Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Expression ofBordetella pertussis toxin subunits inBacillus subtilis

Summary Pertussis toxin subunits (S1–S5) were expressed inBacillus subtilis using a vector with the promoter, Shine-Dalgarno sequence and the sequence coding for the first 7 amino acids of the signal sequence of the -amylase gene fromB. amyloliquefaciens. The use of this vector resulted in a high level of intracellular expression of each pertussis toxin subunit as aggregates in the cytoplasm ofB. subtilis.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

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.     

Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.     

Purification and immunological characterization of HPLC-purified pertussis toxin subunits.

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.     

Production and secretion of pertussis toxin subunits in Bacillus subtilis

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.     

Genetic exchange of the S2 and S3 subunits in pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, produces a complex hetero-oligomeric exotoxin, named pertussis toxin (PTX), which is responsible for several of the clinical manifestations associated with whooping cough. The toxin is composed of five dissimilar subunits, named S1 through S5 and arranged in a hexameric structure with a 1S1:1S2:1S3:2S4:1S5 stoichiometry. Although S2 and S3 share 70% amino acid identity, these two subunits were previously thought not to be able to substitute for each other in toxin assembly/secretion and the biological activities of PTX. Here, we show that toxin analogues containing two S3 subunits and lacking S2 (PTXdeltaS2), or containing two S2 subunits and lacking S3 (PTXdeltaS3), can be produced, assembled and secreted by B. pertussis strains, in which the S2-encoding cistron or the S3-coding cistrons have been inactivated by internal in-frame deletions that avoid downstream effects. In fact, PTXdeltaS3 was produced in higher amounts in the bacterial culture supernatants than natural PTX, whereas PTXdeltaS2 was produced in lower amounts than PTX. The action of the toxin analogues on the clustering of Chinese Hamster Ovary cells was also affected differentially by the S2-S3 substitution. These toxin analogues constitute thus interesting probes for the study of cellular functions, in particular immune cell functions, for which natural PTX has already shown its usefulness.     

Structural relationship between the S1 and S4 subunits of pertussis toxin

Abstract Pertussis toxin, the most important protective antigen of Bordetella pertussis , is a 106-kDa hexameric protein composed of an A-promoter (subunit S1) and a pentameric B-oligomer (S2 + S3 + 2S4 + S5). The most potent mouse-protective monoclonal antibodies against both respiratory and intracerebral infections were specified for either S1 or S4 and competed with each other in binding to epitopes of native pertussis toxin captuted by haptoglobin or in solution, although they did not compete on unfolded pertussin toxin. These data suggest that the protective epitope(s) of S1 and S4 are very closely correlated; they are probably close] together sterically. Non-protective anti-S1 and anti-S4 monoclonal antibodies recognized inner antigenic determinants which are not exposed on the surface o native pertussis toxin and interfered with association of the A-protomer and the B-oligomer. These data suggest that the A-protomer and the S4 subunit of the B-oligomer may be closely associated in the native hexameric pertussis toxin molecule.     

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.     

Genetics of pertussis toxin

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.     

Comparison of the subunits of Torpedo californica acetylcholine receptor by peptide mapping.

The acetylcholine receptor from Torpedo californica electroplax was purified approximately 100-fold by affinity chromatography on alpha-neurotoxin-Sepharose 6B. Four putative subunits (alpha, beta, gamma, delta) of apparent molecular weights of 43,000, 52,000, 58,000, and 63,000 were found when the purified material was analyzed by sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. In some preparations, however, the amount of the gamma polypeptide was small. The presence of N-ethylmaleimide throughout the purification procedure greatly enhanced the amount of the gamma chain. To investigate the possibility that the putative subunits may be structurally related, they were isolated by preparative NaDodSO4 gel electrophoresis and subjected to peptide mapping analyses. The patterns of fragments generated by Staphylococcus aureus V8 protease, papain, or chymotrypsin were different for each of the polypeptides. Thus, it is unlikely that they are derivatives of each other.     

Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.     

Utilization of sialic acid-binding synthetic peptide sequences derived from pertussis toxin as novel anti-inflammatory agents

Pertussis toxin, a virulence factor produced by the organismBordetella pertussis, has been shown to have functional similaritieswith selectins and to bind to similar sialic acid-containingoligosaccharides structures. Previously, we demonstrated thatthe amino-terminal region of the S2 subunit of pertussis toxincontained a short six amino acid sequence (SPYGRC) which displayedreasonable homology to a sequence that constitutes a portionof the sialic acid binding site in wheat germ agglutinin. Syntheticpeptides containing this hexapeptide motif had the ability tobind to sialic acid-containing glycoconjugates including theputative oligosaccharide receptors (sialyl Lewis X and sialylLewis A) for selectins. Control peptides containing randomizedsequences were inactive at inhibiting binding, indicating thatthe hexapeptide motif is important for interacting with sialicacid. Since pertussis toxin-derived peptides demonstrated theability to interact with selectin receptors, we speculated thatthey should antagonize selectin-mediated inflammatory activity.To test this hypothesis, we evaluated the peptides for the abilityto reduce neutrophil binding to activated endothelial cellsas well as the anti-inflammatory activity in the mouse footpadswelling assay. Both S2 peptides were active at reducing neutrophilbinding and footpad swelling, while the randomized control peptideswere inactive. anti-inflammatory agents peptides pertussis toxin     

ADP-ribosylation of transducin by pertussis toxin

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.     

Structure-activity analysis of the activation of pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Construction of the genetically attenuated bacteria Bordetella pertussis devoid of dermonecrotic toxin activity and producing modified nontoxic pertussis toxin form

The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3.