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1.
Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli. 总被引:9,自引:0,他引:9
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Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist. 相似文献
2.
Sharma MK Jani D Thungapathra M Gautam JK Meena LS Singh Y Ghosh A Tyagi AK Sharma AK 《Journal of biotechnology》2008,135(1):22-27
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Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1. 总被引:48,自引:11,他引:37
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N A Strockbine M P Jackson L M Sung R K Holmes A D O''''Brien 《Journal of bacteriology》1988,170(3):1116-1122
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Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. 总被引:42,自引:9,他引:33
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D L Weinstein M P Jackson J E Samuel R K Holmes A D O''''Brien 《Journal of bacteriology》1988,170(9):4223-4230
A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I. 相似文献
7.
A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand. 相似文献
8.
Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins. 相似文献
9.
James Terrell Preeti Yadava Carlos Castro Jeffrey Hughes 《Journal of liposome research》2013,23(1):21-29
Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins. 相似文献
10.
Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1 总被引:6,自引:0,他引:6
The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes. 总被引:5,自引:0,他引:5
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Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes. 相似文献
12.
Julia Ebeling Henriette Knispel Anne Fünfhaus Elke Genersch 《Environmental microbiology》2019,21(8):3091-3106
Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I-IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3-like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full-length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus. 相似文献
13.
Alvin W. H. Lo Kristof Moonens Maia De Kerpel Lea Brys Els Pardon Han Remaut Henri De Greve 《The Journal of biological chemistry》2014,289(36):25374-25381
Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease. 相似文献
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Adenine nucleotides promote dissociation of pertussis toxin subunits 总被引:11,自引:0,他引:11
Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity. 相似文献
15.
C Locht P A Barstad J E Coligan L Mayer J J Munoz S G Smith J M Keith 《Nucleic acids research》1986,14(8):3251-3261
We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough. 相似文献
16.
Structural features of the binding site of cholera toxin inferred from fluorescence measurements 总被引:2,自引:0,他引:2
M De Wolf G Van Dessel A Lagrou H J Hilderson W Dierick 《Biochimica et biophysica acta》1985,832(2):165-174
The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed. 相似文献
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Gzyl A Augustynowicz E Zawadka M Rabczenko D Slusarczyk J 《Medycyna do?wiadczalna i mikrobiologia》2007,59(2):123-135
The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production. 相似文献
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Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans. 总被引:20,自引:2,他引:18
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We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT. 相似文献