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PubMed1973

Rapid, direct tissue culture test for toxigenicity of Corynebacterium diphtheriae.

A method for testing toxigenicity of Corynebacterium diphtheriae in tissue culture is described. The technique, called the colony overlay test (COT), involves inoculating material from an isolated colony of C. diphtheriae to a small area on the surface of an agar medium which overlays a monolayer of toxin-susceptible HeLa cells. If toxin is produced during incubation at 37 C, it diffuses to the tissue monolayer and destroys the cells below the inoculation site. Twenty-four hours after inoculation, organisms are killed and tissue cells are fixed with formaldehyde. The agar overlay is then removed, and the monolayer is stained with crystal violet. Toxin-affected areas fail to stain or stain poorly. A second plate with antitoxin incorporated in the overlay serves as a control for specificity. Forty-eight strains of C. diphtheriae were tested by the COT, guinea pig, and in vitro, gel diffusion tests. The COT is as specific as the other two tests, is easy to read, and can be used to test large numbers of isolates for toxin action more conveniently than by animal inoculation.

Animals↗
PubMed1975

Chemical modulation of diphtheria toxin action on cultured mammalian cells.

Ammonium chloride (4 times 10-3 M) rendered HEp-2 monolayers completely insensitive to the action of diphtheria toxin, as measured by de novo protein synthesis. Total protection was observed even with large amounts of toxin (400 minimum lethal doses/ml). Ammonium chloride did not reduce toxicity by direct action on the protein, nor did it prevent the adsorption of toxin to the cell membrane. Although the ammonium salt did not block the initial interaction between cell and toxin, it did maintain the toxin at a site amenable to neutralization with antitoxin. Surface-adsorbed toxin was inactivated by cellular enzymes or alternatively was desorbed from the membrane during a 12-h incubation in the presence of ammonium chloride. In addition, ammonium chloride provided protection to both toxin-sensitive guinea pig peritoneal macrophages and a partially toxin-resistant strain of HEp-2 cells. Sodium arsenite was effective in protecting cell monolayers from the action of diphtheria toxin; unlike ammonium chloride, its action was not dependent upon continued incubation with cells during exposure to toxin. Inhibitors of energy metabolism abolished toxin action either totally (sodium fluoride) or partially (dinitrophenol and sodium cyanide). Inhibitors of cellular proteases, on the other hand, did not modify toxin activity. The ability of several modifiers of membrane function to alter expression of toxicity for HEp-2 cells was also examined. One compound known to enhance endocytic activity, Tuftsin, had no effect, whereas poly-L-ornithine provided partial protection. Of the two compounds known to alter membrane fluidity, cytochalasin B provided partial protection for HEp-2 cell cultures, whereas colchicine had no effect. Agents that bind to sulfhydryl groups on the cell surface had no apparent effect on toxicity, suggesting that the initial toxin-cell interaction does not involve sulfhydryl groups. Those compounds that provide virtually full protection against the action of diphtheria toxic on cell monolayers (i.e., ammonium chloride, sodium fluoride, and sodium arsenite) had no inhibitory effect on the in vitro enzyme activity associated with fragment A of the toxin.

Ammonium Chloride↗
PubMed1985

Highly toxinogenic but avirulent Park-Williams 8 strain of Corynebacterium diphtheriae does not produce siderophore.

The highly toxinogenic Park-Williams 8 strain of Corynebacterium diphtheriae grows slowly in vitro and is avirulent. C. diphtheriae Park-Williams 8 is defective in iron uptake and does not produce the corynebacterial siderophore corynebactin. Addition of partially purified corynebactin stimulated iron uptake and growth of iron-deprived C. diphtheriae Park-Williams 8 cells.

Biological Transport, Active↗
PubMed1980

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.

Adenosine Diphosphate Ribose↗
PubMed1983

Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin.

Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and were degraded to nonimmunoreactive forms within 1 to 2 h of synthesis. The expression of both gene fragments appears to have originated from the diphtheria toxin promoter.

Bacteriophages↗
PubMed1992

DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

The structural gene encoding DtxR, an iron-dependent diphtheria tox regulatory element, has recently been cloned and sequenced from the C7(-) strain of Corynebacterium diphtheriae (J. M. Boyd, M. Oza, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 87:5972, 1990). We report here the molecular cloning, DNA sequence analysis, and characterization of DtxR from the PW8(-), 1030(-), and C7hm723 strains of C. diphtheriae. While the sequence of dtxR from PW8(-) is identical to that of the C7(-) allele, the sequence of dtxR from the 1030(-) strain is only 91.4% identical; however, the deduced amino acid sequence of DtxR from 1030(-) differs by only 6 of 678 amino acids. Moreover, DtxR from all three strains is shown to regulate expression of beta-galactosidase from a tox promoter-operator (toxPO)-lacZ transcriptional fusion. In contrast, the dtxR allele from the iron-insensitive tox constitutive mutant C7hm723 was found to have a single G----A transition, resulting in a substitution of Arg-47 to His and the loss of tox regulatory activity in recombinant Escherichia coli.

Alleles↗
PubMed1997

Development of a direct PCR assay for detection of the diphtheria toxin gene.

PCR has proved to be a reliable tool for the detection of the diphtheria toxin gene, tox, and its use has allowed for the rapid differentiation between toxigenic and nontoxigenic strains. In this study, this PCR was further developed, evaluated, and standardized to detect this gene directly from clinical specimens. Optimal conditions for collection, transport, and storage of the clinical specimens and isolation and purification of DNA from the clinical specimens were defined. With two sets of primers that detect the A and B subunits of the diphtheria toxin gene, sensitivity levels of 50 and 500 CFU/PCR mixture, respectively, were achieved. This PCR was evaluated with 162 clinical samples collected from patients with diphtheria and other upper respiratory tract infections, as well as from healthy individuals.

Corynebacterium diphtheriae↗
PubMed1981

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production.

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.

Bacteriophages↗
PubMed1981

Regulation of toxinogenesis in Corynebacterium diphtheriae. II. Genetic mapping of a tox regulatory mutation in bacteriophage beta.

The structural gene for diphtherial toxin is present in corynebacteriophage beta. Previous studies located several point mutations within the tox locus and determined the orientation of transcription of the toxin structural gene. The production of maximal quantities of toxin by Corynebacterium diphtheriae C7(beta) occurs only when the bacteria are iron deficient. Mutations in phage beta can affect this control of toxin production by iron. The tox-201 mutation regulates expression of the toxin structural gene in a cis-dominant manner and permits large amounts of toxin to be made under high-iron conditions when phage beta tox-201 infects C. diphtheriae C7. In this study tox-201 was found to be closely linked to the structural gene for toxin. We performed a series of multifactor matings to determine the relative positions of tox-201 and several point mutations within the toxin structural gene. The order of these markers on the vegetative genetic map of phage beta was tox-201-tox-4-tox-2-tox-30. These findings establish that the tox-201 regulatory site is closely linked to the end of the toxin structural gene corresponding to the origin of transcription. This location is consistent with our hypothesis that tox-201 defines a cis-dominant regulatory element, such as an operator, promoter, or attenuator, involved in control of toxinogenesis in C. diphtheriae C7(beta).

Bacteriophages↗
PubMed1985

Detection and physical map of a omega tox+-related defective prophage in Corynebacterium diphtheriae Belfanti 1030(-)tox-.

A library of chromosomal DNA from Corynebacterium diphtheriae Belfanti 1030(-)tox- was cloned in the lambda phage vector EMBL4 and screened for sequences homologous to corynephage omega tox+ and the attB1-attB2 region of the C7(-)tox- chromosome. Two portions of the 1030(-)tox- chromosome, 35 and 30.5 kilobases long which contain, respectively, the entire region homologous to corynephage omega tox+ and the attB1-attB2 sites, were mapped with the restriction endonucleases BamHI and EcoRI. Chromosomal DNA from 1030(-)tox- was shown to contain a 15.5-kilobase region that was homologous to ca. 42% of the corynephage omega tox+ genome. These sequences were found to hybridize to three regions of the phage genome and do not contain either the diphtheria tox operon or the attP site. These sequences are distant from the chromosomal region that contains the attB1-attB2 sites. Moreover, unlike other known defective prophages, the physical map of this prophage starts at the cos site and is colinear with the vegetative phage map. The 30.5-kilobase region of the 1030(-)tox- chromosome, which contains the attB1-attB2 sites, has a central core region that is almost identical to the corresponding region of the C7(-)tox- chromosome; however, the flanking sequences in these two strains of C. diphtheriae are different.

Bacteriophages↗
PubMed1987

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.

Amino Acid Sequence↗
PubMed2006

Evaluation of a diphtheria-tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b vaccine given concurrently with meningococcal group C conjugate vaccine at 2, 3 and 4 months of age.

BACKGROUND AND OBJECTIVE: In view of the possible introduction of diphtheria-tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b (DTaP-IPV-Hib, eg Pediacel) vaccine in the UK, a study of the immunogenicity of Pediacel when given with one of two different meningococcal group C conjugate (MCC) vaccines at 2, 3 and 4 months of age was conducted. METHODS: Randomised controlled study in 241 infants. RESULTS: Post vaccination, the proportion of infants with anti-polyribosylribitol phosphate (PRP) levels > or =0.15 microg/ml was 93.2% (95% confidence interval (CI) 86.6 to 96.7) in the Pediacel group compared with 100% (95% CI 96.4 to 100) in the diphtheria-tetanus-whole-cell pertussis-Haemophilus influenzae type b (DTwP-Hib) group. The anti-PRP response was lower in infants receiving either Pediacel or DTwP-Hib when these vaccines were given concomitantly with meningococcal group C conjugate with diphtheria-derived protein CRM(197) as conjugate protein (MCC-CRM) compared with meningococcal group C conjugate with tetanus toxoid as conjugate protein (MCC-TT). For group C meningococcus, the proportion of infants with serum bactericidal antibody (SBA) titre > or =1:8 in the Pediacel group was 99.0% compared with 100% in the DTwP-Hib group. The MCC SBA geometric mean titre (GMT) was lower in those receiving Pediacel with MCC-TT than in those receiving DTwP-Hib with MCC-TT, although all titres were well above the protective threshold. The MCC SBA GMT was similar in those receiving Pediacel and DTwP-Hib and MCC-CRM. Responses to all other vaccine components were equivalent in the two groups. CONCLUSIONS: Pediacel is immunogenic when given at 2, 3 and 4 months of age. Coadministration of MCC vaccine can influence the Hib response, and the MCC response to a tetanus conjugate can be influenced by the nature of the coadministered DTP-Hib vaccine.

Diphtheria-Tetanus-Pertussis Vaccine↗
PubMed1979

Immunity of children to diphtheria, tetanus, and poliomyelitis.

A survey of titres of diphtheria and tetanus antitoxins and of antibodies to polioviruses in the sera of 291 schoolchildren aged 15, 11, and 7 years showed that high immunisation rates can evoke protective concentrations of tetanus antitoxin in 98% of children and protective levels of the antibodies to diphtheria and all three types of poliomyelitis in 85% of children. Reinforcing immunisation at school entry appeared to be necessary to maintain adequate titres of diphtheria antitoxin in children up to 15 years of age, not essential to maintain adequate titres of tetanus antitoxin, and to have little effect on the titres of antibodies to poliomyelitis.

Adolescent↗
PubMed1987

Diphtheria presenting in the accident and emergency department.

A case of diphtheria encountered in the accident and emergency department is described. In addition, 38 cases of diphtheria have been analysed, and demonstrate that, although now rare in this country, the disease still exists and its clinical features remain unchanged. It presents almost as often in the accident and emergency department as it does to the general practitioner and remains overwhelmingly a disease of children. Diphtheria is still a notifiable disease. The diagnosis must be made clinically and appropriate action taken before the bacteriological confirmation is received. This prevents unnecessary delay in treatment and makes contact tracing less arduous.

Corynebacterium diphtheriae↗
PubMed1973

An outbreak of diphtheria in a hospital for the mentally subnormal.

An account is given of two separate outbreaks of diphtheria amongst mentally subnormal patients and nursing staff. In a total hospital population of about 1000 the number of people involved as carriers or cases was 60 and there were five deaths. The 60 people comprised 56 patients, of whom four were involved in both outbreaks, and four nurses. The organisms isolated were C. diphtheriae mitis but five strains were non-toxigenic. It is postulated that the outbreak began following the conversion of a non-toxigenic organism to a toxigenic one by bacteriophage action. The fatal cases were examples of membranous pharyngo-laryngo-tracheo-bronchial diphtheria with well marked pseudo-casts of the upper air passages.

Adult↗
PubMed1977

Radioimmunodiffusion technique for determining diphtheria antitoxin.

A radioimmunodiffusion technique for detecting low levels of diphtheria antitoxin was developed. Diphtheria toxoid was labelled with 125I to facilitate detection of lines of precipitation by the use of X-ray film, the lower limit of detection being 0.001 unit per millilitre of diphtheria antitoxin.

Diphtheria Antitoxin↗
PubMed1979

Protection of mammalian cells from diphtheria toxin by exogenous nucleotides.

Exogenous nucleotides were found to protect mammalian cells from the lethal effects of diphtheria toxin. Protective potency of a given nucleotide was base specific and phosphate chain length dependent. Full expression of protective potency required an intact nucleotide, but the effect did not appear to be mediated by nucleotide-induced phosphorylation. Nucleotides antagonized the binding of diphtheria toxin to its cell surface receptor in a manner that correlated with the degree of protection. It was concluded that cellular protection from diphtheria toxin by nucleotides results from inhibition of toxin-receptor binding and that nucleotides therefore may serve as valuable research tools for future studies.

Adenine Nucleotides↗