Ecto-5'-Nucleotidase Assay in EPEC-Infected Cells

Bacterial strains used E. coli strains used included laboratory E. coli strain HB101 (O: rough), commensal strain HS (O9: H4), and classic human EPEC strains E2348/69 (serotype O127: H6), B171-8 (O111: NM), and JCP88 (O119: B14) as described in several publications [8 (link)–12 (link)]. EPEC mutants included JPN15, an E2348 derivative which has lost the EPEC adherence factor (EAF) plasmid [13 (link)], UMD874, the espF mutant derived from E2348, which is deficient in host cell killing [3 (link), 14 (link)], and SE1010, with a mutation in sepZ (also called espZ), which is defective in type III secretion [15 (link)]. Bacteria were added to yield a multiplicity of infection (MOI) of 100:1.
Materials The following reagents were obtained from Sigma-Aldrich Chemicals: α,β-methylene-ADP, adenosine, adenosine 5′-monophosphate (AMP), tetramisole (also called levamisole), polymyxin B, neomycin, purified phosphatidylinositol-specific phospholipase C (PI-PLC, from Bacillus cereus), and zinc acetate. BIOMOL (Plymouth Meeting, PA, USA) was the source of the BIOMOL GREEN reagent used in the phosphate release assay for nucleotidase activity and of U73122, a PI-PLC inhibitor. U73122 is 1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione. A cell permeant PI-PLC activator, 3M3-FBS, was from the Calbiochem Division of EMD Biosciences (La Jolla, CA, USA). 3M3-FBS is 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide. Phosphate-free DMEM medium was purchased from MP Biomedicals (formerly ICN Biomedicals, Aurora, OH, USA). UNIFILTER plates were from Whatman (Clifton, NJ, USA).
Bacterial culture E. coli strains were grown overnight in Luria-Bertani (LB) broth at 37°C with 300 rpm shaking, then subcultured for 2 h in serum-free DMEM/F12 medium supplemented with 18 mM NaHCO₃, 25 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, pH 7.4, and 1% D-mannose as previously described [3 (link)]. For experiments with bacteria in minimal medium, bacteria were subcultured at a dilution of 1:2,000 into minimal medium (M9 salts plus casamino acids supplemented with 2 mM glucose). For convenience we used M9-CA liquid broth packets (E. coli Fast Media, MBI-Fermentas, Hanover, MD, USA) and added 2 mM glucose before use.
Cell culture T84 colon cancer cells were grown in DMEM/F12 medium supplemented with 7.5% fetal bovine serum (Gibco/Invitrogen, Grand Island, NY, USA), 18 mM NaHCO₃, 20 μg/ml vancomycin, and 15 μg/ml gentamicin as previously described [16 (link)]. Ussing chamber studies of secretion were performed on T84 cell monolayers grown in Snapwell inserts (Corning Costar, Corning, NY, USA). The Snapwell inserts, which had a 0.4 μm pore size, were coated with 32 μg collagen per well by applying 0.16 ml of 0.2 mg/ml type III collagen (Sigma; dissolved in warm 0.2 M acetic acid) to the Snapwell and allowing it to dry in the tissue culture hood under UV light. T84 cells were seeded onto the Snapwell inserts at ~1.2 × 10⁶ cells per well and allowed to grow to confluency for 7–9 days. At this time the monolayers had transepithelial electrical resistances (TER) of 400–1,000 Ω · cm².
Assay for ecto-5′-nucleotidase by phosphate release An assay for ecto-5′-nucleotidase activity in living cells was developed based on the ability to detect inorganic phosphate (P_i) released from 5′-AMP. This method has been used to detect activity of protein phosphatases such as PTEN [17 (link)] and lipid phosphatases [18 (link)] and is based on sensitive detection of low levels of P_i using the BIOMOL GREEN reagent, an enhanced and stabilized formulation of malachite green. To carry out the assay a phosphate-free buffer was used consisting of (in mM): NaCl, 154; KCl, 2: MgCl₂, 4; NaHCO₃, 18: HEPES, pH 7.4, 25; and glucose, 10. This buffer is referred to as nucleotidase buffer. To measure ecto-5′-nucleotidase activity, the cell monolayer was rinsed once with sterile normal saline, then the medium was replaced with warm nucleotidase buffer. For cells in a 48-well plate, 0.25 ml of nucleotidase buffer were added per well, and the cells were allowed to rewarm to 37°C in the CO₂ incubator. During pipetting the multiwell plate was kept warm using a metal heating block set at 37°, and a stopwatch was used to time the AMP addition and to terminate the assay. The procedure used for measuring monolayer activity was slightly different from that used to measure 5′-nucleotidase activity released into the supernatant, as described below.
Cell-bound or monolayer activity To measure cell-bound ecto-5′-nucleotidase activity in cell monolayers, 5′-AMP was added to yield a final concentration of 0.2 mM to quadruplicate wells. Two other wells were left without addition of AMP (the “no AMP blank”). After a 10-min incubation at 37° an aliquot (usually 50 μl) was removed and quickly transferred to a well of a 96-well plate to terminate the reaction.
E. coli-induced release of nucleotidase activity into the supernatant medium For nucleotidase release experiments, the cell monolayer was changed to warm, phosphate-free DMEM, then infected with an E. coli strain for 35 min to allow adherence, then the medium was changed to nucleotidase buffer and the infection was allowed to continue for 2 or 3 h. Note that in this procedure any nucleotidase activity that is released in the first 35 min is discarded and not detected by our method. However, this two-stage procedure with the medium change was necessary because EPEC bacteria did not adhere normally if they first encountered the host cell in nucleotidase buffer. After a period of infection, supernatant medium was collected with a multichannel pipettor and transferred to the wells of a Whatman UNIFILTER plate (a 96-well with 0.45-μm membrane for sterile filtration). Sterile filtrates were prepared by centrifugation with collection of the filtered medium into another 96-well plate placed beneath the UNIFILTER as previously described [19 (link)]. Once again, experimental conditions were usually done in groups of six, with two wells not receiving any AMP (no AMP blanks) and four wells receiving 0.2 mM AMP. Again, the usual assay condition was 10 min at 37° before the reaction was stopped by addition of 10 μl of 1 M HCl (“stop solution”).
BIOMOL GREEN detection of phosphate released from AMP Stopped samples in a 96-well plate were brought to 100 or 110 μl volume with water if necessary, then treated with 100 μl of BIOMOL GREEN reagent. A standard curve of inorganic phosphate was prepared and run with every experiment; standards and unknown samples were incubated at room temperature for 20 min to allow a green color to develop, then the 96-well plate was read on a multiwell plate spectrophotometer at 620 nm. Unknown values were calculated from the standard curve using a hyperbolic curve fit using GraphPad Prism software, version 4.0. Results of monolayer activity were expressed as nmol of P_i produced/min per 10⁶ cells. For experiments showing nucleotidase release, the results were often expressed as nmol P_i released/ min per well since the assay was done on a cell-free filtrate and because we often noted some detachment of cells during the longer incubations of 2–3 h needed to observe release.Although we believed we were developing a new method for assay of ecto-5′-nucleotidase by phosphate release, during the course of this work another group reported using a virtually identical method, also based on detection of phosphate released from 5′-AMP [1 (link)].
Detection of CD73 by Western immunoblot To prove that the released 5′-nucleotidase activity we measured was of host cell rather than bacterial origin, we performed immunoblots on the supernatants of infected T84 cells with antibodies against CD73. Initial attempts at immunoblotting using a commercially available monoclonal anti-CD73 antibody (Abnova Corp., Taipei, Taiwan) were unsuccessful. Dr. Linda F. Thompson, Oklahoma Medical Research Foundation, kindly sent us mouse monoclonal antibodies against human CD73 which had been generated by Dr. Wolf Gutensohn several years earlier. Of these, the two antibodies that gave the best results were designated CD73.4 and CD73.6 by Dr. Gutensohn; both were of isotype IgG_2b and were used at a concentration of 1 μg/ml. After washings, the secondary antibody was goat anti-mouse IgG_2b conjugated to peroxidase at a dilution of 1:3,000 (Roche Molecular Biochemicals, Indianapolis, IN, USA). Blots were developed by chemiluminescence as previously described [20 (link)].
Ussing chamber studies A Snapwell insert containing a monolayer of T84 cells was placed in the plexiglass “slider” and inserted into the Ussing chamber (Physiologic Instruments, San Diego, CA, USA) at 37°C and continuously short-circuited by a four electrode, automatic voltage-clamp apparatus which measured short-circuit current (I_sc) and transepithelial resistance (TER); chamber fluid resistance was automatically subtracted. Transepithelial resistance was determined by passing 10-s 10-mV current pulses through the tissues. Short-circuit current was measured by passing sufficient current through the tissues via Ag/AgCl electrodes to reduce the spontaneous transepithelial potential to zero. The composition of the tissue bathing solution was (in mM): 140 Na⁺, 124 Cl⁻, 21 HCO₃-, 5.4 K⁺, , 1.2 Mg²⁺, 1.2 Ca²⁺, and 10 glucose. Raw short-circuit current (I_sc) values were converted to μA per cm² by dividing by the area of the Snapwell monolayer (1.13 cm²). Other details of the Ussing chamber methods were exactly as described [16 (link)].
Protein assay Protein assay was by the Coomassie blue dye binding assay method of Bradford, using a Bio-Rad kit [21 (link)].
Expression of ecto-5′-nucleotidase RNA by reverse transcription and real-time polymerase chain reaction (PCR) T84 cells grown in 24-well plates were infected with EPEC for 35 min, then the medium was changed to remove unbound bacteria. Three hours after the medium change, ciprofloxacin was added to 25 μg/ml to kill EPEC and the incubation was continued 1 more hour. Old medium was removed, and the cell monolayer was lysed in extraction buffer with 10% β-mercaptoethanol (RNeasy Kits, Qiagen, Valencia, CA, USA). RNA was subjected to reverse transcription using Invitrogen Superscript III reverse transcriptase; 5 μl of purified RNA was used per 50 μl reaction, and gene-specific primers at 0.2 μM were used. Reverse transcription reaction was at 55° for 1 h. Copy DNA from reverse transcription was diluted 100-fold, then analyzed by quantitative real-time PCR using the same oligonucleotide primers. For ecto-5′-nucleotidase the primers used were 5′-TTC CAC CCT GAA GAA GGC CTT TGA-3′ (forward) and 5′-ATA ACT GGG CAC TCG ACA CTT GGT-3′ (reverse). As a normalizing gene we used glyceraldehyde phosphate dehydrogenase (GAPDH) as described by Khan et al. [22 (link)] except that we redesigned longer primers which were 5′-TCG ACA GTC AGC CGC ATC TTC TTT-3′ and 5′-ACC AAA TCC GTT GAC TCC GACC CTT-3′. PCR was carried out using a MyiQ Single-Color qRT-PCR machine from Bio-Rad (Hercules, CA, USA) using SYBR Green as the dye to monitor the amplification. Relative expression was calculated by the ΔΔC_t (“Livak”) method as described [23 ], where C_t is the number of cycles to threshold. SYBR Green PCR reagents were from Bio-Rad and to reduce the cost, the PCR reaction volume was reduced to 25 μl. PCR was performed using a two-step protocol with an annealing temperature of 58.7° and denaturation at 95° for 30 s each (i.e, no extension step) for 35 cycles. Thermal melt curve analysis was performed at the end of the PCR amplification and showed a single sharp peak for the genes analyzed.
Data analysis and presentation All error bars shown in graphs and error values reported in the text are standard deviations. Significance was tested by one-way analysis of variance (ANOVA) with the Tukey-Kramer post-test for multiple comparisons, using InStat software for the Macintosh from GraphPad software (San Diego, CA, USA). Graphs were prepared using Prism 4.0 software, also from GraphPad. Asterisks shown on graphs indicate a p value of < 0.05.