Ecto-5 ′ - nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli Enteropathogenic Escherichia coli -LRB- EPEC -RRB- triggers a large release of adenosine triphosphate -LRB- ATP -RRB- from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate -LRB- ADP -RRB- , AMP , and adenosine . Adenosine is a potent secretagogue in the small and large intestine . We suspected that ecto-5 ′ - nucleotidase -LRB- CD73 , an intestinal enzyme -RRB- was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea . We developed a nonradioactive method for measuring ecto-5 ′ - nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5 ′ - AMP . EPEC infection triggered a release of ecto-5 ′ - nucleotidase from the cell surface into the supernatant medium . EPEC-induced 5 ′ - nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C -LRB- PI-PLC -RRB- . Ecto-5 ′ - nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate -LRB- α,β-methylene-ADP -RRB- . In the Ussing chamber , these inhibitors could reverse the chloride secretory responses triggered by 5 ′ - AMP . In addition , α,β-methylene-ADP and zinc blocked the ability of 5 ′ - AMP to stimulate EPEC growth under nutrient-limited conditions in vitro . Ecto-5 ′ - nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line , and inhibitors of ecto-5 ′ - nucleotidase , such as α,β-methylene-ADP and zinc , might be useful for treatment of the watery diarrhea produced by EPEC infection . Introduction Ecto-5 ′ - nucleotidase -LRB- CD73 , EC 3.1.3.5 -RRB- is a key extracellular enzyme which catalyzes the hydrolysis of extracellular nucleoside monophosphates to their corresponding nucleosides , with 5 ′ - AMP being the preferred substrate . Ecto-5 ′ - nucleotidase is highly expressed in endothelium , liver , and intestinal mucosa and at varying levels in other tissues including lymphocytes , kidney , and certain cancers . Ecto-5 ′ - nucleotidase is tethered to the extracellular surface of mammalian cells by a glycosylphosphatidylinositol -LRB- GPI -RRB- lipid anchor . Most of the research on this enzyme has been in the cardiovascular system because of its role in the production of adenosine , which protects cells against ischemia by multiple mechanisms including vasodilation -LSB- 1 , 2 -RSB- . Enteropathogenic Escherichia coli -LRB- EPEC -RRB- is a common cause of watery diarrhea in children in developing countries . While some aspects of EPEC infection , such as adherence , are well studied , the way that EPEC triggers watery diarrhea has been obscure , since EPEC produces no toxins . We recently proposed a theory that release of adenine nucleotides from host intestinal cells , followed by breakdown to adenosine , could trigger watery diarrhea by activation of adenosine receptors on intestinal cells -LSB- 3 -RSB- . In the gastrointestinal tract , ecto-5 ′ - nucleotidase was found to be a key enzyme necessary for enterocyte responses to 5 ′ - AMP , the neutrophil-derived secretagogue -LSB- 4 -- 6 -RSB- . At that time the significance of ecto-5 ′ - nucleotidase was felt to be in the setting of invasive , inflammatory pathogens because polymorphonuclear neutrophils -LRB- PMNs -RRB- release 5 ′ - AMP during the process of chemotaxis into areas of infection or inflammation . Since that time , however , we have discovered that a noninvasive pathogen , EPEC , triggers a large release of ATP directly from host intestinal cells without any requirement for PMNs to be present -LSB- 3 -RSB- . Other diarrheal pathogens not considered classically invasive , such as Aeromonas hydrophila , also cause ATP release from enterocytes -LSB- 7 -RSB- . Extracellular ATP is broken down to ADP , AMP , and then adenosine , where it triggers a vigorous fluid secretory response in intestinal epithelium . We suspected that ecto-5 ′ - nucleotidase also played a role in the diarrheal fluid secretion induced by EPEC and initiated this study to determine if it is the case , and , if so , whether inhibitors of ecto-5 ′ - nucleotidase could block fluid secretion triggered by adenine nucleotides and EPEC infection . We used the T84 colon carcinoma cell line as a model because we have used these cells to study EPEC infection , they express ecto-5 ′ - nucleotidase , and they can be studied in the Ussing chamber to measure chloride secretion . In order to facilitate our study we also developed a nonradioactive , non-high-pressure liquid chromatography -LRB- non-HPLC -RRB- method for measuring ecto-5 ′ - nucleotidase in living T84 cell monolayers . Materials and methods Bacterial strains used E. coli strains used included laboratory E. coli strain HB101 -LRB- O : rough -RRB- , commensal strain HS -LRB- O9 : H4 -RRB- , and classic human EPEC strains E2348/69 -LRB- serotype O127 : H6 -RRB- , B171-8 -LRB- O111 : NM -RRB- , and JCP88 -LRB- O119 : B14 -RRB- as described in several publications -LSB- 8 -- 12 -RSB- . EPEC mutants included JPN15 , an E2348 derivative which has lost the EPEC adherence factor -LRB- EAF -RRB- plasmid -LSB- 13 -RSB- , UMD874 , the espF mutant derived from E2348 , which is deficient in host cell killing -LSB- 3 , 14 -RSB- , and SE1010 , with a mutation in sepZ -LRB- also called espZ -RRB- , which is defective in type III secretion -LSB- 15 -RSB- . Bacteria were added to yield a multiplicity of infection -LRB- MOI -RRB- of 100:1 . Materials The following reagents were obtained from Sigma-Aldrich Chemicals : α,β-methylene-ADP , adenosine , adenosine 5 ′ - monophosphate -LRB- AMP -RRB- , tetramisole -LRB- also called levamisole -RRB- , polymyxin B , neomycin , purified phosphatidylinositol-specific phospholipase C -LRB- PI-PLC , from Bacillus cereus -RRB- , and zinc acetate . BIOMOL -LRB- Plymouth Meeting , PA , USA -RRB- 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 - -LRB- 6 - -LSB- 17 beta-3-methoxyestra-1,3,5 - -LRB- 10 -RRB- triene-17-yl -RSB- amino/hexyl -RRB- 1H-pyrroledione . A cell permeant PI-PLC activator , 3M3-FBS , was from the Calbiochem Division of EMD Biosciences -LRB- La Jolla , CA , USA -RRB- . 3M3-FBS is 2,4,6-trimethyl-N - -LRB- m-3-trifluoromethylphenyl -RRB- benzenesulfonamide . Phosphate-free DMEM medium was purchased from MP Biomedicals -LRB- formerly ICN Biomedicals , Aurora , OH , USA -RRB- . UNIFILTER plates were from Whatman -LRB- Clifton , NJ , USA -RRB- . Bacterial culture E. coli strains were grown overnight in Luria-Bertani -LRB- LB -RRB- 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 -LRB- HEPES -RRB- buffer , pH 7.4 , and 1 % D-mannose as previously described -LSB- 3 -RSB- . For experiments with bacteria in minimal medium , bacteria were subcultured at a dilution of 1:2,000 into minimal medium -LRB- M9 salts plus casamino acids supplemented with 2 mM glucose -RRB- . For convenience we used M9-CA liquid broth packets -LRB- E. coli Fast Media , MBI-Fermentas , Hanover , MD , USA -RRB- 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 -LRB- Gibco/Invitrogen , Grand Island , NY , USA -RRB- , 18 mM NaHCO3 , 20 μg/ml vancomycin , and 15 μg/ml gentamicin as previously described -LSB- 16 -RSB- . Ussing chamber studies of secretion were performed on T84 cell monolayers grown in Snapwell inserts -LRB- Corning Costar , Corning , NY , USA -RRB- . 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 -LRB- Sigma ; dissolved in warm 0.2 M acetic acid -RRB- 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 -LRB- TER -RRB- 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 -LRB- Pi -RRB- released from 5 ′ - AMP . This method has been used to detect activity of protein phosphatases such as PTEN -LSB- 17 -RSB- and lipid phosphatases -LSB- 18 -RSB- and is based on sensitive detection of low levels of Pi 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 -LRB- in mM -RRB- : 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 -LRB- the `` no AMP blank '' -RRB- . After a 10-min incubation at 37 ° an aliquot -LRB- usually 50 μl -RRB- 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 -LRB- a 96-well with 0.45-μm membrane for sterile filtration -RRB- . Sterile filtrates were prepared by centrifugation with collection of the filtered medium into another 96-well plate placed beneath the UNIFILTER as previously described -LSB- 19 -RSB- . Once again , experimental conditions were usually done in groups of six , with two wells not receiving any AMP -LRB- no AMP blanks -RRB- 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 -LRB- `` stop solution '' -RRB- . 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 Pi produced/min per 106 cells . For experiments showing nucleotidase release , the results were often expressed as nmol Pi 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 -LSB- 1 -RSB- . 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 -LRB- Abnova Corp. , Taipei , Taiwan -RRB- 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 IgG2b and were used at a concentration of 1 μg/ml . After washings , the secondary antibody was goat anti-mouse IgG2b conjugated to peroxidase at a dilution of 1:3,000 -LRB- Roche Molecular Biochemicals , Indianapolis , IN , USA -RRB- . Blots were developed by chemiluminescence as previously described -LSB- 20 -RSB- . Ussing chamber studies A Snapwell insert containing a monolayer of T84 cells was placed in the plexiglass `` slider '' and inserted into the Ussing chamber -LRB- Physiologic Instruments , San Diego , CA , USA -RRB- at 37 °C and continuously short-circuited by a four electrode , automatic voltage-clamp apparatus which measured short-circuit current -LRB- Isc -RRB- and transepithelial resistance -LRB- TER -RRB- ; 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 -LRB- in mM -RRB- : 140 Na + , 124 Cl − , 21 HCO3 - , 5.4 K + , , 1.2 Mg2 + , 1.2 Ca2 + , and 10 glucose . Raw short-circuit current -LRB- Isc -RRB- values were converted to μA per cm2 by dividing by the area of the Snapwell monolayer -LRB- 1.13 cm2 -RRB- . Other details of the Ussing chamber methods were exactly as described -LSB- 16 -RSB- . Protein assay Protein assay was by the Coomassie blue dye binding assay method of Bradford , using a Bio-Rad kit -LSB- 21 -RSB- . Expression of ecto-5 ′ - nucleotidase RNA by reverse transcription and real-time polymerase chain reaction -LRB- PCR -RRB- 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 -LRB- RNeasy Kits , Qiagen , Valencia , CA , USA -RRB- . 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 ′ -LRB- forward -RRB- and 5 ′ - ATA ACT GGG CAC TCG ACA CTT GGT-3 ′ -LRB- reverse -RRB- . As a normalizing gene we used glyceraldehyde phosphate dehydrogenase -LRB- GAPDH -RRB- as described by Khan et al. -LSB- 22 -RSB- 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 -LRB- Hercules , CA , USA -RRB- using SYBR Green as the dye to monitor the amplification . Relative expression was calculated by the ΔΔCt -LRB- `` Livak '' -RRB- method as described -LSB- 23 -RSB- , where Ct 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 -LRB- i.e , no extension step -RRB- 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 -LRB- ANOVA -RRB- with the Tukey-Kramer post-test for multiple comparisons , using InStat software for the Macintosh from GraphPad software -LRB- San Diego , CA , USA -RRB- . Graphs were prepared using Prism 4.0 software , also from GraphPad . Asterisks shown on graphs indicate a p value of < 0.05 . Results Ecto-5 ′ - nucleotidase activity was readily measurable in T84 cell monolayers using the phosphate release assay method developed in this study . Figure 1 shows the characteristics of the ectoenzyme in this system . Figure 1a shows a curve of activity vs substrate concentration , demonstrating that the enzyme shows Michaelis-Menten kinetics . In four experiments similar to that shown in Fig. 1a , the KM was 229 ± 30 μM and the Vmax was 2.7 ± 1.8 nmol/min per 106 cells -LRB- mean ± SD of 4 experiments -RRB- . While the KM was very consistent over time and from experiment to experiment , the Vmax was more variable as we compared experiments separated by months or years . We noted that highly T84 passaged cells -LRB- passage number of 70 or greater -RRB- showed lower levels of ecto-5 ′ - nucleotidase activity than less passaged cells from the same source -LRB- experiments not shown -RRB- . The KM we observed in intact T84 cells was higher than that reported for the purified enzyme derived from a colon carcinoma cell line , BCS-TC2 cells -LSB- 24 -RSB- . Figure 1b shows the raw data of Pi generated over time in the presence and absence of added AMP substrate and demonstrates that the assay was linear for 10 min ; therefore , 10 min was chosen as the standard assay duration . Fig. 1Characterization of ecto-5 ′ - nucleotidase activity in T84 cell monolayers . 5 ′ - Nucleotidase activity was measured by the release of inorganic phosphate -LRB- Pi -RRB- from AMP in phosphate-free buffer using a colorimetric method as described in `` Materials and methods . '' For a , c , and d , the spectrophotometric reading in the absence of AMP -LRB- `` no AMP blank '' -RRB- was subtracted from the total reading . In b the `` no AMP blank '' was not subtracted and is shown in b as labeled . a Michaelis-Menten curve of enzyme velocity vs concentration of AMP substrate . b Time course of Pi release vs time . c Lack of inhibition by tetramisole , an inhibitor of alkaline phosphatase . d pH dependence of 5 ′ - nucleotidase activity . e , f HPLC traces showing conversion of AMP -LRB- e -RRB- to adenosine -LRB- Ado , f -RRB- in T84 cell monolayers . g Sensitivity of the AMP-hydrolyzing activity to the inhibitor α,β-methylene-ADP , confirming it as ecto-5 ′ - nucleotidase Alkaline phosphatase is another abundant intestinal ectoenzyme which can hydrolyze 5 ′ - AMP -LSB- 25 -RSB- . Alkaline phosphatase can be distinguished from 5 ′ - nucleotidase by its alkaline pH optimum and sensitivity to inhibition by tetramisole . Figure 1c shows that the ectonucleotidase we measured on T84 cells was not inhibited by tetramisole -LRB- also known as levamisole -RRB- . Furthermore , its activity did not increase with increasing pH -LRB- Fig. 1d -RRB- providing evidence that the activity being measured was not alkaline phosphatase . In addition , we used HPLC to confirm that 5 ′ - AMP was being converted to adenosine in the supernatant medium of T84 cells -LRB- Fig. 1e and f -RRB- . Morover , the 5 ′ - nucleotidase activity we measured by phosphate release was sensitive to inhibition by α,β-methylene-ADP , a known inhibitor of ecto-5 ′ - nucleotidase -LRB- Fig. 1g and see also Fig. 4 -RRB- . Lastly , after we initiated this study , other researchers also reported using phosphate release from 5 ′ - AMP as their assay for ecto-5 ′ - nucleotidase -LSB- 1 -RSB- . We have found that the increased throughput of the phosphate release assay increases its usefulness compared to the much slower HPLC methods for ecto-5 ′ - nucleotidase . Figure 2 shows the effect of EPEC infection on ecto-5 ′ - nucleotidase activity released into the supernatant medium during an experimental EPEC infection of cultured T84 cells . Wild-type EPEC strains E2348/69 and JCP88 triggered a release of 5 ′ - nucleotidase into the supernatant medium while nonpathogenic E. coli strains HB101 and HS did not . The supernatant medium was subjected to sterile filtration prior to assay to remove bacterial cells as well as any detached host cells , so that the activity reflects a soluble and not a cell-bound form . In experiments where we determined both the supernatant release and the monolayer activity in the same experiment , supernatant release by wild-type EPEC was 30 -- 40 % of monolayer activity 3 h after the medium change -LRB- Fig. 2a and data not shown -RRB- . To determine if the 5 ′ - nucleotidase could possibly be derived from the E. coli bacteria rather than from the host we also assayed sterile filtrates of E. coli for 5 ′ - nucleotidase activity and it was virtually absent from the culture filtrates of all E. coli strains tested , including EPEC -LRB- Fig. 2b , light gray bars -RRB- . Even after treatment with a concentration of polymyxin B sufficient to cause > 99 % bacterial cell lysis the amount of bacterial nucleotidase-like activity was far less than the amounts we observed in the supernatant in Fig. 2a . In addition , our protocol included a medium change step in order to remove unbound bacteria . Therefore we concluded that the 5 ′ - nucleotidase activity being released in Fig. 2a was of host cell origin . Fig. 2Effect of E. coli infection on 5 ′ - nucleotidase activity in the supernatant medium . a Nonpathogenic E. coli strains -LRB- HB101 and HS -RRB- or EPEC strains E2348/69 and JCP88 were subcultured in DMEM medium for 2 h , then used to infect T84 cells at a multiplicity of infection of 100:1 in phosphate-free DMEM . After 35 min to allow adherence , the medium was changed to nucleotidase buffer and aliquots were collected at various times after the medium change , filtered through a 0.45-μm filter to remove bacterial cells , then assayed for 5 ′ - nucleotidase activity . Since the activity was measured in the cell-free sterile filtrates the activity is expressed as nmol/min per well . bE . coli suspensions were subjected to sterile filtration without treatment -LRB- light gray bars -RRB- or following treatment with 50 μg/ml polymyxin B , a lytic antibiotic -LRB- dark gray bars -RRB- . Then the nucleotidase activity of a 100-μl aliquot was measured . The 100 μl volume was chosen because this was the volume of inoculum needed to achieve an MOI of 100:1 for the slowest growing strain -LRB- E2348/69 -RRB- in a typical 48-well plate of T84 cells To determine if this hypothesis was correct and to try to determine the mechanism of EPEC-induced 5 ′ - nucleotidase release , we compared the 5 ′ - nucleotidase release triggered by wild-type EPEC strain E2348/69 with the plasmid-cured derivative , JPN15 , and its espF mutant , UMD874 -LRB- Fig. 3a -RRB- . The espF mutant is defective in host cell killing and in inducing damage to monolayer integrity -LSB- 14 , 26 -RSB- . Interestingly , the espF mutant was not attenuated in its ability to trigger 5 ′ - nucleotidase release , but instead it consistently outperformed the wild-type strain in this regard . In six experiments 5 ′ - nucleotidase release induced by the espF mutant exceeded that of the wild-type E2348/69 by an average of 2.3 ± 0.14 fold -LRB- p = 0.03 by paired t-test -RRB- , suggesting that host cell death or damage is not the mechanism of EPEC-induced nucleotidase release . Fig. 3a Effect of EPEC mutants and PI-PLC inhibitors and activators on induced 5 ′ - nucleotidase release into supernatant . Release of 5 ′ - nucleotidase activity into supernatant medium was measured as described in `` Materials and methods '' and in the legend to Fig. 2 . Once again , activity in the cell-free sterile filtrates is expressed as nmol/min per well . However , for purposes of comparison 48-well plates contain ~ 0.25 × 106 T84 cells per well at confluency . b U73122 , an inhibitor of PI-PLC , was used at a concentration of 2 μm and was re-added after the medium change ; * significantly decreased compared to the EPEC strain alone , p < 0.05 . cm-3M3-FBS , a cell-permeant sulfonamide activator of PI-PLC , was added at the concentrations and for the times indicated . d Inhibitory effect of neomycin on PI-PLC-induced 5 ′ - nucleotidase release ; this experiment was performed on cells grown in a 24-well plate , with ~ 0.8 × 106 cells per well ; * significantly decreased compared to PI-PLC alone . e PI-PLC was again added to a final concentration of 0.1 U/ml for the times shown ; * significantly different from the corresponding control . This figure is a composite of experiments that were separated in time and with cells of different passage number ; therefore the absolute amount of activity varies among the figure parts . f Effect of EPEC infection on expression of RNA encoding ecto-5 ′ - nucleotidase in T84 cells , by reverse transcription and real-time PCR . T84 cells were infected for 35 min , then the supernatant medium was changed to remove unattached bacteria , then the T84 cell monolayer harvested for RNA extraction 4 h after the medium change . Reverse transcription and PCR conditions were as described in `` Materials and methods . '' Expression of ecto-5 ′ - nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase -LRB- GAPDH -RRB- . f Normalized expression of a single representative experiment -LRB- mean ± SD of 6 PCR wells -RRB- . g Immunoblot analysis of the proteins released into the supernatant after 2 h of infection with EPEC or an EPEC mutant -LRB- SE1010 , sepZ/espZ -RRB- , using monoclonal antibody against CD73 . Lane 1 , supernatant from uninfected , control T84 cells . Lanes 2 and 3 , supernatant from cells infected with wild-type E2348/69 . Lanes 4 and 5 , supernatants from cells infected with the sepZ mutant . GPI-linked CD73 has an apparent molecular size of 72 kDa -LRB- not seen in this blot -RRB- and the GPI-cleaved soluble portion runs at 51 kDa -LRB- heavy band in lanes 2 and 3 -RRB- Ecto-5 ′ - nucleotidase is tethered to the extracellular surface of the cell by a glycosylphosphatidylinositol -LRB- GPI -RRB- lipid anchor -LSB- 24 , 27 -RSB- . GPI lipid anchors are cleaved by phosphatidylinositol-specific phospholipases C -LRB- PI-PLC -RRB- , and EPEC has been shown to activate at least one type of PI-PLC in the host cell , PI-PLC-γ -LSB- 28 -RSB- . Therefore we considered whether PI-PLC activation by EPEC could be a nonlethal mechanism by which EPEC triggered 5 ′ - nucleotidase release from host intestinal cells . Figure 3b shows an experiment in which we tested whether a PI-PLC inhibitor could block EPEC-induced nucleotidase release . Indeed , 2 μM U73122 did reverse the nucleotidase release triggered by wild-type EPEC strains . Conversely , a newly described cell-permeable activator of PI-PLC , called m-3M3-FBS -LSB- 29 -RSB- , triggered nucleotidase release from uninfected cells in a time - and concentration-dependent manner -LRB- Fig. 3c -RRB- . Experiments to test if m-3M3-FBS would enhance EPEC-induced release were inconsistent , with some experiments showing a mild enhancement or additive effect and others showing no enhancement or mild antagonism -LRB- results not shown -RRB- . In addition we also tested whether purified PI-PLC added to the apical surface of the T84 cell monolayer induced 5 ′ - nucleotidase release and it did -LRB- Fig. 3d and e -RRB- . Figure 3d also shows that 50 μg/ml neomycin could partially block 5 ′ - nucleotidase release by purified PI-PLC . In addition to its antimicrobial effects , neomycin is an inhibitor of several types of PLC enzymes including PI-PLC . Figure 3e shows that after a 2-h treatment with PI-PLC not only was 5 ′ - nucleotidase activity increased in the supernatant , but that monolayer activity was depleted as well -LRB- right portion of Fig. 3e -RRB- . In experiments similar to Fig. 3e done with EPEC infection , we could not detect any decrease in monolayer activity 2 -- 3 h after infection , despite the liberation of 5 ′ - nucleotidase activity into the supernatant . We considered the possibility that increased host cell synthesis of ecto-5 ′ - nucleotidase might be occurring in response to the cleavage of the enzyme from the cell surface . We analyzed the abundance of RNA encoding ecto-5 ′ - nucleotidase by reverse transcription and real-time quantitative PCR in response to EPEC infection . Figure 3f and Table 1 show that infection with wild-type EPEC and the EPEC espF mutant -LRB- UMD874 -RRB- increased the abundance of ecto-5 ′ - nucleotidase RNA compared to uninfected control cells . The increase in expression of ecto-5 ′ - nucleotidase RNA by wild-type EPEC was 33 % above control . While this is not a large increase , it does explain the maintenance of normal levels of ecto-5 ′ - nucleotidase on the surface of EPEC-infected cells , because under the most optimum conditions the release of enzyme activity into the supernatant is about 30 -- 40 % of monolayer activity -LRB- e.g. , Fig. 3e -RRB- . Table 1Effect of EPEC infection on expression of RNA encoding ecto-5 ′ - nucleotidase in T84 cells , by reverse transcription and real-time PCRConditionNormalized expression ratioaUninfected control1 .0 , by definition +1 U/ml PI-PLC1.23 ± 0.26HS-infectedb0.66 ± 0.22 E2348/69-infectedb 1.33 ± 0.21 cUMD874 -LRB- espF -RRB- - infectedb1 .40 ± 0.19 caCells were infected for 35 min , then the supernatant medium was changed to remove unattached bacteria , then the T84 cell monolayer harvested for RNA extraction 4 h after the medium change . Expression of ecto-5 ′ - nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase -LRB- GAPDH -RRB- . Data shown are the means ± SD of 5 similar experimentsbBacteria were added with the intent of achieving a multiplicity of infection -LRB- MOI -RRB- of 100:1 ; actual MOIs in the 5 experiments ranged from 71 to 144cSignificantly increased compared to uninfected control by paired t-test -LRB- p = 0.04 -RRB- The 5 ′ - nucleotidase released into the supernatant appeared to be of host cell origin , because it could be triggered by chemical stimuli in the absence of EPEC infection -LRB- Fig. 3c -- e -RRB- . The host origin was also proven using a mouse monoclonal antibody against human CD73 . Wild-type EPEC infection of T84 cells triggered the release into the supernatant of a 51 kDa protein recognized by a mAb against CD73 -LRB- Fig. 3g , lanes 2 and 3 -RRB- . In contrast , the EPEC sepZ/espZ mutant , which is defective in type III secretion , triggered the appearance of a fainter band which was of ~ 55 kDa in size . The origin of this faint band is unknown , but it is possible it is generated by proteolysis rather than PI-PLC activation . For example , E2348/69 expresses EspC , an autotransporter protease whose export is independent of type III secretion . Figure 4 shows that 5 ′ - nucleotidase activity in living T84 cells was susceptible to the same inhibitors as the purified enzyme studied by others . Figure 4a shows that α,β-methylene-ADP inhibited ecto-5 ′ - nucleotidase activity and that its inhibition has the characteristics of a competitive inhibitor . In the presence of α,β-methylene-ADP the enzyme shows an apparent increase in KM but no increase in the Vmax , and the inhibition can be overcome at high concentrations of AMP substrate . In contrast in Fig. 4b one observes that zinc acetate behaves as a noncompetitive inhibitor of the enzyme , with a decrease in the Vmax but no increase in the KM -LRB- in fact there is a small , paradoxical decrease in KM in the presence of zinc -RRB- . Figures 4c and d show the inhibitory dose-response curves at a single 0.2 mM concentration of AMP substrate . For both inhibitors , when concentration is expressed on a logarithmic scale , the curves are well fit by a sigmoidal , single-site model , allowing determination of the inhibitory concentration 50 % -LRB- IC50 -RRB- as shown in Figs. 4c and d . In addition to α,β-methylene-ADP and zinc acetate , ecto-5 ′ - nucleotidase activity was also inhibited by 3 -- 10 mM sodium fluoride as reported for the purified enzyme -LRB- data not shown -RRB- . 5 ′ - Nucleotidase activity released into the supernatant was also sensitive to inhibition by α,β-methylene-ADP and zinc acetate with similar potencies -LRB- data not shown -RRB- . Fig. 4Effect of the 5 ′ - nucleotidase inhibitors on enzyme activity in T84 cell monolayers . a , b Michaelis-Menten curves of enzyme activity vs substrate concentration in the absence -LRB- open symbols -RRB- and presence -LRB- closed symbols -RRB- of the inhibitor shown . Enzyme parameters were calculated by nonlinear curve fitting using GraphPad Prism and the fitted parameters of Vmax and KM are shown below the graphs . c , d Dose-response curves for inhibition of ecto-5 ′ - nucleotidase activity by α,β-methylene-ADP -LRB- c -RRB- and zinc acetate -LRB- d -RRB- using a 0.2 mM concentration of AMP substrate . In all parts of Fig. 4 the inhibitor was added before the AMP , and in c and d the curves were fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50 % -LRB- IC50 -RRB- value derived from the curve fit is shown In Fig. 4 the inhibitor was always added to the cell monolayer prior to the AMP substrate . Using the Ussing chamber apparatus to measure chloride secretion , we realized we could monitor the effects of inhibition of 5 ′ - nucleotidase activity dynamically and assess the ability of inhibitors to reverse a secretory response already underway . In preliminary experiments we found that to reverse an ongoing secretory stimulus we had to add inhibitors at concentrations considerably above their IC50 values . The requirement for addition of inhibitors well above the IC50 is due to the fact that adenosine is a very potent secretagogue in intestinal tissues and a very high degree of inhibition of ecto-5 ′ - nucleotidase is needed to reduce the production of adenosine below the secretory threshold . Figure 5a shows that secretion triggered by 10 μM AMP is reversed by theophylline , an adenosine receptor antagonist . Figure 5a emphasizes the fact that 5 ′ - AMP has no secretory ability unless it is converted into adenosine . Adenosine then acts via apical adenosine A2b receptors to trigger chloride secretion toward the apical side of the monolayer . Figure 5b shows that chloride secretion triggered by 1 μM AMP is partially antagonized by 2 mM zinc acetate even when the inhibitor is added after secretion has started . Addition of 1 μM adenosine restored the short-circuit current -LRB- Isc -RRB- to its previous trend , showing that zinc does not block adenosine receptors or `` poison '' the secretory machinery . Under the same experimental conditions , 200 μM α,β-methylene-ADP produced a much larger degree of reversal of Isc than did zinc -LRB- Fig. 5c -RRB- . As with zinc , α,β-methylene-ADP did not prevent a subsequent , normal Isc response to addition of adenosine -LRB- Fig. 5c , right-hand portion -RRB- , showing that it is acting as a 5 ′ - nucleotidase inhibitor and not an adenosine antagonist . The efficacy of inhibition by α,β-methylene-ADP in this paradigm , however , was strongly dependent on the concentration of AMP used to trigger secretion . At higher concentrations of AMP -LRB- e.g. , 20 μM -RRB- , the degree of inhibition by α,β-methylene-ADP was markedly less -LRB- Fig. 5d -RRB- than when 1 μM AMP was used as the stimulus . In contrast , zinc acetate produced the same , modest inhibition of Isc regardless of the concentration of AMP used . Figure 5e summarizes the effectiveness of the two inhibitors , zinc and α,β-methylene-ADP , when tested against various different concentrations of AMP as the initial stimulus . Figure 5e shows that the effectiveness of α,β-methylene-ADP declines significantly with increasing concentrations of AMP used , whereas zinc 's inhibitory effects are fairly constant over a range of AMP concentrations . The results of Fig. 5 -LRB- b -- e -RRB- vividly illustrate , at the level of of an electrophysiologic response , the difference between a competitive and a noncompetitive mode of action of an enzyme inhibitor -LRB- compare with Fig. 4a and b -RRB- . Fig. 5Effect of 5 ′ - nucleotidase inhibitors on the chloride secretory response to AMP in T84 cells in the Ussing chamber . Short-circuit current -LRB- Isc -RRB- was measured in T84 cells grown in Snapwell inserts as described in `` Materials and methods '' ; in this cell line and electrode configuration , a positive Isc -LRB- upward deflection on the graphs -RRB- represents chloride secretion in the basolateral to apical direction . All of the additions of agonists and inhibitors in these parts of the figure was on the apical -LRB- mucosal -RRB- side of the monolayers only . a Reversal of AMP-induced secretory response by theophylline , an adenosine receptor antagonist . b Partial inhibition of AMP-induced secretion by 2 mM zinc acetate , with restoration of the tracing to its previous trend by adenosine , demonstrating that adenosine receptors are not blocked by zinc . c Substantial reversal of Isc by α,β-methylene-ADP when a low concentration of AMP -LRB- 1 μM -RRB- is used as the stimulus . Note that adenosine fully restores secretion to its previous level . d Lesser degree of reversal of Isc by α,β-methylene-ADP when a higher concentration of AMP -LRB- 20 μM -RRB- is used as stimulus . e Summary of many experiments in which fixed concentrations of zinc and α,β-methylene-ADP were tested for inhibitory efficacy against varying concentrations of AMP as test stimulus . In Fig. 5e the slope of the α,β-methylene-ADP curve was − 1.73 ± 0.5 by linear regression -LRB- 95 % confidence limits of the slope value − 3.27 to − 0.2 , significantly different from zero , p < 0.04 -RRB- . In contrast the slope of the zinc curve was − 0.04 ± 0.23 and did not differ significantly from zero Since the discovery of EPEC-induced ATP release we have hypothesized that EPEC bacteria may gain a nutritional advantage by this inflicting kind of damage on the host cell -LSB- 3 -RSB- . The lumen of the gastrointestinal tract is a purine-limited environment , as demonstrated by in vivo gene expression screens such as in vivo expression technology -LRB- IVET -RRB- -LSB- 30 -RSB- and signature-tagged mutagenesis -LRB- STM -RRB- -LSB- 31 -RSB- . Nucleotides and nucleosides enhance growth of EPEC strains in vitro , and these growth-promoting effects are most obvious when adenosine is added to minimal medium -LRB- Fig. 6 -RRB- . Compared to minimal medium , addition of 30 μM adenosine markedly accelerated growth of EPEC strains such as E2348/69 -LRB- Fig. 6 -RRB- . Addition of 5 ′ - AMP also increased EPEC growth , although not as much as adenosine . Alpha-β-methylene-ADP alone has no effect , stimulatory or inhibitory , on EPEC growth , but it did completely reverse the growth-promoting effects of 5 ′ - AMP -LRB- Fig. 6a and b , right-hand bars -RRB- . Alpha-β-methylene-ADP did not block the enhanced growth seen with adenosine -LRB- not shown -RRB- . Similarly , 100 μM zinc acetate did not affect growth in minimal medium -LRB- Fig. 6c , left-hand bars -RRB- or the enhanced growth seen with adenosine -LRB- Fig. 6c , two middle bars -RRB- , but zinc did abolish the enhancement of growth by AMP . E. coli 5 ′ - nucleotidase is susceptible to inhibition by α,β-methylene-ADP and zinc ; in fact , the crystal structure of the E. coli enzyme was solved with α,β-methylene-ADP bound in the catalytic pocket -LSB- 32 -RSB- . Bacterial utilization of extracellular nucleotides is usually by dephosphorylation followed by uptake of the nucleoside -LRB- in this case adenosine -RRB- and inorganic phosphate -LSB- 33 -RSB- . Although we began studying α,β-methylene-ADP and zinc as inhibitors of the host cell enzyme , CD73 , a side effect -LRB- or side benefit -RRB- of these compounds may be inhibition of bacterial 5 ′ - nucleotidase activity as well , an effect that is able to block the nutritional benefits that E. coli bacteria derive from extracellular AMP -LRB- Fig. 6 -RRB- . Fig. 6Effect of 5 ′ - nucleotidase inhibitors on growth of EPEC strain E2348/69 in minimal medium supplemented with 30 μM adenosine or AMP . Minimal medium here refers to M9 salts , 2 % casamino acids , and 2 mM glucose . Growth was at 37 °C with 300 rpm shaking after a 1 : 2,000 dilution from an overnight culture in LB medium . a , c Culture turbidity by spectrophotometric absorbance at 600 nm . b Bacterial protein content . a , b Growth-enhancing effects of AMP are blocked by α,β-methylene-ADP . c Growth-enhancing effects of AMP are blocked by zinc acetate ; * significantly decreased compared to AMP alone -LRB- p < 0.05 -RRB- Discussion We became interested in ecto-5 ′ - nucleotidase -LRB- CD73 -RRB- because of its likely role in the generation of adenosine from adenine nucleotides released during EPEC infection . This host enzyme has not received a great deal of scrutiny in the context of microbial pathogenesis , and when it was studied its role was felt to be in the setting of a polymorphonuclear neutrophil -LRB- PMN -RRB- response , since activated PMNs release 5 ′ - AMP during migration into an infected area -LSB- 4 , 34 -RSB- . EPEC behaves clinically as a noninvasive pathogen and does not trigger an influx of fecal leukocytes , but nevertheless triggers ATP release from the host . Therefore our results suggest that ecto-5 ′ - nucleotidase plays a role in the response to a broader range of pathogens , including noninvasive enteric pathogens , than was previously thought . One important finding of our study is that EPEC infection triggers a release of ecto-5 ′ - nucleotidase from a cell-bound to a soluble , free form -LRB- Figs. 2 and 3 -RRB- . The significance of this finding is not clear at present but there are several possibilities to be considered . First , studies by other investigators have shown that cleavage of ecto-5 ′ - nucleotidase from its GPI lipid anchor increases its activity significantly via an increase in the Vmax -LSB- 27 -RSB- . This could result in an increase in the activity of 5 ′ - nucleotidase present in an EPEC-infected portion of the intestine , which could be an advantage to EPEC -LRB- see Fig. 6 and additional discussion below -RRB- . The observed increase in transcription of ecto-5 ′ - nucleotidase in response to EPEC infection -LRB- Fig. 3f and Table 1 -RRB- , if accompanied by a similar increase in protein synthesis and surface expression of the enzyme , would replace ecto-5 ′ - nucleotidase lost due to cleavage of the lipid anchor and also contribute to an increase in total activity . Third , release of nucleotidase from its lipid anchor would change its distribution from a mucosal location to a soluble form in the intestinal lumen , where it could possibly be more accessible to substrate . This could be important in the human or animal EPEC infection because expression of ecto-5 ′ - nucleotidase is highest in the intestinal crypts -LSB- 35 -RSB- , whereas EPEC adheres to villus tips as well as crypts . Last , after many days of infection the mucosa could be stripped of ecto-5 ′ - nucleotidase activity , leading to malabsorption of nucleotides in the diet . Indeed , malabsorption of electrolytes has been demonstrated after 7 days of infection in a rabbit model of EPEC infection -LSB- 36 -RSB- . In addition to ecto-5 ′ - nucleotidase , several other important digestive enzymes have GPI lipid anchors , including alkaline phosphatase and aminopeptidase N -LRB- CD13 -RRB- and these also could be liberated by EPEC-induced activation of PI-PLC . Another GPI-linked protein of interest in the intestinal tract is decay-accelerating factor -LRB- CD55 -RRB- , the cellular receptor for adhesion by diffuse adherent E. coli -LSB- 37 , 38 -RSB- and which is also involved in internalization of Salmonella into vacuoles . Several virulent pathogens have been found to secrete their own microbial ATPases or nucleotidases into the extracellular medium , including Entamoeba histolytica -LSB- 39 -RSB- , Trichinella spiralis -LSB- 40 -RSB- , and Vibrio cholerae -LSB- 41 -RSB- . Researchers have speculated that secreted microbial nucleotidases may serve in protection against killing by host immune cells , acquisition of nutrients from the host , or dampening of host immune responses . EPEC does not appear to secrete nucleotidase activity under any conditions we have encountered , but may achieve the same goal by triggering the release of 5 ′ - nucleotidase from the host cell . Ecto-5 ′ - nucleotidase acts on nucleoside monophosphates , with 5 ′ - AMP as the preferred substrate , and is inactive toward ATP . Therefore , the generation of AMP from ATP requires another enzyme or enzymes . CD39 , or ectonucleoside triphosphate diphosphohydrolase , is the major enzyme responsible for this conversion in the cardiovascular system and probably in the gastrointestinal tract as well -LSB- 42 , 43 -RSB- . CD39 is also deserving of further study in the context of microbial pathogenesis . One surprising finding of our study was that the espF EPEC mutant was not attenuated but in fact better than the wild-type EPEC in release of ecto-5 ′ - nucleotidase -LRB- Fig. 3a -RRB- . Although this mutant is defective in host cell killing , its phenotype has been instructive in many ways . For example , the ability of the espF mutant to trigger a robust ATP release led to us the discovery of a new pathway for ATP release from the host cell ; this `` second pathway '' is not dependent on cell death but is dependent on the cystic fibrosis transmembrane regulator -LRB- CFTR -RRB- for ATP efflux -LSB- 19 -RSB- . The competence of the espF mutant in release of ATP and of ecto-5 ′ - nucleotidase from the host cell may help explain why this mutant is only modestly attenuated compared to wild-type in some animal models of EPEC infection , such as Citrobacter rodentium in mice -LSB- 44 -RSB- . On the other hand , the sepZ/espZ mutant is attenuated in its ability to trigger ecto-5 ′ - nucleotidase release -LRB- Fig. 3f -RRB- , showing that type III secretion is necessary for PI-PLC activation and release of the enzyme from its lipid anchor . The role of ecto-5 ′ - nucleotidase in triggering EPEC-induced fluid secretion is highlighted in the Ussing chamber studies shown in Fig. 5 . Even after AMP has induced a chloride secretory response , addition of the nucleotidase inhibitor α,β-methylene-ADP is able to reverse the short-circuit current in a manner similar to that of an adenosine receptor antagonist , theophylline -LRB- compare Fig. 5a and c -RRB- . Tracings such as those in Fig. 5 seem to indicate that to sustain a secretory response there must be on ongoing production of adenosine from AMP . In other words , there seems to be a `` sink '' for depletion of adenosine from the extracellular medium , either by cellular reuptake or by adenosine deaminase . The rapid turnover of adenosine implied in these experiments suggested that inhibitors of ecto-5 ′ - nucleotidase might be able to block EPEC-induced fluid secretion in animal models of infection . In discussions of zinc on ecto-5 ′ - nucleotidase it is worthwhile to note that zinc supplements have been shown to reduce the duration and severity of watery diarrhea in children in developing countries around the world -LSB- 45 -- 48 -RSB- . In most of these trials the microbial etiology of the diarrhea was not carefully investigated , so it is unknown if zinc is particularly efficacious in diarrhea due to certain pathogens , such as EPEC , or if it beneficial `` across the board '' for multiple agents . Likewise , the mechanism by which zinc exerts its beneficial effects has not been carefully studied in the field trials . In earlier reports it was assumed that zinc was acting by correcting a zinc deficiency , but this assumption has been called into question by the finding that zinc supplements have a beneficial effect on diarrhea even in children with normal serum zinc levels at the outset -LSB- 49 -RSB- . Kelleher et al. showed that zinc provided some added benefit beyond a probiotic -LRB- Lactobacillus rhamnosus -RRB- in rhesus monkeys infected with the human EPEC strain E2348/69 ; the monkeys studied were not zinc deficient -LSB- 50 -RSB- . Inhibition of ecto-5 ′ - nucleotidase or K + channels -LSB- 51 -RSB- by zinc could slow diarrhea but this should be considered a pharmacological effect of the mineral rather than repletion of a deficiency . Inhibition of 5 ′ - nucleotidase also blocks the growth-enhancing effects of AMP on EPEC bacteria -LRB- Fig. 6 -RRB- ; since this is a direct effect on bacterial cells this action of zinc would not depend on the nutritional status of the host . The effects of zinc on EPEC infection and on diarrheal diseases in non-zinc-deficient children deserve further study .

O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O 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