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.

Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4.

The ability to colonize the small intestine is essential for the pathogenesis of diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Colonization is mediated by fimbriae (pili), of which there are several antigenically distinct types, including colonization factor antigen I, colonization factor antigen II (CS1, CS2, and CS3), and PCF8775 (CS4, CS5, and CS6). These fimbriae are associated with certain ETEC O serogroups. Serogroup O159 has had no known colonization factor. We found a distinct plasmid-encoded fimbria composed of 19-kilodalton protein subunits associated with ETEC serotype O159:H4. Rabbit antibody against this purified fimbria reacted with a single 19-kilodalton protein band as seen by Western immunoblot of sheared-cell preparations. The rabbit antibody, treated with colloidal-gold-labeled goat anti-rabbit immunoglobulin G, bound specifically to fimbriae when cells were examined with an electron microscope. Of 10 available ETEC O159:H4 strains from Europe, Bangladesh, and Kenya, 6 expressed this type of fimbria; its true prevalence among ETEC strains is unknown. This putative colonization factor of O159:H4 joins other ETEC fimbriae as potentially useful immunogens against human diarrhea.

Cloning of the cytotoxin-hemolysin gene of Vibrio vulnificus.

Genes encoding the cytotoxin-hemolysin of Vibrio vulnificus were cloned in Escherichia coli by using the lytic cloning vector, lambda 1059. Subcloning in plasmid pBR325 resulted in the isolation of a 3.2-kilobase DNA fragment containing the cytotoxin gene. By using this fragment as a DNA probe, homologous gene sequences were detected in all 54 V. vulnificus strains studied; homologous sequences were present in none of 96 isolates from 29 other bacterial species.

Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli.

It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.

Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates.

rns is a trans-acting positive regulatory factor required for expression of the colonization factor antigen II (CFA/II) antigens CS1 and CS2 (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). All 35 CFA/II-positive strains hybridized with a rns gene probe, as did all 10 CFA/I strains and all 4 CS4 strains. Hybridization with rns was detected in 25% of non-enterotoxigenic Escherichia coli strains and was not detected in enteric pathogens with low G + C content.

A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria.

The virulence properties of many pathogenic bacteria are due to proteins encoded by large gene clusters called pathogenicity islands, which are found in a variety of human pathogens including Escherichia coli, Salmonella, Shigella, Yersinia, Helicobacter pylori, Vibrio cholerae, and animal and plant pathogens such as Dichelobacter nodosus and Pseudomonas syringae. Although the presence of pathogenicity islands is a prerequisite for many bacterial diseases, little is known about their origins or mechanism of transfer into the bacterium. The bacterial agent of epidemic cholera, Vibrio cholerae, contains a bacteriophage known as cholera-toxin phage (CTXphi), which encodes the cholera toxin, and a large pathogenicity island called the VPI (for V. cholerae pathogenicity island) which itself encodes a toxin-coregulated pilus that functions as a colonization factor and as a CTXphi receptor. We have now identified the VPI pathogenicity island as the genome of another filamentous bacteriophage, VPIphi. We show that VPIphi is transferred between V. cholerae strains and provide evidence that the TcpA subunit of the toxin-coregulated type IV pilus is in fact a coat protein of VPIphi. Our results are the first description of a phage that encodes a receptor for another phage and of a virus-virus interaction that is necessary for bacterial pathogenicity.

Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes.

Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.

Construction and characterization of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203, a prototype live oral vaccine.

We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.

Purification and analysis of colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae from enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli fimbriae are immunogenic and play a key role in intestinal colonization. Native colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae were purified by a common method involving shearing, differential centrifugation, gel filtration, and density gradient ultracentrifugation. The compositions and N-terminal sequences were determined. Coli surface antigen 3 possesses two N-terminal isoforms, one of which matches the published DNA sequence, except for the previously proposed signal sequence cleavage point.

Milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most commonly isolated pathogen responsible for travelers' diarrhea and the cause of up to 650 million cases of pediatric diarrhea per year in the developing world. As a safe alternative to the prophylactic use of antibiotics, a hyperimmune bovine milk antibody product with specific activity against purified colonization factor antigens (CFAs) was developed and evaluated in a human challenge study. Twenty-five healthy adult volunteers were challenged orally with 10(9) cfu of a virulent CFA/I-bearing ETEC. In the randomized double-blind trial, 7 of 10 volunteers receiving a lactose-free placebo developed clinical diarrhea after challenge, compared with only 1 of 15 cases in volunteers receiving active product (Fisher's exact test, P < .0017). It is concluded that antibodies against CFAs alone are sufficient for protection and that prophylaxis with milk-derived immunoglobulin is a feasible alternative to existing drug interventions.

Strategy for cross-protection among Shigella flexneri serotypes.

Based upon the lipopolysaccharide (LPS) structure and antigenicity of Shigella group B, a strategy for broad cross-protection against 14 Shigella flexneri serotypes was designed. This strategy involves the use of two S. flexneri serotypes (2a and 3a), which together bear the all of the major antigenic group factors of this group. The novel attenuated strains used in these studies were S. flexneri 2a strain CVD 1207 (DeltaguaB-A DeltavirG Deltaset1 Deltasen) and S. flexneri 3a strain CVD 1211 (DeltaguaB-A DeltavirG Deltasen). Guinea pigs were immunized with an equal mixture of these strains and later challenged (Sereny test) with a wild-type S. flexneri serotype 1a, 1b, 2b, 4b, 5b, Y, or 6 strain of demonstrated virulence in the same model. Guinea pigs that were immunized with these two vaccine strains produced serum and mucosal antibodies that cross-reacted with all the S. flexneri serotypes tested (except of S. flexneri serotype 6) as assessed by enzyme-linked immunosorbent assay, immunoblotting, and slide agglutination. Furthermore, the combination vaccine conferred significant protection against challenge with S. flexneri serotypes 1b, 2b, 5b, and Y but not with serotypes 1a, 4b, or (as predicted) 6.

Preliminary structure determination of the capsular polysaccharide of Vibrio cholerae O139 Bengal Al1837.

Vibrio cholerae O139 Bengal has recently been identified as a cause of epidemic cholera in Asia. In contrast to V. cholerae O1, V. cholerae O139 Bengal has a polysaccharide capsule. As determined by high-performance anion-exchange chromatography and 1H nuclear magnetic resonance analysis, the capsular polysaccharide of V. cholerae O139 Bengal strain Al1837 has six residues in the repeating subunit; this includes one residue each of N-acetylglucosamine, N-acetylquinovosamine (QuiNAc), galacturonic acid (GalA), and galactose and two residues of 3,6-dideoxyxylohexose (Xylhex). The proposed structure is [formula: see text]

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.

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.

Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24.

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.

Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR.

An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+. Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E. coli asd strain chi 6212. Oral immunization of mice with nonpathogenic E. coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I. In contrast, oral immunization with a single dose of attenuated S. typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen. Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.

Molecular epidemiology of Haemophilus influenzae within families in Santiago, Chile.

Thirty-six consecutive patients with invasive Haemophilus influenzae type b (Hib) infections at Roberto del Rio Children's Hospital, Santiago, Chile, were enrolled in a prospective study. Throat cultures were obtained from household contacts of each index case, adjacent neighbors, and matched community control households. Colonization rates for H. influenzae were comparable among groups; however, among household contacts 18% of colonizing isolates were Hib, compared with 2% and 3% among neighbor and community controls. When selected isolates were evaluated further by outer membrane protein (OMP) profiles and multilocus enzyme electrophoresis, only one of the four Hib isolates from household members matched the corresponding index case isolate. One serologically nontypeable isolate from a household contact had an OMP profile and electrophoretic type identical to that of the corresponding Hib index case isolate; hybridization studies with a 9-kb capsular gene probe showed a profile consistent with a capsule-deficient mutant. Hib strains were isolated more frequently from household contacts than from control persons living in Santiago, but colonizing Hib strains were often unrelated to the index case strain.