Mutation in the Structural Gene for Diphtheria Toxin carried by Temperate Phage β

Uchida, Tsuyoshi; Dm, Gill; Am, Pappenheimer

doi:10.1038/newbio233008a0

Cited by 194 publications

(124 citation statements)

References 20 publications

Supporting

Mentioning

120

Contrasting

Unclassified

Order By: Relevance

“…The only eukaryotic protein known to be modified by these two toxins is EF-2, and all existing information supports the conclusion that the ADP-ribosylation of EF-2 is responsible for the lethality of these two toxins (2,4). Diphtheria toxin is encoded by a phage gene (2,5), but the location of the structural gene for Pseudononas toxin A is unknown. Approximately 90% of all isolates of Pseudomonas aeruginosa tested produce toxin A (6,7).…”

mentioning

confidence: 99%

Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.

Iglewski¹,

Sadoff²,

Bjorn³

et al. 1978

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

200

149

View full text Add to dashboard Cite

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiola led NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14CJNAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADPribose). Exoenzyme S does not catalyze the transfer of ADPribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor 1. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.Diphtheria toxin and Pseudomonas toxin A inhibit protein synthesis in eukaryotic cells by catalyzing the transfer of the ADP-ribose (ADP-Rib) moiety of NAD+ to elongation factor 2 (EF-2) (1-3). The only eukaryotic protein known to be modified by these two toxins is EF-2, and all existing information supports the conclusion that the ADP-ribosylation of EF-2 is responsible for the lethality of these two toxins (2, 4). Diphtheria toxin is encoded by a phage gene (2, 5), but the location of the structural gene for Pseudononas toxin A is unknown. Approximately 90% of all isolates of Pseudomonas aeruginosa tested produce toxin A (6, 7).In this report we describe an ADP-ribosyltransferase (exoenzyme S) that is present in the culture supernatant fluid of a strain of P. aeruginosa (Ps 388). Ps 388 was consistently negative in an immune precipitation assay (7) using specific toxin A antibody. In the presence of limiting amounts of EF-2, exoenzyme S catalyzed the transfer of far more ADP-Rib from NAD+ than could be accounted for by the production of ADP-ribosylated EF-2. Data are presented to show that exoenzyme S, unlike diphtheria or Pseudomonas A toxins, does not modify EF-2 but some other eukaryotic protein(s). ADP-Rib appears to be present in the modified proteins(s) as monomeric units rather than as poly(ADP-Rib). We also show that exoenzyme S is distinct from Pseudomonas toxin A by several other tests. It is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. (Sigma Chem. Co.). A 25-ml amount of this medium in a 500-ml erlenmeyer flask was inoculated with an overnight culture of Ps 388 to an initial cell density of approximately 5 X 107 cells per ml. The culture was incubated at 320 on a reciprocating shaker (200 linear excursions/min) (Lab.-Line Inst.) for 22 hr. The culture supernatant fluid was obtained by cen...

show abstract

mentioning

confidence: 99%

Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.

Iglewski¹,

Sadoff²,

Bjorn³

et al. 1978

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

200

149

View full text Add to dashboard Cite

show abstract

“…However, other than its proteinaceous nature almost nothing was known of the chemistry of diphtheria toxin or its mode of action. The interpretation of many of Loeffler's astute early observations and questions that puzzled him remained obscure and unanswerable until the discovery of lysogenic conversion to toxinogenicity by Freeman (1951) and the realization that the tox structural gene was carried by a bacteriophage (Uchida, Gill & Pappenheimer, 1971).…”

Section: Introductionmentioning

confidence: 99%

The diphtheria bacillus and its toxin: a model system

Pappenheimer

1984

J. Hyg.

Self Cite

View full text Add to dashboard Cite

Friedrich Loeffler's classical paper entitled ‘Untersuchungen über die Bedeutung der Mikroorganismen für die Entstehung der Diphtheric beim Menschen, bei der Taube und der Kalbe’ (Studies on the significance of bacteria in causing diphtheria in man, pigeons and calves), was published in 1884. In this paper and those which followed during the next few years, Loeffler described the diphtheria bacillus and its isolation in pure culture, and proved its relationship to the disease diphtheria. While the credit for the discovery of diphtheria toxin must go to Roux & Yersin (1888), Loeffler clearly predicted its existence in his original paper. Because, at autopsy, living ‘virulent’ diphtheria bacilli could only be recovered from experimental animals at the site of injection, Loeffler postulated that the bacteria must have released into the bloodstream a ‘chemical poison’ which caused the characteristicsterilehaemorrhagic lesions in remote organs. He even noted early in 1888 that ‘the bacterial poison resembles in its action the poison [now known as abrin] obtained from jaquiriti seeds, which causes inflammation and the production of false membranes when placed on the mucous membranes either of men or animals’ (cited in Loeffler, 1908). Today we know the reason for this astute observation. Both abrin and diphtheria toxin block protein synthesis in sensitive eucaryotic cells (Collier, 1975; Olsnes & Pihl, 1976; Pappenheimer, 1977), albeit by different mechanisms. Almost a quarter of a century after the discovery of the diphtheria bacillus, the British Medical Research Council published a 718-page volume on diphtheria. In the first chapter Loeffler (1908) reviewed the history of the disease and reminisced at some length about his own early work. By 1908 when this book appeared, a good deal was already known about the epidemiology of diphtheria, the bacteriology of the diphtheria bacillus, its mode of spread by healthy adults and the protective effect of antitoxin. However, other than its proteinaceous nature almost nothing was known of the chemistry of diphtheria toxin or its mode of action. The interpretation of many of Loeffler's astute early observations and questions that puzzled him remained obscure and unanswerable until the discovery of lysogenic conversion to toxinogenicity by Freeman (1951) and the realization that thetoxstructural gene was carried by a bacteriophage (Uchida, Gill & Pappenhcimer, 1971).

show abstract

“…Many of the initially identified structure-function relationships of the toxin were revealed by analysis of mutants produced by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (29). Toxins with altered function but with cross-reactivity against polyclonal anti-DT antibodies were generated.…”

mentioning

confidence: 99%

“…Toxins with altered function but with cross-reactivity against polyclonal anti-DT antibodies were generated. These cross-reacting materials (CRMs) generally fall into one of two groups: (a) those which are deficient in binding and/or translocation function (CRMs 30, 45, 228 [30,31], 107, 102, 103 [11,20], 9 [13], and 1001 [7]) and (b) those with altered enzymatic activity (CRM 197,228,and 176 [24,29,30,31]). CRMs have not only provided useful information concerning the structurefunction relationships of the toxin but have also been used for a variety of purposes including immunotoxin construction (CRM 45 [6], 107, 103, 102 [11,16,17], and 9 [22]) and genetic ablation studies (CRM 176 [3]), and as protein carriers for carbohydrate antigens (CRM 197 [1]).…”

mentioning

confidence: 99%

“…Toxins with altered function but with cross-reactivity against polyclonal anti-DT antibodies were generated. These cross-reacting materials (CRMs) generally fall into one of two groups: (a) those which are deficient in binding and/or translocation function (CRMs 30, 45, 228 [30,31], 107, 102, 103 [11,20], 9 [13], and 1001 [7]) and (b) those with altered enzymatic activity (CRM 197, 228, and 176 [24,29,30,31] E162K) and three within DTB (S197G, P378S, and G431S) (18). It has 15 to 20% of the binding activity of native DT and has no ADPRT activity (30, 31).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Identification of a single amino acid substitution in the diphtheria toxin A chain of CRM 228 responsible for the loss of enzymatic activity

Johnson

Nicholls

1994

J Bacteriol

View full text Add to dashboard Cite

CRM 228 (T. Uchida, A. M. Pappenheimer, and R. Greany, J. Biol. Chem. 248:3838-3844, 1973), a mutant form of diphtheria toxin which completely lacks ADP-ribosyltransferase activity, contains five amino acid substitutions. The two amino acid changes that fall within the A chain of the toxin (G79D and E162K) were separately analyzed by substituting a variety of other amino acids at these sites. The substitution at position 79 (G79D) singularly appears to account for the loss of enzymatic activity found in CRM 228.Diphtheria toxin (DT; Mr, 58,342) is secreted by pathogenic strains of Corynebacterium diphtheriae. It is composed of an A and a B subunit; the A subunit (DTA) has ADP-ribosyltransferase (ADPRT) activity, catalyzing the transfer of ADP-ribose from NAD+ to a posttranslationally modified histidine residue, diphthamide, found in elongation factor 2 (12). This results in the inhibition of protein synthesis and ultimately in the death of the intoxicated cell. The toxin B subunit (DTB) is responsible for binding to cell surface receptors and for mediating the translocation of the A subunit through the endosomal membrane into the cytosol.Many of the initially identified structure-function relationships of the toxin were revealed by analysis of mutants produced by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (29). Toxins with altered function but with cross-reactivity against polyclonal anti-DT antibodies were generated. These cross-reacting materials (CRMs) generally fall into one of two groups: (a) those which are deficient in binding and/or translocation function (CRMs 30, 45, 228 [30,31], 107, 102, 103 [11,20], 9 [13], and 1001 [7]) and (b) those with altered enzymatic activity (CRM 197, 228, and 176 [24,29,30,31] E162K) and three within DTB (S197G, P378S, and G431S) (18). It has 15 to 20% of the binding activity of native DT and has no ADPRT activity (30, 31). It was not known whether the lack of ADPRT activity can be attributed to just one or to both of the DTA substitutions. In an attempt to clarify this, the two affected DTA chain sites from CRM 228 (positions 79 and 162) were independently mutagenized; the native amino acid at each of these sites was substituted with a variety of other amino acids, and the relative enzymatic activities of the mutant proteins were compared.Mutagenesis at position 79 or 162 was carried out with a PCR-based mutagenesis system as previously described (14). Random nucleotide substitutions at positions 235 to 237 (encoding amino acid 79) or at positions 484 to 486 (encoding amino acid 162) enabled a variety of amino acids to be substituted at these sites (Fig. 1). PCR products were cloned into the Bluescript KS+ vector in a region downstream of the T7 RNA polymerase promoter, and Escherichia coli XL1 Blue was transformed with the recombinant library. The cloned region of the toxin gene encodes amino acids 1 to 193 corresponding to the enzymatically active form of DTA (32), without DTB, so the construct is in compliance with the restrictions for cloning toxin genes (8).P...

show abstract

Mutation in the Structural Gene for Diphtheria Toxin carried by Temperate Phage β

Cited by 194 publications

References 20 publications

Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.

Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.

The diphtheria bacillus and its toxin: a model system

Identification of a single amino acid substitution in the diphtheria toxin A chain of CRM 228 responsible for the loss of enzymatic activity

Contact Info

Product

Resources

About