Molecular cloning of pertussis toxin genes

Locht, Camille; Barstad, Paul A.; Coligan, John E.; Mayer, Leonard W.; Muñoz, J.; Smith, Susan Gower; Keith, Jerry M.

doi:10.1093/nar/14.8.3251

Cited by 54 publications

(42 citation statements)

References 33 publications

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“…Standard recombinant DNA techniques were used as described by Maniatis et al (21). The recombinant 4pRIT20001 was obtained by isolating the 560-base-pair (bp) Sau3A fragment containing the coding information for rSld from pPTX42 (13) and inserting it into the BamHI site of M13mpl8 (22). The mutant phages were obtained by site-directed mutagenesis of spRIT20001, using the oligonucleotide-directed in vitro mutagenesis system (Amersham) under conditions described by the supplier.…”

Section: Methodsmentioning

confidence: 99%

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Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

Locht¹,

Capiau²,

Feron³

1989

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

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The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADPribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid.Mutations at the Glu-106 position do not significantly reduce the enzymatic activities ofthe S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by

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Section: Methodsmentioning

confidence: 99%

“…The entire PTX gene has recently been cloned (13,14) and sequenced (14,15). To identify potential targets for chemical or genetic manipulation, the primary sequence of the PTX Si subunit was compared to the amino acid sequences of other bacterial ADP-ribosylating toxins.…”

mentioning

confidence: 99%

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

Locht¹,

Capiau²,

Feron³

1989

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

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“…Bordetella pertussis and B. parapertussis are the causative agents of whooping cough in humans, and B. bronchiseptica infects the respiratory tracts of a broad range of mammals (17,26). Numerous virulence factors have been identified in Bordetella spp., including toxins such as pertussis toxin (expressed only in B. pertussis) (25,35,48), adenylate cyclase toxin (24), and dermonecrotic toxin (46) and adhesins such as filamentous hemagglutinin (12, 36), pertactin (37), and fimbriae (31). The expression of these virulence factors is coordinately regulated by the Bordetella virulence gene (bvg) locus (5, 49), which encodes the BvgA/BvgS two-component regulatory system (43).…”

mentioning

confidence: 99%

The Type III Secreted Protein BopD in Bordetella bronchiseptica Is Complexed with BopB for Pore Formation on the Host Plasma Membrane

et al. 2004

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The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.Three major pathogenic species in the genus Bordetella are known. Bordetella pertussis and B. parapertussis are the causative agents of whooping cough in humans, and B. bronchiseptica infects the respiratory tracts of a broad range of mammals (17,26). Numerous virulence factors have been identified in Bordetella spp., including toxins such as pertussis toxin (expressed only in B. pertussis) (25,35,48), adenylate cyclase toxin (24), and dermonecrotic toxin (46) and adhesins such as filamentous hemagglutinin (12, 36), pertactin (37), and fimbriae (31). The expression of these virulence factors is coordinately regulated by the Bordetella virulence gene (bvg) locus (5, 49), which encodes the BvgA/BvgS two-component regulatory system (43). Under growth conditions of 37°C in the relative absence of MgSO 4 or nicotinic acid, the BvgAS phosphorelay is activated and bordetellae grow under Bvg ϩ phase conditions (8). The Bvg ϩ phase is characterized by the expression of various virulence factors, such as toxins and adhesins, and is necessary for respiratory tract colonization in rabbits and rats (1, 10). The Bvg Ϫ phase is avirulent and is characterized by loss of expression of both toxin and adhesin genes and by induction of genes that are not expressed under Bvg ϩ phase conditions. In B. bronchiseptica, Bvg Ϫ phase loci include genes and operons that encode the motility apparatus (2, 3). It has been shown that the type III secretion system in bordetellae is activated in the Bvg ϩ phase (51). Wild-type B. bronchiseptica, but not the type III secretion mutant, was shown to induce cytotoxicity against L2 cells th...

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“…Pertussis toxin is a member of the AB 5 family of toxins, which also includes cholera toxin, Escherichia coli heat-labile toxin, and Shiga toxin. Pertussis toxin has the most complex structure of any bacterial toxin (18,20,24). It is assembled from six subunits encoded by five genes, ptxS1 to -S5.…”

mentioning

confidence: 99%

“…Each of the five subunits possesses a secretion signal peptide, which directs secretion to the periplasm (18,20). In the periplasm, the signal peptide is removed and the subunits fold and assemble (5,9,10,21,26).…”

mentioning

confidence: 99%

Reduced Glutathione Is Required for Pertussis Toxin Secretion by Bordetella pertussis

2003

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The abilities of cysteine-containing compounds to support growth of Bordetella pertussis and influence pertussis toxin transcription, assembly, and secretion were examined. Cysteine is an essential amino acid for B. pertussis and must be present for protein synthesis and bacterial growth. However, cysteine can be metabolized to sulfate, and high concentrations of sulfate can selectively inhibit transcription of the virulence factors, including pertussis toxin, via the BvgAS two-component regulatory system in a process called modulation. In addition, pertussis toxin possesses several disulfide bonds, and the cysteine-containing compound glutathione can influence oxidation-reduction reactions and perhaps disulfide bond formation. Bacterial growth was not observed in the absence of a source of cysteine. Oxidized glutathione, as a sole source of cysteine, also did not support bacterial growth. Cysteine, cystine, and reduced glutathione did support bacterial growth, and none of these compounds caused modulation at the concentrations tested. Similar amounts of periplasmic pertussis toxin were detected regardless of the source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted. Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin. These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium.

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Molecular cloning of pertussis toxin genes

Cited by 54 publications

References 33 publications

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

The Type III Secreted Protein BopD in Bordetella bronchiseptica Is Complexed with BopB for Pore Formation on the Host Plasma Membrane

Reduced Glutathione Is Required for Pertussis Toxin Secretion by Bordetella pertussis

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