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PubMed2001

Regulation of the cytotoxic enterotoxin gene in Aeromonas hydrophila: characterization of an iron uptake regulator.

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.

Aeromonas hydrophila↗

PubMed2001

Enterotoxin plasmid from Clostridium perfringens is conjugative.

Clostridium perfringens enterotoxin is the major virulence factor involved in the pathogenesis of C. perfringens type A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene, cpe, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the cpe plasmid encodes its own conjugative transfer, a C. perfringens strain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to the cpe gene had been replaced by the chloramphenicol resistance gene catP, was used as a donor in matings with several cpe-negative C. perfringens isolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 x 10(-2) to 4.6 x 10(-4) transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype C. perfringens tetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the cpe plasmid occurred in vivo, it would have the potential to convert cpe-negative C. perfringens strains in normal intestinal flora into strains capable of causing gastrointestinal disease.

Clostridium perfringens↗

PubMed2001

Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

Animals↗

PubMed2002

Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli.

We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.

Amino Acid Sequence↗

PubMed2002

Mutational analysis of ganglioside GM(1)-binding ability, pentamer formation, and epitopes of cholera toxin B (CTB) subunits and CTB/heat-labile enterotoxin B subunit chimeras.

Variants of cholera toxin B subunit (CTB) were made by bisulfite- and oligonucleotide-directed mutagenesis of the ctxB gene. Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC). Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM(1) in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies. Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB. RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 + 54, 91, and 95. Tyrosine-12 was identified as a novel residue important for GM(1) binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM(1) in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM(1) binding, based on the crystal structure of CTB and the oligosaccharide of GM(1), were not appreciably altered in their ability to bind GM(1) in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC. Hybrid B genes made by fusing ctxB and the related Escherichia coli heat-labile enterotoxin eltB genes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM(1) as well as wild-type CTB. This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs). Each MAb had a unique pattern of reactivity with the panel of CTB variants. Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope. The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers. Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.

Amino Acid Sequence↗

PubMed2004

Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila.

By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Aeromonas hydrophila↗

PubMed2004

Biological properties of staphylococcal enterotoxin-like toxin type R.

We investigated the biological properties of a novel staphylococcal enterotoxin-like putative toxin, staphylococcal enterotoxin-like toxin type R (SElR). Major histocompatibility complex class II molecules were required for T-cell stimulation by SElR. SElR stimulated T cells bearing receptors Vbeta 3, 11, 12, 13.2, and 14. These results suggested that SElR acts as a superantigen.

Animals↗

PubMed2004

Nasal delivery of antigen with the B subunit of Escherichia coli heat-labile enterotoxin augments antigen-specific T-cell clonal expansion and differentiation.

Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice. These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest. However, the inherent toxicity mediated by the A subunit precludes its widespread use. This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit. The ability of the B subunit of E. coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry. Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens. The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production. Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation. Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained. Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation. Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.

Adjuvants, Immunologic↗

PubMed2005

The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila.

Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

Aeromonas hydrophila↗

PubMed1973

A paracrystalline inclusion formed during sporulation of enterotoxin-producing strains of Clostridium perfringens type A.

A large paracrystalline inclusion is formed by certain strains of Clostridium perfringens type A during spore morphogenesis. In most cell thin sections, the inclusion appeared rod-shaped when sectioned at an angle perpendicular to its longer axis, and circular or oval-shaped when sectioned at an angle parallel to its longer axis. Measurements performed on electron micrographs of inclusions sectioned to reveal the rod shape indicated a fairly consistent thickness (width) of 192 +/- 23 nm. The length of the inclusions varied considerably with a maximum of approximately 2,120 nm being observed. Ultrastructurally, the inclusion was composed of closely packed, periodically spaced, parallel layers. Usually a single inclusion was randomly located in the cytoplasm of the cell. Two inclusions per cell were rarely observed. The inclusion was formed only by ent(+) strains of C. perfringens. Mutants of the ent(+) strain NCTC 8798 that were altered in their sporulating and enterotoxin-producing capacities and revertants of these mutants were tested for inclusion formation. The results indicate that, as with the ent(+) trait, a direct relationship exists between inclusion formation and spore formation. The synthesis of enterotoxin, formation of a morphologically distinct inclusion, and the initial deposition of discontinuous coat fragments around the forespore appear to be events closely related in time during spore morphogenesis.

Clostridium↗

PubMed1975

Genetic and physical characteristics of an enterotoxin plasmid.

We are engaged in the genetic and physical characterization of an enterotoxin (Ent) plasmid, Ent P307, which contains genes for the production of a hear-labile and a heat-stable enterotoxin. We are using an Escherichia coli K-12 strain, 711 (P307), constructed by S. Falkow, which contains no other plasmids besides Ent P307. Our genetic studies have shown that the plasmid is incompatible with the sex factor F, both in the integrated (Hfr) and the autonomous (F-prime) state. Ent P307 can thus be assigned to incompatibility group FI. An R factor, R386, which belongs to the same incompatibility group, was also found to be incompatibile with Ent P307, whereas five other R factors belonging to different incompatibility groups were compatible with Ent P307. In the presence of Ent P307, conjugal transfer and sensitivity to a male-specific phage of a derepressed F-like R factor, R1drd19, were repressed. Ent P307 is, thus, finO+. Presumably, it also causes repression of its own transfer genes since conjugal transfer of Ent P307 could not be demonstrated. Unlike F, it does not restrict the growth of female-specific phage phiII. From physical studies on extracted deoxyribonucleic acid, the molecular weight of Ent P307 was determined to be 54 X 10(6). By electron microscope heteroduplex analysis, the plasmid was found to be homologous with F in four regions, encompassing about half of its length. One long region and two short ones contain genes for conjugal transfer; the other short region carries genes for replication and incompatibility.

Conjugation, Genetic↗

PubMed1980

Clostridium perfringens type A: in vitro system for sporulation and enterotoxin synthesis.

Polysomes were isolated from an enterotoxigenic strain of Clostridium perfringens during vegetative growth and at 1-h intervals after transfer into Duncan-Strong sporulation medium. During vegetative growth, about 67% of the ribosomes were in polysomal complexes. This proportion decreased to about 20% during the first 2 h in sporulation medium and then gradually increased to a maximum of 45% at 6 h. Ribosomes isolated from cells in vegetative or in sporulation phase could equally translate vegetative, sporulation, and natural viral R17 messenger ribonucleic acid with either vegetative or sporulation initiation factors. When polysomes were allowed to complete their nascent chains with labeled amino acids in vitro, most of the polypeptides synthesized by the vegetative phase and by the sporulation phase polysomes appeared to be identical. There were, however, notable differences upon further investigation. Specifically, when antiserum against the enterotoxin was reacted with the completed polypeptides, no counts were precipitated from the vegetative products. On the other hand, up to 12% of the total labeled protein was precipitated from the products obtained with the sporulation phase polysomes. Upon electrophoresis on sodium dodecyl sulfate, the putative enterotoxin synthesized in vitro ran as a major band with a molecular weight of 35,000, and as two minor bands with molecular weights of 17,000 and 52,000, respectively.

Bacterial Proteins↗

PubMed1980

Nucleotide sequence homology between the heat-labile enterotoxin gene of Escherichia coli and Vibrio cholerae deoxyribonucleic acid.

Isolated deoxyribonucleic acid fragments encoding the heat-labile enterotoxin of Escherichia coli were used to probe for homologous sequences in restricted whole-cell deoxyribonucleic acid from Vibrio cholerae. Significant sequence homology between the heat-labile enterotoxin gene and V. cholerae deoxyribonucleic acid was demonstrated, and apparent differences were observed in the organization of the cholera toxin gene among different strains of V. cholerae.

Base Sequence↗

PubMed1981

Catabolite repression of Escherichia coli heat-stable enterotoxin activity.

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.

Bacterial Toxins↗

PubMed1981

Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli.

Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E. coli. Two polypeptide species related to the B subunit of the toxin were expressed in the minicells. One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin. Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit. The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form. This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.

Bacterial Toxins↗

PubMed1982

Mapping of a plasmid, coding for colonization, factor antigen I and heat-stable enterotoxin production, isolated from an enterotoxigenic strain of Escherichia coli.

The non-autotransferring plasmid NTP113 codes for production of colonization factor antigen I and heat-stable enterotoxin, NTP113, which has a molecular weight of 58 X 10(6), was digested with BamHI, EcoRI, and HindIII and combinations of these restriction endonucleases, and the products of these digestions were analyzed by agarose gel electrophoresis. The results were used to construct a partial restriction map of NTP113. Transposons coding for resistance to ampicillin, kanamycin, and tetracycline were inserted into NTP113, and we obtained a series of deletion mutants, as determined by the loss of tetracycline or kanamycin resistance from strains carrying the insertion mutants. A number of plasmid mutants obtained by insertion or deletion did not code for colonization factor antigen I, but most of these mutants still coded for heat-stable enterotoxin production. The position of the inserted transposons and of the deletions were determined on the restriction map. Two regions of NTP113 were required for the expression of colonization factor antigen I, and the two sites were separated by a length of DNA corresponding to a molecular weight of about 25 X10(6).

Antigens, Bacterial↗

PubMed1982

Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production.

Earlier studies have suggested the involvement of a small 1.3-kilobase plasmid, pSN2, in the production of enterotoxin D by certain Staphylococcus aureus strains. On the basis of extensive biochemical studies on pSN2, including the determination of its coding properties and its primary nucleotide sequence, we conclude that this plasmid is not in act involved in enterotoxin B production in S. aureus: although the toxin genes are apparently chromosomal, it is probable that they are part of a special genetic system such as a hitchhiking transposon.

Bacterial Proteins↗

PubMed1983

Plasmids of enterotoxigenic Escherichia coli H10407: evidence for two heat-stable enterotoxin genes and a conjugal transfer system.

Three species of plasmids, associated with virulence and conjugal transfer, were identified in a clinically isolated enterotoxigenic Escherichia coli strain, H10407 (serotype O78:H11). pCS1, a non-self-transmissible plasmid species with a molecular weight of 62 X 10(6) and a 47 mol% guanine-plus-cytosine content, specified colonization factor antigen I and heat-stable enterotoxin (ST) production, as reported by others previously. A second non-self-transmissible plasmid species, designated pJY11, with a molecular weight of 42 X 10(6) and a 51 mol% guanine-plus-cytosine content, specified ST and heat-labile enterotoxin production and manifested T5/T6 phage restriction. The third plasmid species, pTRA1, also had a molecular weight of 42 X 10(6) and had a guanine-plus-cytosine content of 51 mol%; this species was self-transmissible and promoted transfer of both pCS1 and pJY11 to other bacterial cells. pCS1 may have originated from species of bacteria with a lower guanine-plus-cytosine content than E. coli. Finally, although demonstrating some heterogeneity with each other, both STs encoded by pCS1 and pJY11 belonged to the STa group.

Base Sequence↗