The Production of Diphtheria Toxin

Park, W. H.; Williams, Anna W.

doi:10.1084/jem.1.1.164

Cited by 59 publications

(28 citation statements)

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“…The C7(Ϫ) and PW8 strains of C. diphtheriae are two of the oldest strains available to researchers and are the main standard strains utilized in analysis of C. diphtheriae pathogenesis and vaccine production (10,20,32,46,(48)(49)(50)(51). The C7(Ϫ) strain, which can infect humans (3), was considered to lack most PAIs but still showed signs of pathogenicity, whereas the nonpathogenic (32) PW8 strain retained more PAIs but showed much reduced signs.…”

Section: Discussionmentioning

confidence: 99%

“…8 (PW8), originally isolated from a very mild diphtheria case during the 1890s (46), has been widely used for toxoid vaccine production because of its great ability to secrete diphtheria toxin into the culture supernatant (46). As PW8 is effectively the only strain employed for vaccine production, its importance in public health and the vaccine industry is incomparable.…”

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confidence: 99%

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Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains

Iwaki¹,

Komiya²,

Yamamoto³

et al. 2010

Infect Immun

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Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(؊) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(؊). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(؊) genome. Despite this, the C7(؊) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains

Iwaki¹,

Komiya²,

Yamamoto³

et al. 2010

Infect Immun

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“…This difference suggests considerable evolutionary divergence-convergence between C. diphtheriae (belfanti) 1030(-) and the C7(-) and PW8(-) strains of C. diphtheriae. It should be noted that more than 50 years had passed between the isolation of the PW8 strain in New York and that of the C7(-) strain from a patient in California (3,20). Nonetheless, the nucleic acid sequences of their respective diphtheria tox alleles carried by corynebacteriophage X and have been shown to be identical (9,23).…”

Section: Discussionmentioning

confidence: 99%

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

1992

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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 87:5968-5972, 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 1-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.We have recently described the use of a diphtheria tox promoter-operator (toxPO)-lacZ transcriptional fusion in the molecular cloning of an iron-dependent tox regulatory element (dtxR) from Corynebactenum diphtheriae (4). DtxR is a 25,316-Da protein which was shown to be an iron-dependent negative control element in the expression of 3-galactosidase from the toxPO-lacZ fusion in recombinant Escherichia coli. This study was recently confirmed and extended by Schmitt and Holmes (25), who demonstrated that dtxR controlled the iron-dependent expression of tox and the siderophore genes in C. diphtheriae.Different strains of C. diphtheriae which are lysogenic for tox+ corynebacteriophage may under optimal conditions produce different yields of diphtheria toxin. The final yield of toxin produced may be influenced by the ability of a given strain to continue growth following exhaustion of iron from the medium (18) and the number of integrated tox+ corynebacteriophage genomes (22). Regardless of the final yield of toxin, the expression of the tox structural gene in C. diphtheriae is repressed by the addition of iron to the culture medium.In contrast, the expression of diphtheria toxin from the C7hm723(,tox+) strain of C. diphtheriae is insensitive to iron-mediated repression (11). The mutant C7hm723 strain was isolated after nitrosoguanidine mutagenesis and was postulated to carry a defect in the diphtheria tox regulatory gene. In addition, Cryz et al. (6) were able to demonstrate that, relative to the parental C7 strain, C7hm723 also had a decreased rate of iron uptake. In order to gain additional insight into the regulation of diphtheria tox expression in both wild-type and mutant strains of C. diphtheriae, the dtxR alleles of the PW8(-), 1030(-), and C7hm723(-) strains were cloned and sequenced and the tox regulatory activity of * Corresponding author. t Present address:

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“…While detected both high-affinity (KD -30 pM) and low-affinity (KD -1 nM) receptors on murine haemopoeitic cells, Park et al (1986a) detected only a single receptor class of KD = 1-3 nM. By contrast, only high affinity receptors have been described on human haemopoietic and endothelial cells (KD = 30 pM) (Gasson et al, 1986;Bussolino et al, 1989;Park et al, 1986b) although a receptor of lower affinity has been described on monkey COS cells (Cocita Baldwin et al, 1989). The basis for this variable expression of high and low affinity GM-CSF receptors is at present unknown.…”

Section: Introductionmentioning

confidence: 99%

“…The (Gasson et al, 1986;Park et al, 1986b;Kelleher et al, 1988). However, we have consistently detected both high and low affinity GM-CSF receptors on human bone marrow cells, primary human myeloid leukaemic cells and the human promyelocytic leukaemic cell line, HL-60 (Figure 1 and N.A.…”

Section: Introductionmentioning

confidence: 99%

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor

1989

Cytokine

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Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor (hGM-CSF-R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach. The cloned GM-CSF-R precursor is a 400 amino acid polypeptide (Mr 45 000) with a single transmembrane domain, a glycosylated extraceliular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin-6, erythropoietin and interleukin-2 ( (-chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity (KD = 2-8 nM) and specificity for human GM-CSF but not interleukin-3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding, and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.

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The Production of Diphtheria Toxin

Cited by 59 publications

References 0 publications

Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains

Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains

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

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor

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