Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen

Kimura, Alan; Mountzouros, K T; Schád, Péter; Cieplak, Witold; Cowell, J L

doi:10.1128/iai.58.10.3337-3347.1990

Cited by 30 publications

(13 citation statements)

References 58 publications

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“…is required to ascertain that none of the potent biological activities associated with PT are due to this component. Our results, which are consistent with previously published results (15,17), suggest that the B oligomer as part of a genetically attenuated holotoxin exhibits neither the LP nor the HS activity of native toxin. Therefore, these activities are likely due to the ADP-ribosyltransferase activity of the toxin.…”

Section: Discussionsupporting

confidence: 93%

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin

et al. 1991

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An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.

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Section: Discussionsupporting

confidence: 93%

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin

et al. 1991

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“…Although many biological activities of PTX depend on its Sl-catalyzed ADP-ribosyltransferase activity, some, such as mitogenicity, are independent of this enzymatic activity (15). In fact, reduction in enzymatic activity may sometimes result in increased mitogenic activity (16). The PTX-CRM with impaired receptor-binding abilities, however, presents an undetectable level of T cell mitogenicity, as indicated by its inability to stimulate II~2 secretion by spleen cells exposed to the holotoxin, regardless of the toxin's ability to catalyze ADP-ribosylation.…”

Section: Discussionmentioning

confidence: 99%

“…However, some biological activities are independent of the ADPribosyltransferase activity (15). Of concern is PTX-assodated mitogenicity, one of the activities that are not reduced by inactivation of the $1 subunit (16). Since it is unclear which precise molecular activities especially cause the rare but severe side effects of current pertussis vaccines, it is desirable to abolish all measurable biological activities of the PTX-CRM molecules used in the future vaccines.…”

mentioning

confidence: 99%

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Lobet

Feron

Dequesne

et al. 1993

The Journal of Experimental Medicine

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SummaryPertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The $1 subunit is responsible for the ADPribosyltransferase activity of the toxin. The B moiety, composed of $2 through $5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits $2 and $3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Ash 105 in $2 and of Lys 105 in $3 resulted in the more drastic reduction of binding to haptogiobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the $1 subunit led to the production of a toxin analog with reduced ADPoribosyltransferasedependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monodonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the $1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.tel/a pertuss/s, the etiologic agent of whooping cough, duces a number of toxins responsible for the local and systemic manifestations of this important childhood disease (1). Among these toxins, pertussis toxin (PTX) 1 plays a major role in the pathogenesis, in that it expresses a wide variety of biological activities, such as histamine sensitization, activation of insulin secretion, promotion of lymphocytoffs, and others (2). In addition, immunization with PTX has been shown to protect mice against subsequent lethal challenge with virulent R pertuss/s (3). It is therefore considered an important constituent in vaccines against pertussis, and is the major component of the new acellnlar vaccines currently being tested or used in several countries (4,5).Paradoxically, this toxin may itself be responsible for the harmful side effects associated with the current vaccines (6). These side effects range in severity from minor local reactions and simple flushing, to permanent neurological damage t Abbre~aons used in this paper: CRAM, cross-reactive mutant; NAD, nicotinamide adenine dinudeotide; FTX, txn'tussis toxin; WT, wild type. and, in some rare cases, death. Before PTX can effectively and safely be used in pertussis vaccines, complete and irreversible detoxification is therefore required.The toxin is composed of five different subunits, named $1 to $5, based on their decreasing molecular weights. The subunits associate in a molar ratio of 1S1:1S2:1S3:2S4:1S5 to form the holotoxin. Functionally, FTX can be...

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“…PT, PT* and peptide-tagged derivatives were prepared from B. pertussis culture supernatants by the fetuin affinity method (Kimura et al, 1990), dialysed against PBS and stored in 10% glycerol at -80°C until use.…”

Section: Protein Purificationmentioning

confidence: 99%

Retrograde transport of pertussis toxin in the mammalian cell

Plaut

Carbonetti²

2008

Cell Microbiol

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SummaryPertussis toxin (PT), an AB5 exotoxin and important virulence factor of Bordetella pertussis, is hypothesized to traffic along a retrograde transport pathway in mammalian cells. This pathway includes endosomal uptake, transport to the Golgi complex and endoplasmic reticulum (ER), dissociation of the holotoxin in the ER and translocation of the A subunit (S1) to the cytosol, where it ADP-ribosylates its G protein targets. In this study, PT was visualized in the Golgi complex by immunofluorescence microscopy, but transport beyond the Golgi could not be detected by this method. To gain evidence for the retrograde pathway, peptide tags with target sites for tyrosine sulfation (a trans-Golgi network-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained in vitro enzymatic and cellular activity as assessed by ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation, but not that of S1, was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. When cells were pretreated with methyl-bcyclodextrin, sulfation of PT subunits and PT cytotoxicity were reduced, suggesting that PT transport is dependent on cellular cholesterol content. These data support a retrograde pathway for PT intracellular transport.

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Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen

Cited by 30 publications

References 58 publications

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Retrograde transport of pertussis toxin in the mammalian cell

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