Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

Cieplak, Witold; Burnette, W N; Mar, V L; Kaljot, KT; Morris, Charles F.; Chen, K K; Sato, Hiroko; Keith, Jerry M.

doi:10.1073/pnas.85.13.4667

Cited by 44 publications

(41 citation statements)

References 38 publications

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“…Whereas the overall homology of the toxin with proteins in the database was low, a strong regional homology was found between the 100-kDa toxin and the S1 subunit of pertussis toxin (31). The homologous sequence corresponds in part to a region of the pertussis toxin (residues 8 to 15 of the S1 subunit) found to be essential for activity (11) and previously observed to be related to the A subunits of both cholera toxin and E. coli heat-labile toxin (23,31). These three toxins act by ADP-ribosylation of G proteins.…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1

et al. 1991

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A cosmid library was prepared from a partial BamHJ digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons TnSOl and Tn2l. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADPribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.Bacillus sphaericus is an aerobic spore-forming Bacillus species, several strains of which are pathogenic for mosquito larvae (13). B. sphaericus SSII-1 was isolated in 1973 from infected mosquito larvae collected in India (36). This strain was considerably more toxic than the other B. sphaericus strains known at that time. Early studies indicated that the toxin was retained within or attached to the bacterial cells and that the toxic activity was markedly unstable. Storage of cells under refrigeration or heating at 80°C resulted in the loss of activity. Toxin production occurred predominantly in the vegetative phase of growth before the onset of sporulation (27,29).Further studies on B. sphaericus SSII-1 declined rapidly with the discovery of strains, such as 1593 (36a), 2362 (40), and 2297 (41), which had higher toxicities. In contrast to strain SSII-1, these strains develop relatively stable, high toxicity at the onset of sporulation (7). To date, two types of toxin have been characterized in the highly toxic strains: a pair of proteins of 51.4 and 41.9 kDa associated with the parasporal crystal (3,5,19) and a toxin of 110 kDa related to a 125-kDa surface layer protein (5, 6). Genes encoding the 51.4-and 41.9-kDa proteins were shown to be absent from strain SSII-i (4). In the same study, Baumann et al. showed that sequences inserted in their group C clones were also absent from strain SSII-1. The group C clones were later shown to contain sequences corresponding to the gene encoding the 125-kDa protein (6). Davidson (12) Construction of the cosmid library. To prepare total genomic DNA from B. sphaericus SSII-1, we lysed cells in the presence of 1% sodium dodecyl sulfate and 0.1 mg of proteinase K per ml. DNA was purified from the lysate by the method of Ausubel et al. (2). In brief, hexadecyltrimethyl ammonium bromide was added, and the mixture was incubated at 65°C for 20 min prior to extraction with chloroformisoamyl alcohol. DNA was precipitated with ethanol from the aqueous phase and purified by banding on a cesium chloride gradient coqtaining ethidium bromide. DNA was partially digest...

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Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1

et al. 1991

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“…Recent reports suggest that the dominant protective epitope on the S1 subunit is discontinuous (32,35,37), perhaps assuming its native conformation after association with the B oligomer. That recombinant S1 protein and the B oligomer combine to yield a functional holotoxin suggests that this important antigenic determinant may also be formed in this complex.…”

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confidence: 99%

“…For these purposes, the individual subunit polypeptides were expressed at high levels in Escherichia coli (36), and a region of the recombinant Si subunit (Val8-Pro'5) was identified as both necessary for enzyme activity and critical for the formation of its neutralizing, protective epitope (37). Selective site-directed mutagenesis of the subcloned S1 cistronic element permitted us to derive and analog polypeptide (Arg9 -k Lys) retaining the protective epitope, but with strikingly reduced ADP-ribosyltransferase activity (38).…”

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Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

Bartley

Whiteley²,

Mar³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced byBordeteUapertussis, is believed to be due to the ADP-ribosyltransferase activity of the Si subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymaticaily deficient form of the Si subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant Si subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant Si displayed greatly diminished cytopathicity.These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.In an effort to create a fully efficacious yet less reactogenic pertussis vaccine, we have focused on the recombinant production of PTX subunit proteins, detoxification of the Si moiety by site-specific mutagenic inactivation of enzyme activity, and the in vitro assembly of a genetic "holotoxoid." For these purposes, the individual subunit polypeptides were expressed at high levels in Escherichia coli (36), and a region of the recombinant Si subunit (Val8-Pro'5) was identified as both necessary for enzyme activity and critical for the formation of its neutralizing, protective epitope (37). Selective site-directed mutagenesis of the subcloned S1 cistronic element permitted us to derive and analog polypeptide (Arg9 -k Lys) retaining the protective epitope, but with strikingly reduced ADP-ribosyltransferase activity (38). We wished to assess the biological activity of this genetically deactivated S1 mutant subunit and evaluate its potential to participate in the production of a recombinant holotoxoid. To this end, we have assembled holotoxin molecules in vitro that are composed of native PTX B oligomer and recombinant SI subunits of either native or mutant sequence and evaluated their relative abilities to elicit cytotoxic responses in cultured cells.Pertussis toxin (PTX) is both a major virulence factor of Bordetella pertussis (1), the etiologic agent of whooping cough (2), and an important antigenic element in vaccines for immunoprotection against disease (3-7). Certain clinical manifestations of infection (1,8,9) and minor reactions to vaccination (10,11) are attributable to the effects of this complex exotoxin. The origin of the extremely rare adverse neurologic events temporally related to pertussis immunization remains controversial (11-15). Chemic...

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“…The subcloning, recombinant expression, and mutagenesis of PT subunits was performed in Escherichia coli as described (23)(24)(25). A 647-base-pair (bp) (Nde I/Hindll) fragment of the S2 gene was removed from expression vector pCFM4722 (23) and subcloned into pUC19.…”

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Pertussis toxin has eukaryotic-like carbohydrate recognition domains.

Saukkonen

Burnette

Mar

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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127

116

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Bordetela peinussis is bound to glycocoijugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite >80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.During the clinical course of whooping cough, Bordetella pertussis specifically attaches to the cilia of the respiratory epithelium (1), establishes an intracellular state within alveolar macrophages (2), and produces systemic disease by elaborating several toxins (3). At least two adhesins, filamentous hemagglutinin and pertussis toxin (PT), are known to mediate bacterial attachment to human cells (4, 5). Both adhesins are required for attachment to cilia, but each can act independently in the adherence of the organism to macrophages. These adhesins have several unusual features of interest. They are large, nonfimbrial molecules that contain multiple binding domains (1, 5) and, when shed from the cell surface, retain the ability to mediate adherence even for other bacterial species (6).PT is a major virulence determinant of B. pertussis, which induces metabolic changes in the host (7), alters immune responses (8), and is the only toxin known to bind whole bacteria to eukaryotic cells (4). The toxin is a hexameric protein with an A-B architecture (9). The A protomer is composed of a single S1 subunit (Mr, 26,026) containing the catalytic site for toxic ADP-ribosylation of cellular signaltransducing guanine nucleotide regulatory proteins. The B oligomer possesses the cellular recognition domains; it is a complex pentamer containing subunits S2 (Me,21,925), S3 (Mr,21,873), S4 (Mr,12,059), and S5 (Mr, 11,013) in a respective molar ratio of 1:1:2:1 (9). By interacting with glycoproteins and glycolipids on many types of eukaryotic cells (4,(10)(11)(12)(13)(14)(15)(16), the B oligomer serves several distinct and independent functions (4, 15): mitogenicity for T lymphocytes, adherence of the bacteria to eukaryotic cells, and delivery of the toxic S1 subunit to its target. Several findings suggest that the B oligomer contains at least two distinct binding specificit...

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Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

Cited by 44 publications

References 38 publications

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1

Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

Pertussis toxin has eukaryotic-like carbohydrate recognition domains.

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