Vitamin A as adjuvant to the mouse immune response to pertussis, tetanus and diphtheria vaccines

Vitamin A was used as adjuvant, comparatively with Al(OH)₃, in pertussis, tetanus and diphtheria vaccines. Both groups induced a primary immune response in mice, and one single booster dose elevated the antibodies titers in average 554 times to vitamin A groups and 104 times to Al(OH)₃. These antibodies titers correlate with sera IL-4 in immunized animals, suggesting a Th2 response. Other cytokines detected in the sera and/or lymphocytes culture supernatants (IL-2 and IFN-) indicated that vitamin A could also modulate a Th1 response in DPT and acellular pertussis vaccines.     

Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes.

The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT''s function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1''N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.     

Common structure of the catalytic sites of mammalian and bacterial toxin ADP-ribosyltransferases

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding and ADP-ribose transfer.     

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Immune responses to the pertussis toxin in Balb/c and C57B1/6 mice

Abstract Immunization of Balb/c and C57B1/6 mice with the pertussis toxin (Ptx), purified from the culture supernatant of Bordetella pertussis (the whooping cough bacillus) resulted in different immune reactions in these genetically different strains of mice. Antibody responses to Ptx were detected only in Balb/c, whereas both Balb/c and C57B1/6 produced anti-Ptx antibodies when immunized with detoxified Ptx. Also, delayed-type hypersensitivity reactions differ strongly according to the use of Ptx or detoxified Ptx as eliciting antigen.     

Studies of the diphtheria toxin receptor on chinese hamster cells

Concanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β-N-acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell-surface receptor analogue. Diphtheria toxin-resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10-to 15-fold more resistant to diphtheria toxin than wild-type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild-type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell-surface receptor.     

Recombinant acellular pertussis vaccine—from the laboratory to the clinic: improving the quality of the immune response

Abstract Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.     

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein–coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.     

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies,molecular dynamics,and docking simulations

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30’s molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π–π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.     

Inhibition of oxytocin-stimulated phosphoinositide turnover in rat myometrium by pertussis and cholera toxins may involve protein kinase a activation

Both pertussis and cholera toxins inhibit oxytocin-stimulated phosphoinositide turnover in rat myometrium. The actions of pertussis and cholera toxins as well as those of CPTcAMP are reversed by H-8, an inhibitor of protein kinase A. H-8 does not have a major effect on cAMP elevation by the toxins in the presence of oxytocin. The results suggest that the stimulation by oxytocin of phosphoinositide turnover does not involve direct obligatory coupling to a pertussis toxin-sensitive GTP-binding protein. Rather, indirect effects on protein kinase A activation may contribute to the inhibitory effects of both cholera and pertussis toxins. This study suggests that caution must be exercised in interpreting inhibition of phosphoinositide turnover by pertussis toxin in whole cell experiments as indicative of direct involvement of a toxin-sensitive GTP-binding protein.     

Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.     

An integrative bioinformatics pipeline to demonstrate the alteration of the interaction between the ALDH2*2 allele with NAD+ and Disulfiram

Alcohol use disorder (AUD) is a multifactorial psychiatric behavior disorder. Disulfiram is the first approved drug by the Food and Drug Administration for alcohol-dependent patients, which targets the ALDH2 enzyme. Several genes are known to be involved in alcohol metabolism; mutations in any of these genes are known to be associated with AUD. The E504K mutation in the ALDH2 of the precursor protein or the E487K of the mature protein (E504K/E487K; ALDH2*2 allele) is carried by approximately 8% of the world population. In this study, we aimed to test the known inactive allele ALDH2*2, to validate the use of our extensive computational pipeline (in silico tools, molecular modeling, and molecular docking) for testing the interaction between the ALDH2*2 allele, NAD^+, and Disulfiram . In silico predictions showed that the E504K variant of ALDH2 to be pathogenic and destabilizing with the maximum number of prediction in silico tools. Consequently, we studied the effect of this mutation mainly on the interaction between NAD⁺-E504K and Disulfiram-E504K complexes using molecular docking technique, and molecular dynamics (MD) analysis. From the molecular docking analysis with NAD⁺, we observed that the interaction affinity of the NAD⁺ decreases with the impact of E504K variant. On the other hand, the drug Disulfiram showed similar interaction in both the native and mutant ALDH2 proteins. Further, the comprehensive MD analysis predicted that the E504K destabilizes the protein and influences the NAD⁺ and Disulfiram interactions. Our findings reveal that the interaction of NAD⁺ to the protein is disturbed by the E504K/E487K variant whereas the drug Disulfiram has a similar effect as both native ALDH2 and ALDH2 bearing E504K/E487K variant. This study provides a platform to understand the effect of E504K/E487K on the molecular interaction with NAD⁺ and Disulfiram.     

An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.     

Comparison of high-resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation-anion binding site.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is a divalent-metal activated repressor of chromosomal genes responsible for siderophore-mediated iron-uptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized iron-dependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation-anion pair discovered in its first metal-binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100-fold higher than other divalent cations. In the presently reported 1.85 A resolution Co-DtxR structure at 100K, the sulfate anion in the cation-anion-binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion. A comparison of the 1.85 A Cobalt-DtxR structure at 100K and the 2.4 A Zinc-DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the properties of the intriguing second metal-binding site. Our studies do, however, provide a high resolution view of the cationanion-binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.     

A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Abstract Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) at doses as low as 0.8 ng·ml⁻¹, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertusis , containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for PT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

Immunochemical comparison of pertussis toxin substrates in brain and peripheral tissues

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified -subunit of G₀ from bovine brain or against synthetic peptides predicted from cDNAs for distinct G_i subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophilis. Brain contained both G₀ and G₁. G_il was also found to be abundant in heart. All peripheral tissues contained tissues contained readily detectable amounts of G₁₂, whereas only barely detectable amounts of G_i2 were found in brain. G_i3 was found to be prominent in erythorocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, G_i2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.