICC168 (Uniprot proteome id UP000001889- C. rodentium ache ICC168 downloaded 12/12/2016) proteomes depending on the samples, with carbamidomethylation of cysteine set as a anchored modification. Searches were performed with Trypsin or Lys-C break specificity depending on the experiment. Two miscleavage contest were allowed, as able-bodied as the capricious modifications of blaze of methionine, N-Acetylhexosamine accession to arginine (Arg-GlcNAc) and acetylation of protein N-termini. Forerunner accumulation altruism was set to 20 parts-per-million (ppm) for the aboriginal chase and 10 ppm for the capital search, with a best apocryphal appraisal amount (FDR) of 1.0% set for protein and peptide identifications. The Bout Amid Runs advantage was enabled with a forerunner bout window set to 2 minutes and an alignment window of 10 minutes. For label-free quantitation, the MaxLFQ advantage aural Maxquant41 was enabled, in accession to the re-quantification module. Peptide outputs were candy application the Perseus (v18.104.22.168)42 appraisal ambiance to abolish about-face matches and accepted protein contaminants with missing ethics accepted and the peptide intensities z-scored. MS/MS annotations were undertaken application the Interactive Peptide Spectral Annotator43. Accumulation spectrometry proteomics abstracts were deposited to the ProteomeXchange Consortium via the PRIDE accomplice athenaeum with the dataset identifier PXD015752.
In vitro glycosylation assays were performed as declared previously17 application 200 nM of NleB1 or its orthologs with 1 μM of either WT GshB or GshB R256A in 50 mM Tris-HCl pH 7.4, 1 mM UDP-GlcNAc, 10 mM MnCl2, and 1 mM DTT. Afterwards 2 h evolution at RT, samples were subjected to western blotting application an anti-R-GlcNAc monoclonal antibiotic (Abcam). In vivo glycosylation assays were performed application C. rodentium and S. enterica strains electroporated with plasmids cogent His-GshB or His-GshB(R256A). Transformed bacilli were developed brief in the attendance of 0.5 mM IPTG, harvested application centrifugation, and afresh His-tagged proteins were antiseptic as declared above, and afresh subjected to western blotting application an anti-R-GlcNAc monoclonal antibiotic (Abcam).
Overnight cultures were acclimated to bathe 50 ml of LB medium. H2O2 (2.4 mM for C. rodentium or 12.0 mM for S. enterica) was added to cultures back they able an OD600 of 0.3 and bacterial advance was monitored for 10–16 h at 37 °C.
Bacteria were harvested by centrifugation, lysed in 50 mM Tris-HCl pH 7.4 supplemented with 0.5 ug/ml lysozyme, and recentrifuged. The afloat was incubated with 1 mM CDNB and 1 µM GST. Absorbance was recorded at 340 nm every 20 seconds for 10 minutes and OD340 abstracts were adapted into molar concentrations of GSH application a GST accepted curve24.
His-GshB or His-GshB(R256A) were antiseptic over Ni-NTA slurries afterwards they were appropriately co-expressed with either NleB-FLAG or NleB(AAA)-FLAG in E. coli BL21(DE3) cells. An in vitro GSH appraisal was afresh able by aboriginal incubating GshA (1 µM) with 5 mM glycine, 5 mM cysteine, and 5 mM glutamate in a acknowledgment absorber absolute 50 mM Tris HCl pH 7.4, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, and 3% DMSO. Afterwards 1 h evolution at 37 °C, the antiseptic GshB proteins (50 nM), forth with 100 nM GST and 1 mM CDNB were added, and glutathione accumulation was monitored by account the absorbance at 340 nm as a action of time.
Bacterial advance assays were analyzed application non-linear corruption followed by Dunn’s assorted allegory testing. GSH assays were analyzed application beeline regression. p-values <0.05 were advised significant.
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