The subunit S1 is important for pertussis toxin secretion

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.     

Temporal expression of pertussis toxin and Ptl secretion proteins by Bordetella pertussis

Pertussis toxin is an AB(5) toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.     

Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway

Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli. The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G(M1) ganglioside binding site and that LT binds lipopolysaccharide and G(M1) concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoid-associated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G(M1)-independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell.     

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.     

Characterization of enterotoxin and soluble hemagglutinin from Vibrio mimicus: identity with V. cholerae O1 toxin and hemagglutinin

Abstract Eight strains of Vibrio mimicus isolated from patients with diarrhoea in Bangladesh were all found to produce an extracellular toxin identical to cholera toxin produced by Vibrio cholerae O1 bacteria, with regard to subunit structure and immunological properties. Like cholera toxin, but in contrast to heat-labile enterotoxin from Escherichia coli most of the toxin from V. mimicus was found extracellularly and was proteolytically 'nicked' in its A subunit. This may relate to the finding that V. mimicus also produced an extracellular hemagglutinin which was immunologically indistinguishable from the soluble hemagglutinin/nicking protease of V. cholerae O1.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.     

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.     

Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin

We have investigated the assembly of the heat-labile enterotoxin (LT) subunits after their processing and segregation into the periplasmic space as mature LT A and LT B polypeptides. LT B starts associating into oligomers during or immediately after translocation through the cytoplasmic membrane. Binding to LT A occurs immediately after oligomerization. Over 80% of the LT B subunits have oligomerized, and over 50% have associated with LT A into holotoxin within 1 min after synthesis. The fate of newly synthesized LT A is totally different. There is an extensive overproduction of LT A relative to LT B and after membrane translocation it becomes part of a periplasmic pool of free LT A. It is then bound by LT B oligomers or degraded at such a rate that the free periplasmic LT A disappears from the pool with a half-time of 20-25 min. About half of the LT A is incorporated into holotoxin, while the other half is degraded. We conclude that LT subunits are translocated and processed in a ratio of about 2 A to 5 B. Since free LT A is either degraded slowly or bound to newly synthesized LT B oligomers, the net result is a steady state of 1.4 to 1.7 A subunits to 5 B subunits in the periplasm. About 60% of this LT A is bound by LT B to form periplasmic holotoxin with a subunit ratio of about 1 A to 5 B. The remaining 40% of periplasmic LT A occurs free.     

MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.     

Membrane-bound enterotoxin of Vibrio cholerae

The mode of transport of the complex toxin molecule of Vibrio cholerae (which has a mol. wt of 84000 and consists of several subunits) across the inner and outer membranes of V. cholerae is not known. In this study we found two peptides in the outer and inner membranes of V. cholerae which may be the form in which the toxin subunits are transported across the membrane. We examined two growth conditions: aerobic growth at 37 degrees C, when most of the synthesized toxin is membrane-bound; and anaerobic growth at 37 degrees C, when little toxin remains membrane-bound, the toxin being released into the growth medium. When V. cholerae was grown aerobically at 37 degrees C, the outer and the inner membranes contained two peptides with mol. wts of approximately 22000 and 6000 which were not found in the outer or the inner membrane of anaerobically grown cells. Sodium deoxycholate, which releases membrane-bound toxin, released several peptides including the 22000 and the 6000 mol. wt peptides. Trypsin also released the 22000 and 6000 mol. wt peptides. Purified cholera toxin had three kinds of peptides, of mol. wt 21000 (A1 peptide), 11000 (B subunit) and 5000 (A2 peptide). We postulate that the membrane peptides may be precursors of the A subunit of the toxin molecule.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

N-terminus of mature heat-labile enterotoxin chain B is critical for its extracellular secretion in Vibrio cholerae

The effects of addition of a few amino acids to the amino- and carboxy-terminal regions of the mature portion of the heat-labile enterotoxin chain B (LTB) of Escherichia coli on protein export, secretion and assembly were investigated. In E. coli, LTB (secretory protein) with or without the extension at the N- or C-terminus accumulated in the periplasmic fraction. For Vibrio cholerae, LTB with the extension at the C-terminus was exported to the periplasm followed by secretion to the extracellular milieu. However, LTB with the N-terminus extension was exported to the periplasm only. Our findings suggest that in the case of V. cholerae, the N-terminus of the mature LTB plays an important role in its secretion to the extracellular milieu.     

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.     

Contribution of the Disulfide Bond of the A Subunit to the Action of Escherichia coli Heat-Labile Enterotoxin

Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.     

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.     

Overexpression of a Mutant B Subunit in Toxigenic Vibrio cholerae Diminishes Production of Active Cholera Toxin In Vivo

A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae. The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of B^G33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains. Received: 29 January 1998 / Accepted: 30 April 1998     

The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.     

Escherichia coli heat-labile enterotoxin genes are flanked by repeated deoxyribonucleic acid sequences.

The enterotoxin regions of the heat-labile and heat-stable enterotoxin (LT+ ST+) plasmid, pJY11, originating in a clinically isolated Escherichia coli strain, have been isolated as various-sized deoxyribonucleic acid (DNA) fragments by using cloning vehicles. The structure of the LT+ region and its neighboring DNA regions was studied by utilizing these recombinant plasmids. The LT+ region consisted of at least two genes, toxA and toxB, which could complement each other in trans. The toxA- and toxB-encoded polypeptides (LT subunits A and B, respectively) were identified by their immunological cross-reactivity with Vibrio cholerae enterotoxin subunit A or B. These tox genes and the promoter(s) were localized with respect to the restriction endonuclease cleavage map. The LT+ region was flanked by repeated DNA sequences (designated as beta). Another tox gen(s), encoding ST (designated as toxS), which was also flanked by inverted, repeated DNA sequences (designated as alpha), was located between one of the beta sequences and the LT+ region. These novel DNA structures (beta-alpha-toxS-alpha-toxA-toxB-beta) suggest the possibility that the LT+ region is on a transposon containing an ST transposon within the structure.