Pertussis Toxin

Protein-based AB5-type exotoxin Pertussis toxin (PT) is produced by the bacterium Bordetella pertussis. PT can cause whooping cough.

In vitro: PT has the effect on innate immune response. The effects of PT on human monocyte-derived DC (MDDC), including maturation, are regulated by cAMP [1].

In vivo: Pertussis toxin did not affect the relaxation and contraction properties of isolated NIH or BALB/c mouse trachea, but significantly declined KCl or noradrenaline-induced maximal contraction and decreased sensitivity to noradrenaline in isolated male Wistar rat small mesenteric resistance arteries [2].

Clinical trial: Pertussis toxin was used as vaccine, acellular pertussis booster immunizations, to young and older persons in order to reduce the community transmission and to enhance the protection. Older persons with infections are mainly asymptomatic. Acellular pertussis boosters provide protection against symptomatic pertussis and give protection against mild and asymptomatic infections. The use of boosters may reduce transmission rate, especially in infant population [3].

References:
[1] Bagley KC, Abdelwahab SF, Tuskan RG, Fouts TR, Lewis GK.  Pertussis toxin and the adenylate cyclase toxin from Bordetella pertussis activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a cAMP-dependent pathway. J Leukoc Biol. 2002 Nov;72(5):962-9.
[2] van Meijeren CE, Vleeming W, van de Kuil T, Manni J, Kegler D, Hendriksen CF, de Wildt DJ.  In vivo pertussis toxin treatment reduces contraction of rat resistance arteries but not that of mouse trachea. Eur J Pharmacol. 2004 Mar 19;488(1-3):127-35.
[3] Ward JI, Cherry JD, Chang SJ, Partridge S, Keitel W, Edwards K, Lee M, Treanor J, Greenberg DP, Barenkamp S, Bernstein DI, Edelman R; APERT Study Group.  Bordetella Pertussis infections in vaccinated and unvaccinated adolescents and adults, as assessed in a national prospective randomized Acellular Pertussis Vaccine Trial (APERT). Clin Infect Dis. 2006 Jul 15;43(2):151-7. Epub 2006 Jun 5.

Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis.[Pubmed:28335677]

Infect Dis (Lond). 2017 Aug;49(8):594-600.

BACKGROUND: The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and Pertussis Toxin enzyme-linked immunosorbent assay (ELISA). METHODS: The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to Pertussis Toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups. RESULTS: The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres >/=1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population. CONCLUSIONS: Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.

Stability, structural and functional properties of a monomeric, calcium-loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis.[Pubmed:28186111]

Sci Rep. 2017 Feb 10;7:42065.

Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.

Reporter cell lines for detection of pertussis toxin in acellular pertussis vaccines as a functional animal-free alternative to the in vivo histamine sensitization test.[Pubmed:28129894]

Vaccine. 2017 Feb 22;35(8):1152-1160.

Detoxified Pertussis Toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual Pertussis Toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST.

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin.[Pubmed:28097085]

FEBS Open Bio. 2016 Dec 9;7(1):25-34.

Adenylate cyclase toxin domain (CyaA-ACD) is a calmodulin (CaM)-dependent adenylate cyclase involved in Bordetella pertussis pathogenesis. Calcium (Ca(2+)) and magnesium (Mg(2+)) concentrations impact CaM-dependent CyaA-ACD activation, but the structural mechanisms remain unclear. In this study, NMR, dynamic light scattering, and native PAGE were used to probe Mg(2+)-induced transitions in CaM's conformation in the presence of CyaA-ACD. Mg(2+) binding was localized to sites I and II, while sites III and IV remained Ca(2+) loaded when CaM was bound to CyaA-ACD. 2Mg(2+)/2Ca(2+)-loaded CaM/CyaA-ACD was elongated, whereas mutation of site I altered global complex conformation. These data suggest that CyaA-ACD interaction moderates CaM's Ca(2+)- and Mg(2+)-binding capabilities, which may contribute to pathobiology.

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha.[Pubmed:2496748]

Biochemistry. 1989 Jan 24;28(2):611-6.

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the Pertussis Toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.

ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin.[Pubmed:3135265]

Infect Immun. 1988 Aug;56(8):1934-41.

The ADP-ribosyltransferase activity of Pertussis Toxin resides within the S-1 subunit of the toxin. Deletion mapping of a recombinant S-1 subunit produced in Escherichia coli showed that amino acids 2 through 180 are required for ADP-ribosylation of Gi protein. Mutants of the S-1 subunit which lacked either amino acids 2 through 22 or amino acids 153 through 180 failed to express enzyme activity, implicating a functional or structural role for these residues in catalysis. The catalytic carboxy-terminal S-1 deletion, C-180, was found to be more soluble than the recombinant S-1 subunit, making it a useful construct for future structure-function studies on enzyme catalysis. Four independent single-amino-acid substitutions which decreased ADP-ribosyltransferase activity were constructed in the recombinant S-1 subunit. Substitution of Asp-11 by Ser, Arg-13 by Leu, or Trp-26 by Ile decreased enzyme activity to below detectable levels (less than 1.0% of that of the recombinant S-1 subunit). The Glu-139-to-Ser substitution reduced ADP-ribosyltransferase activity to 15% of that of the recombinant S-1 subunit. Both the oxidized and reduced forms of the recombinant S-1 subunit and recombinant S-1 subunits containing single-amino-acid substitutions were degraded through identical immunoreactive tryptic peptides, suggesting that the conformations of the mutants are similar to that of the recombinant S-1 subunit. Identification of noncatalytic forms of the S-1 subunit of Pertussis Toxin which have conserved protein structure is an initial step in the generation of a recombinant noncatalytic form of Pertussis Toxin which may be tested as a candidate for an acellular vaccine against Bordetella pertussis.

Identification of the predominant substrate for ADP-ribosylation by islet activating protein.[Pubmed:6296122]

J Biol Chem. 1983 Feb 25;258(4):2072-5.

Islet activating protein (IAP), a toxin isolated from Bordetella pertussis, blocks the ability of inhibitory hormones to attenuate adenylate cyclase activity and enhances the ability of stimulatory hormones to activate the enzyme. The toxin appears to act by catalyzing the transfer of ADP ribose from NAD to a 41,000-dalton protein in target cell membranes. A protein purified from rabbit liver membranes, apparently composed of 41,000- and 35,000-dalton subunits, is shown to be a specific substrate for IAP. Cholera toxin does not ADP-ribosylate this protein. In contrast, the purified guanine nucleotide-binding regulatory component of adenylate cyclase (G/F), which is ADP-ribosylated by cholera toxin, is not covalently modified by IAP. Equilibrium binding studies and photoaffinity labeling experiments demonstrate that the 41,000-dalton subunit of the IAP substrate has a specific binding site for guanine nucleotides.

Pertussis Toxin is a protein-based AB5-type exotoxin produced by the bacterium Bordetella pertussis, which causes whooping cough.

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