Pertussis Toxin S1 Mutant with Reduced Enzyme Activity and a Conserved Protective Epitope

Burnette, W N; Cieplak, Witold; Mar, V L; Kaljot, KT; Sato, Hiroko; Keith, Jerry M.

doi:10.1126/science.2459776

Cited by 105 publications

(72 citation statements)

References 35 publications

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“…The protective epitopes on PTx are conformational and thus particularly sensitive to chemical modification; indeed, we observe significant losses in 1B7 and 11E6 binding to PTx after chemical inactivation. In contrast, no loss in binding is observed for the genetically detoxified PTx R9K/E129G variant, PTg (7,27), and overall titers of serum samples for PTg are indistinguishable from PTx titers. Thus, we propose that vaccination with PTg is likely to induce stronger responses to these two potently neutralizing epitopes than vaccination with PTd.…”

mentioning

confidence: 81%

“…To determine whether the route of PTx exposure, to native PTx during an infection or to PTd during vaccination, could explain the differences in 1B7-and 11E6-like titers in exposed and vaccinated adults, we prepared PTd with procedures similar to those used during vaccine production using formaldehyde. A genetically detoxified variant of PTx, containing two amino acid changes in the S1 subunit (R9K and E129G; PTg) was developed in the late 1980s as an alternative to chemical toxoiding (7,26) but was not included in the acellular vaccine due to patent issues.…”

Section: Resultsmentioning

confidence: 99%

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Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Sutherland

Chang

Yoder

et al. 2011

Clin. Vaccine Immunol.

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Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.Pertussis vaccines, widely introduced as an inactivated whole-cell vaccine in 1950, have been responsible for a dramatic decline in pertussis-related morbidity and mortality but have been unable to eradicate disease despite 95% coverage in many areas. Disturbingly, rates of confirmed pertussis cases in industrialized countries have increased steadily

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confidence: 81%

Section: Resultsmentioning

confidence: 99%

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Sutherland

Chang

Yoder

et al. 2011

Clin. Vaccine Immunol.

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“…In the process of trying to genetically produce pertussis toxoid for vaccine purposes, several laboratories independently produced dozens of strains expressing mutant forms of S1 (4,13,17,18,24,25). In many cases, only recombinant S1 produced by E. coli was characterized.…”

Section: Figmentioning

confidence: 99%

Mutations in the S1 Subunit of Pertussis Toxin That Affect Secretion

2000

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Pertussis toxin is a member of the AB 5 family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the ptl operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into Bordetella pertussis and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the ptl genes, suggesting that this region may have a role in pertussis toxin secretion.Pertussis toxin is a major virulence factor of Bordetella pertussis, the gram-negative bacterium that is the causative agent of whooping cough. It is a member of the AB 5 family of toxins, consisting of five subunits, S1, S2, S3, S4, and S5, present in a 1:1:1:2:1 ratio (18,20,27). S1 is the A or enzymatic subunit and catalyzes the ADP-ribosylation of G proteins in the target mammalian cell. Subunits S2 to S5 form the B pentamer, which delivers the S1 subunit to the target mammalian cell. Several of the mammalian cells targeted by pertussis toxin (including lymphocytes, macrophages, and neutrophils) are important effectors of the immune system, and toxin treatment compromises their ability to function, contributing to the severity of the disease (22,23,29).Pertussis toxin assembly and secretion is a complex process. Each subunit is synthesized with a signal peptide (20), which mediates secretion to the periplasm via the equivalent of the Sec-mediated secretion machinery of Escherichia coli. Folding and assembly of the subunits occurs in the periplasm, and the ptl (pertussis toxin liberation) operon is required for efficient secretion of assembled toxin past the outer membrane (7, 9, 31). The ptl secretion machinery is specific for pertussis toxin, since secretion of other known virulence factors occurs normally in ptl mutant strains (31, 32). The secretion machinery appears to discriminate between assembled and unassembled pertussis toxin, since only assembled toxin is efficiently released from the bacteria. Since secretion involves substrate recognition, the Ptl secretion machine...

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“…The S-1 subunit contains 235 amino acids (18) and catalyzes the covalent transfer of the ADP-ribose portion of NAD to Gi protein, the negative regulatory component of the adenylate cyclase complex (12). The remaining five subunits (S-2 through S-5) compose a pentamer which is responsible for delivering the S-1 subunit to the Gi protein.We and others (1,3,5,6,20) have begun to define the functional residues of the S-1 subunit of PT with the goal of generating noncatalytic but immunologically conserved forms of the subunit amenable for administration as an acellular vaccine. The subunits of PT have been expressed in E. coli (2,13,17) and Bacillus slibtilis (21) showed that residues 2 through 180 possessed ADP-ribosyltransferase activity (1,13,20).…”

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confidence: 99%

Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli

1989

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The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C1801-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C1801-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.Bordetella pertussis, the etiologic agent of whooping cough, produces several factors which contribute to its pathogenesis (26). Two of these factors, pertussis toxin (PT) and filamentous hemagglutinin, also act as immunogens which stimulate production of protective antibodies against the bacterium (22). Present investigations have focused on the development of an acellular vaccine to replace the whole cell vaccine currently administered in the United States. One approach to the development of an acellular vaccine would be to use a nontoxic form of PT and filamentous hemagglutinin, alone or in concert, as the immunogen. The cloning of the PT operon in Escherichia coli (14,18) has allowed the use of recombinant DNA techniques to attempt to modify PT such that the protein is nontoxic but retains immunogenic properties, allowing its use as an immunogen.PT (molecular weight, 105,060 [18]) is composed of six noncovalently bound subunits designated S-1 through S-5 (24). There is one copy of each subunit, except for S-4, which is present in two copies. The S-1 subunit contains 235 amino acids (18) and catalyzes the covalent transfer of the ADP-ribose portion of NAD to Gi protein, the negative regulatory component of the adenylate cyclase compl...

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Pertussis Toxin S1 Mutant with Reduced Enzyme Activity and a Conserved Protective Epitope

Cited by 105 publications

References 35 publications

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Mutations in the S1 Subunit of Pertussis Toxin That Affect Secretion

Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli

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