Vol. 60, No. 2

INFECTION AND IMMUNITY, Feb. 1992, p. 406-415 0019-9567/92/020406-10$02.00/0 Copyright C 1992, American Society for Microbiology

Role of Vibrio cholerae Neuraminidase in the Function of Cholera Toxin JAMES E. GALEN,"2 JULIAN M. KETLEY,1t ALESSIO FASANO,lt STEPHEN H. RICHARDSON,3 STEVEN S. WASSERMAN,1 AND JAMES B. KAPERl 2* Center for Vaccine Development, Department of Medicine,1 and Department of Microbiology and Immunology,2 University of Maryland School of Medicine, Baltimore, Maryland 21201, and Department of Microbiology and Immunology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 271033 Received 19 July 1991/Accepted 9 November 1991 Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vectormediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture ifitrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ ifitrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 104 to 108 CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 109 CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions. erae NANase and that GM1 is resistant to further hydrolysis

The intestinal pathogen Vibrio cholerae must colonize the gastrointestinal tract and excrete a potent enterotoxin to cause disease (23, 30). This 84-kDa cholera enterotoxin (CT) is composed of the catalytically active A subunit and five identical B subunits that constitute the binding region of the toxin (24). Binding of CT to the GM1 ganglioside receptors of enterocyte microvilli is followed by internalization of the toxin, which catalyzes the activation of adenylate cyclase, causing a rise in intracellular cyclic AMP (cAMP) (10, 14, 24). Sodium and chloride transport into villus epithelial cells is inhibited, and chloride is secreted into the lumen from crypt cells. Since water flows passively with electrolytes in response to osmotic gradients, CT causes the absorption of water through villi to cease and amplifies the secretion of water from crypt cells, resulting in copious diarrhea (1, 8). In addition to CT, V. cholerae 01 strains produce a neuraminidase (EC 3.2.1.18; NANase) which catalyzes the conversion of higher-order gangliosides to GM1 (25). It has been hypothesized that NANase may produce locally high concentrations of the GM1 receptor for CT in vivo, thereby enhancing the binding and internalization of CT and resulting

by NANase; (ii) isolated rabbit small-intestine brush borders exposed to NANase bound twice as much 125I-CT as did untreated control tissues (16); (iii) treatment of cultured mouse neuroblastoma cells with NANase increased the accessibility of GM1 receptors and binding of 1251I-labeled CT five- to sevenfold (37); (iv) exposure of isolated rat adrenal tumor cells to NANase prior to incubation with CT caused up to a sevenfold increase in intracellular cAMP depending on the concentration of CT used (19). When the influence of NANase on toxin-induced secretion was examined in vivo by using canine ligated ileal loops perfused with CT, secretion increased fourfold in loops pretreated with NANase relative to that in untreated control loops (48). These results suggest a synergistic influence of V. cholerae NANase and CT on secretion in which NANase enhances toxin binding to increase the severity of a diarrheal response. However, Holmgren et al. (25) were unable to confirm this proposed enhancement of CT binding and penetration by NANase with ligated rabbit ileal loops. The objective of this investigation was to examine the role of V. cholerae NANase in the binding and penetration of CT into intestinal epithelial cells. The potential role of NANase in colonization was not addressed. We recently reported the cloning and preliminary characterization of the nanH gene coding for V. cholerae NANase (12, 50). Here we report the sequence of this gene and the construction of isogenic V. cholerae strains whose only known difference is the ability to excrete active NANase. Comparisons of the biological effects of these isogenic strains made with both in vitro and in vivo techniques will be described; these comparisons indicate a subtle but significant role for NANase in the binding and uptake of CT by susceptible cells under defined conditions.

in greater efflux of water and electrolytes (25, 28). Evidence supporting the role of V. cholerae NANase in the binding and penetration of CT comes primarily from in vitro results using purified NANase and CT with a variety of substrates: (i) Svennerholm (49) first showed that GM1 can be generated in vitro from complex gangliosides hydrolyzed by V. chol* Corresponding author. t Present address: Department of Genetics, Adrian Building, University Road, University of Leicester, Leicester LE1 7RH, Great Britain. t Present address: Cattedra di Pediatria, Ospedale Pugliese, Universita di Catanzaro, Via Pio X88100, Catanzaro, Italy.

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TABLE 1. Bacterial strains and plasmids used in this study Relevant genotype or phenotype

Organism and Strain

V. cholerae (classical biotype, 01 serotype) Ogawa 395 CVD395.1 CVD395.2 CVD101 CVD101.2 E. coli HB101

DHSa SY327Xpir SMlOXpir

CT' NANase+ CT' NANase+ CT' NANaseCT- NANase+ CT- NANase-

Reference 27 This study This study 27 This study

F- hsdS20 (rB- mB-) recA13 leuB6 ara-14 proA2 lacYl galK2 rpsL20 (Smr) xyl-5 mtl-l supE44AF- 480dlacZAM15 A(lacZYA-argF)U169 endAl recAl hsdR17 (rK MK+) deoR thi-l supE44 gyrA96 relAl XA(lac pro) argE(Am) rif nalA recA56 (XpirR6K) thi thr leu tonA lac Y supE recA::RP4-2-Tc:: Mu

32

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34 35 12, 50 12, 50 This study This study This study

11

35 35

(XpirR6K) Kmr

Plasmids

pJBK54.1a pGP704 pCVD361

ori R6K mob RP4 Apr oriEI nanH::mini-kan Apr Kmr oriEl NANase+ Apr oriEI nanH::mini-kan Cmr Kmr oriEl AnanH Cmr ori R6K mob RP4 AnanH Apr

pCVD364 pCVD382 pCVD383 pCVD383.1

a pJBK54.1 is a derivative of pBR325 containing a 622-bp deletion between the unique HindIII and Sall sites. In addition, the HindIlI site has been changed by linker addition to a Sail site, and the PstI site within the ampicillin gene has been removed (34).

MATERIALS AND METHODS Genetic procedures. Subcloning and sequencing of the nanH gene was performed as previously described (12, 50). Plasmids and bacterial strains used in this study are listed in Table 1. The nanH deletion mutation to be mobilized into the chromosome of V. cholerae 395 was constructed in vitro from a previously mutagenized copy of nanH inactivated by insertion of a 1.9-kb mini-kan transposon to create pCVD361 (12, 50; Table 1). This nanH::mini-kan mutation, carried on a 14-kb Sall restriction fragment from pCVD361, was subcloned into the unique SalI site of pJBK54.1 to create pCVD382. As shown in Fig. 2, two BamHI sites, positioned 66 bp within either terminus of the mini-kan insert, were cleaved with BamHI to remove the transposon prior to deletion of flanking nanH sequences with exonuclease Bal 31 (38). Following treatment with Klenow polymerase I, the DNA was religated with T4 DNA ligase. Nucleotide sequence analysis of the internal nanH deletion carried on the resulting construct, designated pCVD383, revealed the deletion to be 124 bp in length (see Fig. 1). This mutation causes a frameshift that is predicted to produce a truncated 44.8-kDa NANase protein. A 4.8-kb BglII fragment from pCVD383 was ligated into the BglII-digested suicide vector pGP704 to produce the chimeric suicide vehicle pCVD383.1, which contains the nanH deletion mutation. pCVD383.1 was recovered by transformation of the ligation mixture into competent cells of the permissive Escherichia coli strain SY327Apir. The pir gene within the phage A lysogen of SY327 encodes the rr protein necessary for the function of the R6K origin of replication within pCVD383.1. However, since no conjugative functions are present within the SY327 chromosome, pCVD383.1 was retransformed into the permissive E. coli donor strain SMlOApir for mobilization into nonpermissive V. cholerae recipients. pCVD383.1 was not

recovered from ligation mixtures transformed directly into SMlOApir because of the low transformation frequency of this strain. Bacterial matings were accomplished by using donor and recipient strains freshly grown overnight from frozen stocks on L agar (32). Selection where appropriate for Apr colonies was carried out on L agar containing 200 ,ug of ampicillin per ml. All incubations were carried out at 37°C. Donor strains were E. coli SMiOApir lysogens carrying nanH deletion mutations within chimeric suicide vehicles. Recipient strains were either wild-type V. cholerae Ogawa 395 or the vaccine strain CVD101 deleted for the A subunit of CT. Plate matings (cross-streak matings) were performed on L agar containing 15 U of polymyxin B and 200 ,ug of ampicillin per ml incubated for 16 h. To resolve merodiploids, Apr V. cholerae transconjugants were subsequently grown in L broth without selection on an orbital shaking incubator at 210 rpm until the optical density at 600 nm (OD6w) reached 1.2 to 1.6 (-2 x 109 to 3 x 109 CFU/ml). Cultures were diluted 106-fold in L broth and plated on L agar. Aps colonies were identified by replica plating, streaked for isolation on L agar, and tested for their abilities to produce active NANase. In order to minimize genetic variability, isogenic strains were frozen at -70°C in 50% glycerol immediately after their creation; a single frozen stock of each strain was used for all of the in vitro and in vivo testing of the isogenic strains described in this work. Characterization of isogenic strains. NANase production was quantitated by measuring hydrolysis of the fluorogenic substrate 2'-(4-methylumbelliferyl)a-D-N-acetylneuraminic acid (39) with an SLM 8000 fluorescence spectrophotometer (SLM Instruments, Champaign, Ill.). Production of CT was measured by the GM1 enzyme-linked immunosorbent assay (ELISA) as described previously (43). The 18-base synthetic

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GALEN ET AL.

oligonucleotide C13A (5'-GAAGAGCATCGATTCACA-3', which corresponds to bases 158 to 175 of the DNA sequence reported in Fig. 1) was used with Southern blots as indicated elsewhere (27) to demonstrate the absence of large uncharacterized chromosomal deletions or rearrangements near the nanH locus. Strains were also probed with the 17-base synthetic oligonucleotide 363K (5'-TCGGAGCACTAA CACCG-3', which is complementary to bases 1473 to 1489 of the sequence reported in Fig. 1) to confirm that NANaseisogenic strains no longer carried this sequence. The sequence of 363K is contained within the region of nanH deleted in the construction of the isogenic strains. Sequencing of the AnanH chromosomal mutation. In addition to Southern blot analysis, the integrity of the nanH deletion within the chromosome of V. cholerae CVD395.2 was confirmed by direct sequencing of size-fractionated chromosomal DNA with the 16-base synthetic primer 380A (5'-TCTCCTGCGATACTTC-3', which is complementary to bases 1612 to 1627 of Fig. 1). Chromosomal DNA (200 ,ug) was cleaved with the restriction endonuclease EcoRI and electrophoresed into 0.3% agarose. Restriction fragments of approximately 3 kb were excised and purified by electroelution and ethanol precipitation by standard techniques. Purified DNA was denatured by the alkaline denaturation method of Chen and Seeburg (3) and analyzed by the dideoxynucleotide sequencing technique (46) with Sequenase (United States Biochemical Corp., Cleveland, Ohio). Sequence reactions were carried out by following protocols recommended by U.S. Biochemical Corp. with several modifications: (i) approximate molar ratio of DNA/primer was 1:1; (ii) labeling mixture was diluted 1:10; (iii) labeling and extension reactions were carried out for 3 min at room temperature with [a-32P]dATP (Amersham; -800 Ci/mmol). Flow cytometry. Bacterial culture filtrates were prepared with fresh isolates grown on L agar from frozen isogenic stocks. Cells were inoculated into 3 ml of L broth in culture tubes (17 by 100 mm) to an initial OD600 of 0.42 (1-cm path length) and grown at 37°C with shaking at 210 rpm to an OD6. of 2.03 (-3 x 109 CFU/ml). Cultures were not used if cell clumping was observed. Bacteria were removed by centrifugation at 12,000 x g for 10 min, and the supernatants were filter sterilized by passage through a 0.22-,um-pore-size Millipore Millex-GV low-protein-binding filter. Culture filtrate from CVD395.1 contained 400 U of NANase and 23.0 ng of CT per ml; CVD395.2 filtrate contained 14.4 ng of CT per ml but no detectable NANase activity. Simian virus 40-transformed C3H (15) or C57BL/6 (42) mouse fibroblast cell lines were used for flow cytometry. Monolayers were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and harvested by incubation with 0.25% trypsin at 37°C for 2 to 5 min. Washed cells were resuspended in Dulbecco's phosphate-buffered saline to a density of -1.75 x 105 cells per ml. Fluorescencebinding studies were carried out by using CT conjugated to fluorescein isothiocyanate (CT-FITC; List Biochemicals). Fibroblasts (3.5 x 105) were either stained directly with 100 ng of CT-FITC for 2 h at 4°C or pretreated with NANase prior to CT-FITC addition. Pretreated cells were incubated for 30 min at 37°C with 0.2 U of purified NANase (Calbiochem) or 500 ,l1 of culture filtrates. For each binding experiment, 40,000 CT-FITC-labeled fibroblasts were analyzed on a FACS 440 instrument (Becton Dickinson). Ussing chambers. Bacterial culture filtrates were prepared as described above for flow cytometry analysis. L-broth filtrates were obtained from cultures grown at 37°C to an OD6. of 1.91 + 0.03 (mean ± standard error [SEM]). When

INFECT. IMMUN.

quantitated by GM1-ELISA, CVD395.1 produced 26.7 + 5.8 ng of CT and 530 ± 22.3 U of NANase per ml; CVD395.2 produced 16.0 ± 1.7 ng of CT per ml with no observable NANase activity. Analysis by the two-tailed Student's t test confirmed no significant difference in CT production in CVD395.1 and CVD395.2. In some experiments, culture filtrates were prepared from bacteria grown at 25°C in 2x colonization factor broth with sialic acid (2xCFBN medium), a modified 2xCFB medium (21) supplemented with 1.0 mg of sialic acid (Sigma Chemical Co.; type VI) per ml to induce the production of NANase. Cultures were grown to an OD6. of 1.98 ± 0.28 (mean ± SEM). Both isogenic strains cultured under these conditions produced median values of 2.7 p.g of CT per ml, ranging from 0.6 to 14.0 p.g/ml for CVD395.1 and from 0.4 to 15.4 ,ug/ml for CVD395.2; 584 ± 16 U (mean ± SEM) of NANase per ml were present in filtrates from CVD395.1, with no observable activity in CVD395.2 filtrates. Spontaneous fluctuations in the transepithelial potential difference (PD), tissue resistance, and short-circuit current (ISC) were measured in conventional Lucite Ussing chambers (1.12-cm2 opening) using New Zealand White male rabbit ileal tissue, as described elsewhere (18). Increases in ISC are presented as AIsc, where AIsc = ISC at time x - ISC at time zero. The tissue mounted into the chambers was stripped of its serosal and muscular layers to remove any influence on PD not attributable to enterocyte active transport (7). The mucosal tissue used was therefore composed of the epithelial cell layer, basement membrane, lamina propria, and muscularis mucosae. Measurements were made under constant conditions of ionic strength, temperature, oxygenation, and circulation of the solutions bathing the ileal tissue, with equivalent hydrostatic pressure present on either side of the tissue. Culture filtrates (300 ,ul) were added to the mucosal and serosal sides of tissues. Tissues were therefore exposed to the following mean concentrations of CT (per milliliter of Ringer solution) and NANase (also per milliliter of Ringer solution) from L-broth filtrates: [CT]cvD395.1 = 0.8 ± 0.2 ng/ml, [CT1CVD395.2 = 0.5 ± 0.1 ng/ml; [NANase]CVD395.1 = 15.9 ± 0.7 U/ml, with no observable NANase activity produced by CVD395.2. Final concentrations of CT and NANase from 2xCFBN filtrates added to chambers were [CTICVD395.l = [CT1cvD395.2 = 81.1 ng/ml and [NANase]CVD395.1 = 17.5 + 0.5 U/ml, with no detectable NANase activity produced by CVD395.2. Theophylline was added to the serosal side to a final concentration of 5 mM after the maximum AIsc was observed, and all experiments were carried out within 4 h. Data were analyzed by using two-tailed Student's t tests. FA studies in suckling mice. The secretory response evoked by each of the isogenic strains was evaluated by using a sealed infant mouse model, as described by Kothary and Richardson (29). For the experiments involving unstarved mice, isolated colonies from each test strain were inoculated into 20 ml of L broth in a 250-ml flask and grown as described above to a viable cell concentration of 2 x 109 to 3 x 109 CFU/ml. Cells from 10 ml of culture were collected by centrifugation and suspended in 1 ml of fresh L broth containing 0.05% Evans blue dye. Approximately 107, 108, or 109 CFU in a volume of 50 ,ul was inoculated intragastrically into nonfasted 5-day-old CD1 suckling mice sealed at the anus with a cyanoacylate adhesive just prior to inoculation. Colony counts were determined by tube dilution on L agar to confirm the number of viable organisms in the inoculum. Mean colony counts for CVD395.1 and CVD395.2 never differed by more than 1.3-fold. Litters of at least 15

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ROLE OF V. CHOLERAE NEURAMINIDASE IN FUNCTION OF CT

pups were used to compare CVD395.1, CVD395.2, and CVD101.2, with at least four pups per strain inoculated. Inoculated mice were not returned to their mothers and were sacrificed 18 h later for removal of the gastrointestinal tract. Fluid accumulation (FA) ratios were calculated by using the following formula: FA = (weight of stomach + weight of intestines)/[body weight - (weight of stomach + intestines)]. For experiments involving starved mice, isolated colonies from each test strain were inoculated into 3 ml of L broth in a 14-ml polystyrene culture tube and incubated at 37°C and 220 rpm for 1.5 to 2.0 h. Cultures were then diluted 1:500 in 4 ml of fresh L broth and incubated at 30°C and 220 rpm for 16 h. Cells from 2 ml of these cultures were then collected by centrifugation and resuspended at 1:1 in fresh L broth containing Evans blue dye. Approximately 105, 106, or 107 CFU was inoculated into 7- to 8-h-fasted suckling mice, and FA ratios were determined as described above. Mean colony counts were different by 2.5-fold at 105 CFU but by less than 1.3-fold for inoculation doses of 106 and 107 CFU. Results from both fasted and nonfasted mouse experiments were statistically analyzed by two-tailed Student's t tests. Nucleotide sequence accession number. The nucleotide sequence reported in Fig. 1 has been submitted to GenBank and assigned accession no. M83562. RESULTS Sequence and organization of the nanH gene. Nucleotide sequence analysis revealed an open reading frame 2,346 bp in length (Fig. 1). The predicted N-terminal amino acid sequence of this open reading frame has previously been compared with that obtained from automated Edman degradation amino acid sequence analysis of purified extracellular V. cholerae 395 NANase (12, 50); the complete nucleotide sequence reported here corresponds to that of nanH, which encodes the V. cholerae 395 NANase protein. The predicted molecular mass of the NANase precursor polypeptide is 85.6 kDa. Upon cleavage of the 24-residue leader peptide (12, 50), the predicted molecular mass of extracellular NANase is reduced to 83.0 kDa. The arrangement within NANase of the repeated sequence Ser-X-Asp-X-Gly-X-Thr-Trp is also indicated in Fig. 1. This repeated sequence, whose function is unknown, has been reported to be conserved among the NANases of Clostridium perfringens A99 (44), Clostridium sordellii G12 (44), Salmonella typhimurium LT-2 (44), and Bacteroides fragilis (45) and is arranged within these proteins in a configuration similar to that of the V. cholerae NANase. No TTTTGAT binding site for the V. cholerae transcriptional activator Tox R (36) was found. However, an unusual arrangement of three putative catabolite repression protein (CRP)-binding sites (4) involving both sense and antisense DNA strands was found 5'-proximal to the nanH gene (Fig. 1). In addition to these putative transcriptional control signals, a Shine-Dalgarno sequence was identified within a sequence, AAAGGGAG, that is also found within the 5'proximal region of the V. cholerae ctx operon (31, 33). The significance of this homology is unknown. Characterization of isogenic strains CVD395.1 and CVD395.2. As described in Materials and Methods, a 124-bp deletion mutation was obtained within the nanH gene of pCVD383.1 (Fig. 2), which was then mobilized into the chromosome of V. cholerae 395. Apr V. cholerae transconjugants containing pCVD383.1 integrated into the chromosome by single homologous crossovers were readily isolated from plate matings. Upon passage of transconjugants for several generations without selective pressure, V. cholerae

409

Aps revertants were recovered at a frequency of 1 in 250. Of four revertants isolated, one proved to be deficient in NANase production. From these revertants, the isogenic V. cholerae strains CVD395.1 (CT' NANase+) and CVD395.2 (CT' NANase-) were chosen for further study. The construction of CVD395.1 and CVD395.2 is represented schematically in Fig. 3. This 124-bp nanH deletion mutation was also mobilized by identical manipulations into the nontoxigenic V. cholerae vaccine strain CVD101 (CT subunits A-B+), creating the negative control strain CVD101.2 used in experiments described below. CVD395.1 and CVD395.2 produced similar levels of CT, as indicated by GM1-ELISA; as expected, production of active NANase was observed only for CVD395.1 (see in vitro results below). Southern blot analysis was used to demonstrate the integrity of these isogenic strains (data not shown). Results from the 18-base synthetic oligonucleotide probe C13A are consistent with a single genetic locus for nanH in V. cholerae 395 and ruled out extensive rearrangements within the chromosome of either CVD395.1 or CVD395.2 in the regions flanking nanH. The 17-base synthetic oligonucleotide 363K, derived from a nucleotide sequence deleted at the AnanH locus, was used to confirm that NANase- isogenic strains no longer carried this sequence. As expected, probe 363K hybridized to DNA from CVD395.1 but did not hybridize to DNA from CVD395.2, in which homologous sequences were deleted. pGP7O4 was also used as a probe to confirm that vector nucleotide sequences were absent from all strains used in these studies. Direct chromosomal sequencing of DNA isolated from CVD395.2 by using the 16-base synthetic primer 380A confirmed the presence within the chromosome of an internal nanH deletion identical to that carried on the plasmid pCVD383.1, as shown in Fig. 4. In vitro analysis of wild-type and AnanH V. cholerae isogenic strains. To test the hypothesis that NANase increases the binding of CT to cell membranes, tissue culture cells were first studied by flow cytometry. C57BL/6 mouse fibroblasts were exposed to 0.2 U of purified V. cholerae NANase and then to CT-FITC. Control C57BL/6 cells were exposed only to CT-FITC. Cell suspensions were then analyzed by flow cytometry to monitor changes in fluorescence. As can be seen in Fig. 5A, fluorescence due to the binding of CT-FITC to C57BL/6 fibroblasts increased fivefold when cells were first exposed to purified NANase (peak 3 versus peak 4). C57BL/6 cells were then exposed to crude NANase present in culture filtrates. Exposure of C57BL/6 fibroblasts to CVD395.1 culture filtrate again increased the fluorescence due to CT-FITC binding by fivefold relative to CT-FITC binding induced by CVD395.2 filtrate (Fig. SB, peak 3 versus peak 4). When these experiments were repeated with a C3H mouse fibroblast cell line, the increase in binding of CT-FITC due to exposure of cells to purified or crude supernatant NANase was once again observed. As can be seen in Fig. 5C, fluorescence due to the binding of CT-FITC to C3H fibroblasts increased 18-fold when cells were first exposed to 0.2 U of purified NANase (peak 3 versus peak 4). C3H cells exposed to crude NANase present in CVD395.1 culture filtrate (identical to that used for the C57BL/6 experiments) increased in fluorescence due to CT-FITC binding eightfold relative to CT-FITC binding induced by CVD395.2 filtrate (Fig. SD, peak 3 versus peak 4). These experiments support the hypothesis that NANase increases the binding of CT to the surfaces of susceptible cell lines, although neither specificity of binding nor extent of toxin penetration can be addressed by using these data.

168_~ GAT

INFECT. IMMUN.

GALEN ET AL.

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A T T 2601 GTTAAGCGATAGCACIATCAAGTGCTATCA 25212 CTASCAAGM GCTITGAAACTC= >TAAACGTACGT FIG. 1. Nucleotide sequence and deduced amino acid sequence of the V. cholerae 395 nanH gene. Amino acids are given in standard one-letter code, with the stop codon represented by an asterisk. The -10 and -35 regions of a putative promoter are shown. Open arrows (centered at bases 102, 145, and 168) represent putative CRP-binding sites, with dots over DNA bases (or under bases for binding sites on the opposite strand) denoting bases homologous to the E. coli CRP consensus sequence. The double underbar centered at base 200 identifies a proposed ribosome-binding site. The leader peptide for NANase is marked with a bold line originating at base 208. Open rectangles show the positions of the repeated amino acid sequence motif reported conserved among bacterial NANases (44); circled residues are homologous to the highly conserved sequence Ser-X-Asp-X-Gly-X-Thr-Trp. The wavy underline centered at base 1434 indicates the insertion site for the mini-kan transposon used to mark nanH; the bold arrows extending to either side of the mini-kan insertion site delineate a 124-bp Bal 31 deletion mutation created within nanH (Fig. 2). The boundaries of this deletion were determined by DNA sequence analysis, as shown in Fig. 4. S.D., Shine-Dalgarno sequence.

The ability of NANase to increase the binding and subse-

quent internalization of CT produced by the isogenic V. cholerae strains was quantitated by using rabbit smallintestine tissue mounted in Ussing chambers. Ussing cham-

bers were used to measure fluctuations in the spontaneous PD across rabbit ileal tissue exposed to culture filtrate from CVD395.1, CVD395.2, or CVD101.2. Since CT blocks the

absorption of Na+ and Cl- through small-intestine villus enterocytes and increases net secretion of Cl- from crypt cells, a net flow of negative charge from the serosal to the mucosal side of the tissue will be electrogenic and result in increased PD. If NANase increases the binding and subsequent penetration of CT present in culture filtrates, then the PD measured for filtrates from CVD395.1 (NANase+)

ROLE OF V. CHOLERAE NEURAMINIDASE IN FUNCTION OF CT

VOL. 60, 1992 BHI

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FIG. 2. Schematic diagram of the construction of pCVD383.1, carrying AnanH. pCVD382 carries the nanH gene (open rectangle) marked by insertion of a mini-kan transposon (hatched rectangle); flanking vibrio DNA is represented with bold lines. pCVD382 was cleaved with the restriction endonuclease BamHI, which removed the mini-kan transposon, and treated with Bal 31 to delete flanking nanH sequences. Following treatment with the Klenow fragment of E. coli DNA polymerase I, the DNA was religated by using T4 DNA ligase. The resulting plasmid, pCVD383, contains a 124-bp deletion within nanH, indicated by an arrowhead. pCVD383 was cleaved with BglII, generating a 4.7-kb fragment containing AnanH, which was ligated into the suicide vector pGP704 cleaved with BglII and treated with bacterial alkaline phosphatase (BAP). The resulting suicide vehicle, pCVD383.1, contains the origin of replication ori R6K (filled rectangle), which functions only in the presence of the 'T protein produced by the pir gene. Therefore, pCVD383.1 was recovered by transformation of the ligation mixture into the readily transformable strain E. coli SY327Xpir. Abbreviations: BG II, BglII; BH I, BamHI; SA I, Sall; bla, ,B-lactamase; cat, chloramphenicol acetyltransferase; mob RP4, plasmid mobilization region from pRP4.

should be higher than the PD resulting from CVD395.2 (NANase-) filtrates. Since Ohm's Law states that ISc = PDIR, where R is tissue resistance, an increase in PD is expected to cause an increase in Isc. As shown in Fig. 6, when AIsc was measured, tissues exposed to L-broth filtrates from CVD395.1 produced a significant increase in Alsc (59.53 ± 6.10 p.Amp/cm2) relative to tissues exposed to CVD395.2 filtrates lacking NANase activity (36.08 ± 3.56 p.Amp/cm2; P < 0.001). A tendency for CVD395.2 culture filtrates to produce elevated AIsc responses relative to those of CVD101.2 supernatants lacking both active CT and NANase (36.08 ± 3.56 versus 29.81 + 2.50 pLAmp/cm2) was observed, but this difference was not statistically significant (P > 0.10). Such changes in ISC were demonstrated by the addition of theophylline to be related to CT action on ion fluxes (6). Theophylline inhibits cAMP phosphodiesterases, which modulate the intracellular concentration of cAMP produced by adenylate cyclase (2). If CT is present to activate adenylate cyclase, the effect of theophylline addition on the rise of intracellular cAMP and the concomitant rise in ion fluxes will be reduced relative to the effects in tissue exposed to theophylline only. Therefore, when tissues were treated with CVD395.1 filtrate (CT+) and then exposed to theophylline, the ISc rose 81.96 ,uAmp/cm2,

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FIG. 3. Schematic diagram for construction of the isogenic strains CVD395.1 (NANase+) and CVD395.2 (NANase-). For clarity, the deletion within nanH is represented in this figure by a cross-hatched region within the nanH gene of pCVD383.1. Homologous DNAs within the V. cholerae chromosome and pCVD383.1 are represented by bold lines, and designated crossover points are arbitrary. The nanH deletion mutation was mobilized into V. cholerae by matings with E. coli SMlOApir(pCVD383.1), as shown in step A. Apr V. cholerae transconjugants (merodiploid intermediates; 47) containing pCVD383.1 integrated into the chromosome by single homologous crossovers were readily isolated from plate matings (Step B). Upon passage of transconjugants for several generations without selective pressure, V. cholerae Aps revertants were recovered at a frequency of 1 in 250. These revertants result from spontaneous excision of the suicide vehicle from the chromosome by homologous recombination (site 1 or 2 in step C) followed by curing of the resulting plasmids from the nonpermissive V. cholerae background. Of four revertants isolated, one proved to be deficient in NANase production. From these revertants, the isogenic V. cholerae strains CVD395.1 (wild type) and CVD395.2 (NANase-) were chosen for further study (step D).

but tissues exposed to CVD101.2 filtrates (CT-) rose 114.25 ,xAmp/cm2 (as shown in Fig. 6). To determine if these differences in the Alsc responses of CVD395.1 and CVD395.2 were dependent on the concentration of CT present in filtrates, bacterial culture conditions were varied by growing the isogenic strains in 2xCFBN medium. 2 x CFBN medium was used because of its capacity to support elevated levels of CT production (21; see Materials and Methods). Ussing chamber measurements with these 2 x CFBN filtrates demonstrated that when the production of CT was increased at least 100-fold, the difference in AISC between isogenic strains was no longer significant (Fig. 6; P > 0.20). In addition, the AIsc of 58.76 ± 8.23 j±Amp/cm2

INFECT. IMMUN.

GALEN ET AL.

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produced from CVD395.2 2 x CFBN filtrates (containing high concentrations of CT) became equivalent to the AIsc of 59.53 ± 6.10 R.Amp/cm2 produced from wild-type CVD395.1 filtrates of L-broth cultures (containing low concentrations of CT). FA responses of isogenic strains in suckling mice. To investigate whether V. cholerae NANase influences secretory responses in vivo, CVD395.1, CVD395.2, and CVD101.2 were inoculated intragastrically into 5- to 6-day-

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FIG. 5. Flow cytometric analysis of the effect of NANase on the binding of CT-FITC to C57BL/6 and C3H mouse fibroblasts. Simian virus 40-transformed mouse fibroblasts were treated as follows. (A and C) Fibroblasts were exposed to purified V. cholerae NANase, CT-FITC, or both. Peak 1, untreated cells; peak 2, NANase only; peak 3, CT-FITC only; peak 4, NANase plus CT-FITC. (B and D) Fibroblasts were exposed to an isogenic strain culture filtrate, CT-FITC, or both. Peak 1, CVD395.1 filtrate only; peak 2, CVD395.2 filtrate only; peak 3, CVD395.2 filtrate plus CT-FITC; peak 4, CVD395.1 filtrate plus CT-FITC.

VOL. 60, 1992

ROLE OF V. CHOLERAE NEURAMINIDASE IN FUNCTION OF CT

tested between 106 and 109 CFU. The FA ratio for CVD395.1 increased from 0.0955 ± 0.0051 at 107 CFU to a plateau value of 0.1246 ± 0.0048 at 108 CFU and remained constant at 0.1264 ± 0.0045 at 109 CFU. In contrast, the FA ratio for CVD395.2 rose from 0.0839 ± 0.0038 at 107 CFU to a peak of 0.1301 ± 0.0043 for 108 CFU but dropped unexpectedly to 0.1061 ± 0.0029 when 109 CFU was inoculated. At the inoculating dose of 107 CFU, only a small difference in FA between toxigenic strains and the nontoxigenic negative control CVD101.2 was observed (P = 0.01). Toxigenic and nontoxigenic strains were clearly distinguishable at inoculating doses of 108 CFU and greater (P = 0.0001); however, no difference between the FA ratios for CVD395.1 and CVD395.2 was observed for an inoculating dose of 107 or 108 CFU. Although a highly significant (P = 0.0002) difference in secretory response was detected for CVD395.1 and CVD395.2 at a dose of 109 CFU, it is not clear why the FA response for CVD395.2 at 109 CFU decreased relative to the response of CVD395.2 at 108 CFU. In an effort to increase the sensitivity of the assay and reduce the number of inoculated organisms necessary to distinguish CT' from CT- strains, isogenic strains were inoculated into suckling mice starved for 7 to 8 h. CVD395.1 FA ratios increased from 0.0951 ± 0.0057 at 105 CFU to 0.1088 + 0.0037 at 106 CFU and 0.1107 ± 0.0047 at 107 CFU inoculated; the FA ratio of CVD395.2 also maintained a steady increase from 0.0831 ± 0.0034 at 105 CFU through 0.1119 ± 0.0036 at 106 CFU up to 0.1281 ± 0.0122 at 107 CFU. The sensitivity of the assay increased such that toxigenic strains were clearly distinguished from nontoxigenic strains at an inoculating dose of 106 CFU (P = 0.0007), which can be compared with the dose of 108 CFU required with unstarved suckling mice. However, no statistically significant differences were observed for FA ratios for CVD395.1 and CVD395.2 when a dose of 105, 106, or 107 CFU was inoculated into starved mice. Although the results from in vitro experiments supported a role for NANase in the increased binding and penetration of CT, this could not be confirmed with a suckling mouse model.

DISCUSSION CT binds via its B subunits to GM1 receptors present on the lumenal membranes of enterocytes. Subsequent penetration of the A subunit leads to the activation of adenylate cyclase in the basolateral membrane of enterocytes, which ultimately triggers a secretory response. Initiation of a diarrheal response is therefore a function of the concentration of CT near the surface of intestinal epithelial cells and the number of functional receptors available. With a given concentration of CT, the efficiency of penetration will be dependent on the accessibility of GM1 ganglioside receptors. Since NANase can catalyze the conversion of complex gangliosides to GM1, the hypothesis that in the presence of NANase CT will bind to and penetrate enterocytes more efficiently naturally follows. The objective of the work reported here was to construct isogenic nonrevertible deletion mutants of V. cholerae 395 differing only in their abilities to produce active NANase. The hypothesis that production of NANase increases the biological activity of CT produced by these strains would then be investigated both in vitro and in vivo. The DNA sequence of nanH was determined first, thus allowing the extent of any deletion mutation created within nanH to be defined exactly. As shown in Fig. 1, analysis of the 5'flanking sequence of nanH revealed a putative promoter

413

sequence exhibiting poor homology to the consensus sequence for E. coli promoters (22). In addition, three putative CRP-binding sites were identified, two of which may occlude RNA polymerase and inhibit transcription. The role of these putative CRP-binding sites remains to be determined by specific mutation. Interestingly, a similar arrangement has been identified upstream of the pertussis toxin gene, in which a putative CRP-binding site overlaps the -10 region of the toxin promoter (17). A 124-bp nonrevertible deletion mutation within nanH was constructed in vitro by using Bal31. This deletion mutation is predicted to produce a truncated NANase protein missing 45% of its predicted molecular mass. Although it has been confirmed by Western blots (immunoblots) that the 83.0-kDa wild-type NANase protein is not produced by CVD395.2, the predicted 44.8-kDa truncated protein encoded by AnanH has not been observed (20). The mutagenesis technique employed here to mobilize AnanH into the chromosome of V. cholerae 395 involved integration of a suicide vehicle carrying AnanH into the chromosome, causing a duplication of the nanH locus (35). The resulting NANase+/NANase- partial diploid (merodiploid) resolved upon subculturing to produce both NANase+ and NANase- daughters from the same merodiploid parent (47). Any cryptic mutations arising elsewhere in the chromosome prior to resolution of the merodiploid were passed on to the resulting daughter strains. It is therefore concluded that CVD395.1 and CVD395.2 are as isogenic as is experimentally possible and that the involvement of NANase in the binding and penetration of CT can be meaningfully investigated by using pathogenesis models of increasing complexity. One could postulate three possible cases in which the influence of NANase on secretion depends on the concentration of CT present. In case 1, in which the concentration of CT is far below the biological threshold of detection, the presence or absence of NANase becomes irrelevant, and no secretory response would be expected. In case 2, in which the concentration of CT is far above the level at which GM1 receptors are saturated for CT binding, the presence or absence of NANase again becomes irrelevant; with saturating concentrations of CT, a maximum secretion response would be expected. This response would be diminished as the concentration of CT is diminished. Studies by Levine et al. (30) support case 2 and demonstrate that oral administration of 25 jig of purified CT to healthy adult volunteers is sufficient to induce cholera gravis; administration of 5 ,ug of purified CT induced a diarrheal response, but cholera gravis was not observed. Case 2 is strengthened by the Ussing chamber data presented in the work reported here, which demonstrated that as the amount of CT allowed to bind to mucosal tissue increased, a maximum Alsc response of approximately 60 pLAmp/cm2, which was independent of NANase activity (Fig. 6, 2 x CFBN culture filtrates), was observed. In case 3, in which the concentration of CT is above the biological threshold of detection and below gross saturation, one could postulate that an influence of NANase on CT binding and penetration might be observable. The conditions defined for the flow cytometry experiments reported here are apparently representative of case 3 and demonstrate at least a fivefold enhancement of the binding of CT-FITC by NANase present in the isogenic strain culture filtrates, regardless of the mouse cell line used (Fig. 5). Since similar increases in the binding of CT-FITC were also observed with cells treated with purified NANase instead of culture filtrates, it is concluded that the observed increases in binding of CT-FITC were due to the activity of NANase and not to

414

GALEN ET AL.

the influence of any other component present in the crude culture filtrates used. Untreated C57BL/6 fibroblasts bound approximately fivefold more CT-FITC than untreated C3H fibroblasts. However, after treatment with NANase (either purified or in culture filtrates), C57BL/6 cells bound only two- to threefold more CT-FITC than C3H cells. These data suggest that NANase pretreatment of C3H cells produces more of an increase in CT-FITC binding than does NANase pretreatment of C57BL/6 cells. The mechanism for such an effect is not clear. It would appear that C57BL/6 cell receptors are more accessible than C3H receptors, assuming that C57BL/6 and C3H cells have roughly the same number of CT binding sites per cell. However, upon treatment with NANase, this difference in receptor availability is diminished and the binding of CT-FITC becomes more equivalent in the two cell lines. If functional receptors for CT are more accessible for C57BL/6 fibroblast cell lines than for C3H cell lines and if these differences in accessibility are also present within the gastrointestinal tract of intact C57BL/6 and C3H mice, then C57BL/6 mice would be more susceptible to the biological effects of CT than C3H mice, i.e., would experience higher levels of fluid secretion. Richardson and Kuhn (41) previously confirmed this hypothesis by measuring mean FA ratios of C57BL/6 and C3H mice exposed to defined saturating doses of crude or purified CT; mean FA ratios for C57BL/6 mice were twofold higher than those for C3H mice. The flow cytometry results of the present study support the hypothesis that NANase increases the binding of CT to receptors on the surfaces of susceptible cells but do not demonstrate that CT-FITC is binding specifically to GM1, the functional receptor for CT. Data from Pacuszka and Fishman (40) indicate that binding of CT to membrane surfaces is necessary but not sufficient for penetration of the toxin. For penetration to occur, CT must bind to the functional receptor GM1, which presumably allows for intimate binding proximal to the membrane bilayer favorable to toxin penetration (5, 9). The conditions defined for the Ussing chamber experiments of this study demonstrate that NANase increases both the binding and the penetration of isogenic-strain filtrate CT into intact intestinal tissue. Filtrates containing low levels of CT plus NANase increased Isc responses of rabbit ileal tissue almost twofold relative to responses with filtrates containing low levels of CT without NANase, which supports case 3 described above (Fig. 6, L-broth culture filtrates). Preliminary experiments in which purified NANase (Sigma) has been added to filtrates from CVD395.2 demonstrate that AIsc is restored to the levels observed when unaltered filtrates from CVD395.1 were used (data not shown). However, as predicted by case 2, when production of CT in culture filtrates increases (Fig. 6, 2xCFBN filtrates), the effect of NANase is diminished. When examined in vivo with a suckling mouse model, the observed effect of NANase on CT penetration does not conform to the case scenario outlined above. Contrary to the prediction that the effect of NANase might be observed at CT levels below receptor saturation, the only observed enhancement of FA by CVD395.1 (NANase+) occurred as levels of FA were leveling off. In fact, FA ratios of the CT' NANase+ strain remained fairly constant between inoculating doses of 108 and 109 CFU but dropped as doses of the CT' NANase- strain increased from 108 to 109 CFU. The reason for this decrease is unclear. Perhaps in addition to CT and NANase, another extracellular factor modulates the combined influence of CT and NANase on the secretory

INFECT. IMMUN.

response. Young and Broadbent (51) have described an alkaline protease which is produced in vitro in the stationary phase of growth for V. cholerae and degrades CT. At high doses of V. cholerae inoculated into suckling mouse intestinal tracts, perhaps production of such a protease reduces the available concentration of CT produced by V. cholerae such that enhanced penetration by NANase of remaining CT produces higher secretion responses for CVD395.1 which would not be observed for CVD395.2. The effect of such a protease would be missed in in vitro experiments in which the levels of CT and NANase present in the assay have been optimized and are presumed to be constant over time. Such a factor could easily account for the discrepancies observed between the effect of NANase when studied in vitro and its effect when studied in vivo. It is concluded from the data presented in this study that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions. However, deletion of NANase activity does not necessarily decrease the biological effect of CT elaborated by mutant strains relative to the effect of CT produced by wild-type V. cholerae 01 strains. Miller and Mekalanos (35) have shown that production of CT as well as expression of the V. cholerae toxin-coregulated pilus (TCP) required for colonization are regulated by diverse environmental signals including osmolarity, pH, temperature, and the presence of certain amino acids. The results reported here suggest that when environmental conditions do not promote optimal expression of CT (or the TCP pilus), the ability of NANase to enhance the binding of CT could contribute to the pathogenic process. Our results may explain the observation that El Tor strains of V. cholerae produce a higher ratio of asymptomatic (or mild) to symptomatic infections than do classical strains (13); Kabir et al. (26) reported that El Tor strains produce less NANase with lower specific activity than that produced by classical strains. Thus, NANase may be an accessory virulence factor of V. cholerae which enhances pathogenicity when the influence of primary factors is reduced. ACKNOWLEDGMENTS We are grateful to M. C. O'Neill, G. Losonsky, J. Czeczulin, R. Hall, J. R. Lakowicz, and D. Jacoby for invaluable technical

assistance. Fluorescence-activated cell-sorting analysis was carried

out in the FCM core facility of the Bowman Gray Oncology Research Center. This work was supported by grants A119716 (J.B.K.) and DK38783 (S.H.R.) from the National Institutes of Health. REFERENCES 1. Armstrong, W. M. 1987. Cellular mechanisms of ion transport in the small intestine, p. 1251-1265. In L. R. Johnson (ed.), Physiology of the gastrointestinal tract, vol. 2, 2nd ed. Raven Press, Inc., New York. 2. Butcher, R. W., and E. W. Sutherland. 1962. Adenosine 3',5'phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-phosphate in human urine. J. Biol. Chem. 237:1244-1250. 3. Chen, E. Y., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165-170. 4. de Crombrugghe, B., S. Busby, and H. Buc. 1984. Cyclic AMP receptor protein: role in transcription activation. Science 224: 831-838. 5. Dwyer, J. D., and V. A. Bloomfield. 1982. Subunit arrangement of cholera toxin in solution and bound to receptor-containing model membranes. Biochemistry 21:3227-3231.

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ROLE OF V. CHOLERAE NEURAMINIDASE IN FUNCTION OF CT

6. Field, M., D. Fromm, Q. Al-Awqati, and W. B. Greenough III. 1972. Effect of cholera enterotoxin on ion transport across isolated ileal mucosa. J. Clin. Invest. 51:796-804. 7. Field, M., D. Fromm, and I. McColl. 1971. Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current. Am. J. Physiol. 220:1388-1396. 8. Field, M., M. C. Rao, and E. B. Chang. 1989. Intestinal electrolyte transport and diarrheal disease, part I. N. Engl. J. Med. 321:800-806. 9. Fishman, P. H. 1980. Mechanism of action of cholera toxin: events on the cell surface, p. 85-106. In M. Field, J. S. Fordtran, and S. G. Schultz (ed.), Secretory diarrhea. American Physiological Society, Baltimore. 10. Fishman, P. H. 1982. Role of membrane gangliosides in the binding and action of bacterial toxins. J. Membrane Biol. 69:85-97. 11. Focus. 1986. BRL pUC host: E. coli DHSa competent cells. Focus 8(2):9. 12. Galen, J. E., E. R. Vimr, L. Lawrisuk, and J. B. Kaper. 1990. Cloning, sequencing, and expression of the gene, nanH, for V. cholerae neuraminidase, p. 143-153. In R. B. Sack and Y. Zinnaka (ed.), Advances in research on cholera and related diarrheas, vol. 7. KTK Scientific Publishers, Tokyo. 13. Gangarosa, E. J., and W. H. Mosley. 1974. Epidemiology and surveillance of cholera, p. 381-403. In D. Barua and W. Burrows (ed.), Cholera. The W. B. Saunders Co., Philadelphia. 14. Gill, D. M., and R. Meren. 1978. ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl. Acad. Sci. USA 75:3050-3054. 15. Gooding, L. R. 1977. Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells. J. Immunol. 118:920-927. 16. Griffiths, S. L., R. A. Finkelstein, and D. R. Critchley. 1986. Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders. Biochem. J. 238:313-322. 17. Gross, R., and R. Rappuoli. 1989. Pertussis toxin promoter sequences involved in modulation. J. Bacteriol. 171:4026-4030. 18. Guandalini, S., A. Fasano, M. Migliavacca, G. Marchesano, A. Ferola, and A. Rubino. 1987. Effects of berberine on basal and secretagogue-modified ion transport in the rabbit ileum in vitro. J. Pediatr. Gastroenterol. Nutr. 6:953-960. 19. Haksar, A., D. V. Maudsley, and F. G. Peron. 1974. Neuraminidase treatment of adrenal cells increases their response to cholera enterotoxin. Nature (London) 251:514-515. 20. Hall, R. H. Unpublished data. 21. Hall, R. H., A. P. D. Silveira, and P. A. Vial. Submitted for

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