4 methylumbelliferyl β d glucuronide

Previously standardized nonenrichment and selective enrichment culture protocols were used to isolate O157 with slight modifications [49 (link)–51 (link)]. Briefly, per the protocol, 10 g fecal sample was added to 50 ml Trypticase soy broth (BD Bioscience, San Jose, Ca.) supplemented with cefixime (50 μg/liter; U.S. Pharmacopeia, Washington D.C), potassium tellurite (2.5 mg/liter; Sigma-Aldrich Corp., St. Louis, Mo.), and vancomycin (40 mg/liter; Alfa Aesar, Haverhill, Ma.) (TSB-CTV) and mixed well. Serial dilutions of each sample were prepared with sterile saline (0.15 M NaCl) both before and after overnight incubation of the TSB-CTV-fecal suspension at 37°C with aeration. The dilutions prepared before incubation were spread plated onto sorbitol MacConkey agar (BD Biosciences) containing 4-methylumbelliferyl-β-d-glucuronide (100 mg/liter; Sigma) (SMAC-MUG) (nonenrichment cultures). SMAC-MUG supplemented with cefixime (50 μg/liter), potassium tellurite (2.5 mg/liter), and vancomycin (40 mg/liter) (SMAC-CTMV) was used to plate the dilutions prepared after overnight incubation (selective-enrichment cultures). Both SMAC-MUG and SMAC-CTMV plates were read after overnight incubation at 37°C, and colonies that did not ferment sorbitol or utilize 4-methylumbelliferyl-β-d-glucuronide (nonfluorescent under UV light) were further evaluated to be O157 serologically.

The following O157 strains were used in peptide susceptibility assays: (i) EDL933 (ATCC 43895; stx1⁺, stx2⁺, eae⁺, hlyA⁺; American Type Culture Collection/ATCC, Manassas, VA) and (ii) 86–24 (NADC 6103; stx2⁺, eae⁺). O157 strains EDL933 and 86–24 displayed similar results in initial peptide susceptibility assays, and due to EDL933 having both stx genes, only strain EDL933 was used in subsequent assays. Bacterial freezer stocks were streaked individually on Luria-Bertani (LB) Lennox agar (Sigma-Aldrich, St. Louis, MO) and incubated overnight at 37˚C. Bacterial cultures were prepared by growing each isolate from a single colony in 5 mL LB broth (Sigma-Aldrich) per assay for 20 h at 37˚C with constant shaking at 150 rpm. Assay isolates were streaked on Difco sorbitol MacConkey agar (BD Biosciences, Franklin Lakes, NJ) containing 4-methylumbelliferyl-β-d-glucuronide (100 mg/L; Sigma-Aldrich) (SMAC-MUG) or SMAC-MUG supplemented with cefixime (50 μg/L), potassium tellurite (2.5 mg/L), and vancomycin (40 mg/L) (SMAC-CTMV) agar [32 (link), 33 (link)]. Plates were read after overnight incubation at 37˚C and colonies that did not ferment sorbitol or utilize 4-methylumbelliferyl-β-d-glucuronide (non-fluorescent under UV light) were confirmed to be O157 via latex agglutination (E. coli O157 latex, Oxoid Diagnostic Reagents, Oxoid Ltd., Hampshire, UK) [32 (link), 33 (link)].

4x10⁵ neutrophils/well in HBSS+0.2 mM BSA +1mM HEPES were incubated with 100 μl Cytochalasin B (30µM) in 96 well plates for 15 minutes on ice, followed by 15 minutes of incubation at 37°C and 5% CO₂. After centrifugation, supernatants were removed and the pellets were stimulated for 10 minutes (37°C, 5% CO₂) with 100 μl of diluted bacterial culture supernatants (0.75% and 0.36%), fMLF or PSMs to induce degranulation. The stimulation was stopped via centrifugation for 10 minutes at 250 x g and 4°C. 20 μl of the supernatants were then transferred into a black 96 well flat bottom plate and 20 μl of a 10 mM 4-Methylumbelliferyl β-d-glucuronide hydrate stock solution in 0.1 M Sodium Acetate (pH 4.0) were added and mixed briefly. After 15 minutes at 37°C and 5% CO₂ the reaction was stopped by adding 250 μl of stop solution (50 mM glycine, 5 mM EDTA in ddH₂O). Degranulation results in the release of β-glucuronidase, and its activity can be measured in a microplate reader by monitoring cleavage of the substrate 4-Methylumbelliferyl β-d-glucuronide (Sigma), which releases the fluorescent moiety 4-Methylumbelliferyl (excitation wavelength of 365 nm and an emission wavelength of 460 nm).

Activity of lysosomal enzyme of interest (β‐glucuronidase and β‐hexosaminidase) was measured as previously described (Bartolomeo et al, 2017). Briefly, cells were lysed in extraction buffer (50 mM NaHPO₄ pH 7.0, 10 mM 2‐mercaptoethanol, 10 mM Na₂ EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X‐100) and protein concentration was measured using the colorimetric BCA protein assay kit (Pierce Chemical). 200 μg of proteins was incubated with 200 μl of fluorogenic substrate (4‐methylumbelliferyl‐β‐D‐glucuronide 2 mM Sigma‐Aldrich for β‐glucuronidase; 4‐methylumbelliferyl‐N‐acetyl‐β‐D‐glucosaminide 6 mM Sigma‐Aldrich for β‐hexosaminidase) for 1 h at 37°C. The reaction was stopped by adding 200 μl of the carbonate stop buffer (0.5 M NaHCO₃/0.5 M Na₂CO₃ pH 7.0), and the fluorescence was measured in a fluorimeter (GloMax‐Multi Detection System, Promega) using 365 nm excitation and 460 emission.

For histochemical staining of GUS, excised tobacco leaf and apple peel discs were immediately treated with 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronide in 100 mM phosphate buffer pH 7.0, 10 mM EDTA, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 [77 (link)] at 37 °C for 24 h. Stained samples were bleached with 70% (v/v) ethanol, and GUS activity was determined by measuring the fluorescence of 4-methylumbelliferone produced by GUS cleavage of 4-methylumbelliferyl-β-D-glucuronide (Sigma) as described previously [78 (link)]. The protein concentration in the supernatant was determined using the Bradford procedure with bovine serum albumin (Sigma) as a standard. Three independent biological replications were performed for each experiment.

β-Glucuronidase (GUS) activity in L. crescens cells (carrying pLF057; hol::uidA) was examined using the fluorogenic substrate MUG (4-methylumbelliferyl-β-d-glucuronide; Sigma-Aldrich, St. Louis, MO). One-milliliter bacterial cultures were harvested at 2,013 × g for 15 min at 4°C, and the bacteria were resuspended in 30 µl GUS extraction buffer (50 mM Na₂HPO₄ [pH 7.0], 10 mM β-mercaptoethanol, 10 mM Na₂EDTA [pH 8.0], and 0.1% Triton X-100) and incubated for 10 min at 37°C. Following the lysis reaction, 90 µl substrate solution (1 mM MUG in GUS extraction buffer) was added per 10 µl lysate and incubated at 37°C. Reaction aliquots (100 µl) were withdrawn at 10, 40, and 70 min, stopped with 50 µl of 0.2 M Na₂CO₃, and measured for fluorescence. The data were normalized against a standard curve prepared using the purified GUS enzyme (type IX-A from E. coli; Sigma-Aldrich). The data presented are mean values derived from two biological assays run in triplicate.

GUS histochemical location was conducted as previously described [1 (link)]. Each photograph in Fig. 2f is representative of at least nine tissues from three replicated experiments. Fluorometric quantitative analysis was measured according to Jefferson’s method [16 (link)]. The control or transgenic samples were analysed to determine GUS activities with the substrate of 1 mM 4-methyl umbelliferyl β-d-glucuronide (Sigma-Aldrich, Shanghai, China). Fluorescence values were recorded with a Hitachi 850 Fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The protein concentration was determined as described by Bradford [17 (link)].