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A Fasano

Publications and source records attributed to A Fasano.

At least 91 records · Page 5Linked to original sources
PubMed1995

Calcium-dependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model.

BACKGROUND & AIMS: Vibrio parahaemolyticus is a major agent of seafood gastroenteritis that induces intestinal secretion in the rabbit through its thermostable direct hemolysin. The aim of this study was to characterize the enterotoxicity of purified hemolysin in vitro. METHODS: Rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after addition of hemolysin. Intracellular calcium concentrations in the nontumoral rat crypt-derived cell line IEC-6 were measured using microspectrofluorometry. RESULTS: In Ussing chamber experiments, mucosal toxin addition up to 50 hemolytic units per milliliter induced a proportional increase of the electrical parameters in normal but not Cl(-)-free Ringer's solution. The response to the toxin was not additive to that of calcium ionophore A23187 and was eliminated by preloading the tissue with 1-2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA), a calcium buffer. In IEC-6 cells, a 10-fold increase in intracellular calcium level was found after addition of hemolysin. Such an increase was totally quenched by BAPTA. Finally, preincubation with trisialoganglioside GT1b, but not monosialoganglioside GM1, eliminated toxin-induced increases in potential difference and short-circuit current. CONCLUSIONS: These data support the hypothesis that the thermostable direct hemolysin induces intestinal chloride secretion using GT1b as a putative receptor and Ca2+ as a second messenger.

Animals↗
PubMed1995

Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes.

Shigella enterotoxin 1 (ShET1) is a novel, iron-dependent, toxin encoded by chromosomal genes (set1). To determine the prevalence of this enterotoxin, 172 Shigella clinical isolates (and 10 enteroinvasive Escherichia coli [EIEC]) from distant areas worldwide, representing all 4 groups and 45 serotypes of Shigella, were screened for set1 by DNA colony hybridization and polymerase chain reaction amplification. set1 was present in all 22 Shigella flexneri 2a strains tested but was rare in isolates of other Shigella serotypes (3.3%, 5/150) and not found in EIEC (0/10). That ShET1 is found almost exclusively in S. flexneri 2a may help explain the epidemiologic predominance of this serotype in the developing world.

Bacterial Toxins↗
PubMed1995

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.

Amino Acid Sequence↗
PubMed1995

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

Amino Acid Sequence↗
PubMed1995

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

Actins↗
PubMed1994

Effects of deconjugated bile acids on electrolyte and nutrient transport in the rabbit small intestine in vitro.

To obtain new information on the poorly investigated mechanisms of the diarrheogenic effects of bile acids, we investigated the effects of the unconjugated bile acids chenodeoxycholate and ursodeoxycholate on the small intestine transport of electrolytes and nutrients in the rabbit jejunal and ileal mucosa mounted in vitro in Ussing or influx chambers. When added to the ileal mucosa at a concentration of 1 mM, both bile acids induced a secretory shift in ion transport; absorption of Na and Cl was abolished and secretion of bicarbonate was enhanced. No changes in short circuit current or electrolyte transport were observed when the bile acids were added to the jejunal mucosa. The addition of 1 mM chenodeoxycholate to the luminal side of the ileum induced a marked inhibition in the uptake of glucose, phenylalanine, and glutamic acid. A similar inhibition, though of a lesser magnitude, was also induced by both bile acids on jejunal nutrient transport. Further studies on the inhibitory effect of chenodeoxycholate on glucose influx showed that the inhibition was dose-dependent; at 1 mM it was maximal and resulted in the complete abolition of the active component, with only the diffusional pathway still operative. We conclude that unconjugated bile acids act as secretagogues in the ileum and significantly blunt the active transport of nutrients in the small intestine. Both effects may well play a role in the diarrheogenic action of these bile acids.

Animal Nutritional Physiological Phenomena↗
PubMed1993

Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette.

Vibrio cholerae causes the potentially lethal disease cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying intercellular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarity to eukaryotic ion-transporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette."

Amino Acid Sequence↗
PubMed1993

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Amino Acid Sequence↗
PubMed1993

Safety and immunogenicity of live oral cholera vaccine candidate CVD 110, a delta ctxA delta zot delta ace derivative of El Tor Ogawa Vibrio cholerae.

The current pandemic of cholera is caused primarily by Vibrio cholerae O1 of the El Tor biotype. Live attenuated classical biotype V. cholerae vaccine strains prevent severe and moderate cholera due to either biotype in challenged volunteers but may provide less protection against mild cholera due to El Tor organisms. CVD 110, a new ctxA-deleted vaccine strain derived from an El Tor Ogawa parent, lacks zona occludens toxin (Zot), accessory cholera enterotoxin (Ace), and hemolysin/enterotoxin. Ten healthy adult volunteers were given 10(8) cfu of CVD 110 with buffer; 7 developed diarrhea (mean stool volume, 861 mL). Vaccine organisms were shed in stool by all vaccines and were recovered from duodenal fluid in three-quarters of vaccinees. After vaccination, the geometric mean peak reciprocal vibriocidal titer among vaccinees was 17,829. CVD 110 is a powerful immunogen but insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Another unrecognized toxin or colonization alone may be responsible for diarrhea after ingestion of this strain.

Administration, Oral↗
PubMed1993

CVD110, an attenuated Vibrio cholerae O1 El Tor live oral vaccine strain.

The recent expansion of the seventh cholera pandemic into South America emphasizes the need for a safe, long-lasting, protective, and nonreactogenic vaccine for this disease. Since the predominant Vibrio cholerae O1 strains in the world today are of the El Tor biotype, a bivalent vaccine containing both classical and El Tor biotypes may be desirable. We have constructed a new oral vaccine candidate, V. cholerae CVD110 El Tor, Ogawa, from which all toxin genes so far identified in V. cholerae have been deleted. Three of these genes, those encoding cholera toxin (ctx), zonula occludens toxin (zot), and accessory cholera enterotoxin (ace), are located on a 4.5-kb virulence cassette flanked by repetitive sequences (RS1 elements). Homologous recombination between these RS1 elements resulted in the deletion of this virulence cassette to yield V. cholerae CVD109. Insertion of genes encoding mercury resistance (mer) and the cholera toxin B subunit (ctxB) into the hemolysin locus (hlyA) produced CVD110. This insertion serves three purpose. (i) It genetically tags the vaccine strain so as to distinguish it from wild-type V. cholerae O1. (ii) It produces cholera toxin B subunit in order to elicit antitoxic immunity. (iii) It inactivates the hemolysin gene, rendering the strain nonhemolytic on sheep erythrocyte plates. Supernatants from V. cholerae CVD110 cultures are nonreactogenic when assayed in Ussing chambers.

Bacterial Toxins↗
PubMed1993

Assessment of enterotoxin production by Yersinia enterocolitica and identification of a novel heat-stable enterotoxin produced by a noninvasive Y. enterocolitica strain isolated from clinical material.

Twenty-eight clinical isolates of Yersinia enterocolitica were investigated for their abilities to produce heat-stable enterotoxin (YST). All 21 invasive strains (serogroup O3 biotype 4) carried the previously described gene for YST (yst), with toxin detectable in culture supernatants from 20 strains. One of seven noninvasive, biotype 1A strains also had enterotoxic activity, despite failure to hybridize with a probe for yst. The toxin produced by this noninvasive (serogroup O6) strain resembled YST in terms of molecular size, heat stability, and solubility in methanol. It differed from YST, however, with respect to regulation of its production by temperature and its mechanism of action, which did not appear to involve cyclic GMP.

Animals↗
PubMed1992

Multifocal corneal topographic changes with excimer laser photorefractive keratectomy.

Excimer laser photorefractive keratectomy can flatten the central cornea, thereby eliminating myopic refractive errors; in older patients, however, presbyopia limits satisfaction. Computer-assisted topographic analysis of corneas after refractive surgery indicates that a minority of patients achieve a multifocal lens effect, such that they maintain reasonable acuity over a range of defocus. We have purposefully attempted to create a multifocal refractive effect and have analyzed the subsequent topographies quantitatively to determine if multifocality was achieved. In corneas not operated on and plastic hemispheres, a fairly small range of corneal powers is observed; the range of powers is increased after a monofocal ablation. After multifocal ablations, a greater spread of surface powers is observed, often with a bimodal distribution, indicative of an apparent multifocal effect. These observations suggest that in some patients undergoing photorefractive keratectomy for myopia, it may be possible to reduce symptoms of presbyopia, although a decrease in image contrast or monocular diplopia may complicate this approach.

Animals↗
PubMed1992

Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

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 vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates 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 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) 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.

Amino Acid Sequence↗
PubMed1992

Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae.

Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin-producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin.

Amino Acid Sequence↗
PubMed1992

Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin.

Vibrio parahaemolyticus produces a thermostable direct hemolysin (TDH) that has been implicated in the pathogenesis of diarrheal disease caused by this organism. However, previous studies attempting to demonstrate the contribution of the hemolysin to virulence have been inconclusive. We investigated this putative virulence factor by using an isogenic TDH-negative (TDH-) strain constructed by specifically inactivating the two copies of the tdh gene encoding TDH. The enterotoxigenicities of the parent strain (AQ3815) and the mutant strain were tested by adding sterile culture supernatants to rabbit ileal tissue mounted in Ussing chambers. The culture filtrate of the parent strain produced a significant increase in short-circuit current (Isc), compared with the change induced by the TDH- mutant. The capacity of the culture filtrate of AQ3815 to increase the Isc was reduced by neutralization with anti-TDH serum, and the return of the cloned tdh gene to the TDH- mutant restored the ability to increase the Isc. These results were corroborated by rabbit ileal loop assays in which AQ3815 caused fluid accumulation but the TDH- mutant did not. No microscopic damage was seen in mucosal tissues exposed to the culture filtrate of either strain. These results indicate that TDH has an enterotoxigenic effect on rabbit small intestine and could be responsible for the watery diarrhea seen with V. parahaemolyticus.

Animals↗
PubMed1992

Multifocal corneal topographic changes after radial keratotomy.

Quantitative analysis of the central corneal refractive powers of both eyes of three patients demonstrated that, after radial keratotomy, the corneas tended to have a greater range of refractive powers than preoperatively, and that the corneas with a multifocal effect had a wider distribution of refractive powers than the corneas of eyes without this effect. It may be possible to intentionally achieve a multifocal lens effect by designing a refractive procedure to predictably widen the distribution of central corneal refractive powers.

Cornea↗
PubMed1991

Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.

Attenuated Vibrio cholerae vaccine strains specifically mutated in genes encoding cholera toxin (CT) are still capable of causing mild to moderate diarrhea. Culture supernatants of V. cholerae strains, both CT-positive and CT-negative, were examined in Ussing chambers, and a toxin was found that increases the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens. The activity of this toxin is reversible, heat-labile, sensitive to protease digestion, and found in culture supernatant fractions containing molecules between 10 and 30 kDa in size. Production of this factor (named ZOT for zonula occludens toxin) correlates with diarrheagenicity of V. cholerae strains in volunteers and may represent another virulence factor of infectious diarrhea that must be eliminated to achieve a safe and effective live oral vaccine against cholera.

Animals↗