Production Of Diphtheria Toxin Research Articles

Abstract 649: Skeletal Muscle Satellite Cells Play a Role in Regulating Angiogenesis and Arteriogenesis

Satellite cells are myogenic stem cells that play a critical role in skeletal muscle repair by proliferating and differentiating into myoblasts to repair muscle fibers. However, their role in reestablishing vascular supply following injury is not well defined. We hypothesized that satellite cells promote vascular growth through paracrine signaling induced following muscle injury or ischemic damage from diseases such as peripheral artery disease. We used a murine model of hind limb ischemia and found that satellite cells increased 3.4 fold (p<0.01) in response to this ischemic insult. We used a co-culture system to determine that satellite cells led to a 3.5 fold and 2.8 fold increase in smooth muscle and endothelial cell migration (p<0.0001). These results demonstrate the satellite cells produce paracrine factors which drive cell migration required for both angiogenesis and arteriogenesis. To test the potential therapeutic capability, alginate encapsulated satellite cells were delivered in the hind limb ischemic model. We found the encapsulated cells were viable for up to 2 weeks and mice that received satellite cells had significantly increased perfusion (28%, p<0.05) at 2 weeks as measured by Laser Doppler imaging and a 1.9 fold (p<0.5) increase in capillaries and small vessels measured by histological staining. To examine the role of satellite cells in a physiological setting and determine if they are critical to robust recovery in our model, we used a Cre-Lox system in which recombination results in the production of diphtheria toxin a (DTA) to deplete Pax7 specific cells. Mice which lacked satellite cells had decreased perfusion by Laser Doppler imaging (p<0.05) and capillary density compared animals with intact satellite cells. A cytokine array and genomics analysis show that several factors related to angiogenesis and cell migration are upregulated in satellite cells in response to ischemia. In conclusion, we have found that satellite cells proliferate in response to ischemia, produce factors that drive cell migration, and their delivery or depletion affect vascular growth in vitro. We believe that our studies show that satellite cells play an important role vascular growth and are a novel potential therapy for peripheral artery disease.

The association between tuberculosis and diphtheria.

This research investigates the long-forgotten relationship between diphtheria and tuberculosis. Historical medical reports from the late 19th century are reviewed followed by a statistical regression analysis of the relationship between the two diseases in the early 20th century. Historical medical reports show a consistent association between diphtheria and tuberculosis that can increase the likelihood and severity of either disease in a co-infection. The statistical analysis uses historical weekly public health data on reported cases in five American cities over a period of several years, finding a modest but statistically significant relationship between the two diseases. No current medical theory explains the association between diphtheria and tuberculosis. Alternative explanations are explored with a focus on how the diseases assimilate iron. In a co-infection, the effectiveness of tuberculosis at assimilating extracellular iron may lead to increased production of diphtheria toxin, worsening that disease, which may, in turn, exacerbate tuberculosis. Iron-dependent repressor genes connect both diseases.

The Association Between Tuberculosis and Diphtheria

This research investigates the now-forgotten relationship between diphtheria and tuberculosis. Historical medical reports from the 19th century is reviewed followed by a statistical regression analysis of the relationship between the two diseases in the early 20th century. Historical medical records show a consistent association between diphtheria and tuberculosis that can increase the likelihood and severity of either disease in a coinfection. The statistical analysis uses historical weekly public health data on reported cases in five American cities over a period of several years, finding a modest but statistically significant relationship between the two diseases. No current medical theory explains the association between diphtheria and tuberculosis. Alternative explanations are explored with a focus on how the diseases assimilate iron. In a co-infection, the effectiveness of tuberculosis at assimilating extracellular iron can lead to increased production of diphtheria toxin, worsening that disease, which may in turn exacerbate tuberculosis. Iron-dependent repressor genes connect both diseases.

Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8

In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174–239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

PRODUCTION OF RECOMBINANT PROTEIN CRM197 IN ESCHERICHIA COLI

The CRM197 is a non-toxic mutant of diphtheria toxin having a single amino acid substitution of a glycine for a glutamic acid in position 52. Being naturally nontoxic, CRM197 is a promising adjuvant and ideal carrier protein for conjugate vaccines. Typically, production of diphtheria toxin and some of the non-toxic proteins are carried out by Corynebacterium diphtheriae. Production of recombinant protein CRM197 in Escherichia coli is advantageous. It is simple, cheap and permits production of the target protein in a short time using a non-pathogenic microorganism. In this study patented high-yield-producing E. coli strain was used. As a part of the study the following steps were taken: protocol adjustment for induction of crm197 gene, production and purification of recombinant CRM197 by ion-exchange, hydrophobic and gel-filtration chromatography. The purity of the final preparation reached 97%.

Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine

Background: Diphtheria is a highly communicable disease caused by toxin-producing strains of Corynebacterium diphtheriae. Objectives: To evaluate the efficacy of A and B subunits of diphtheria toxin (DT-A, DT-B) as potential vaccines against C. diphtheriae. A culture of C. diphtheriae (strain PW 8) was grown on Loeffler plates while Lingood medium was used for production of diphtheria toxin (DT). Materials and Methods: DT was purified and digested to obtain pure DT-A and DT-B and detoxified to obtain diphtheria toxin. Four groups of mice were immunised with different antigens (Ag) of C. diphtheriae. Results: The antibody (Ab) titres were significantly increased with immunised groups subsequent to three injections. On the other hand, Ab titres were estimated after the three immunisations and the levels of different Ab isotypes were comparatively measured. The levels of various isotypes immune responses showed variation between immunised groups where the IgG subclasses were significantly increased mainly with DPT immunised group. The IgM and IgA were significantly increased with DT-A more than others. Additionally, the evaluation of the cellular immune responses demonstrated that spleen cells from DPT and DT-A groups gave highly significant proliferative response with production of high levels of IL-2 and IFN-γ (Th1/Th2). Separation and purification of DT gene were performed using polymerase chain reaction (PCR) and sub-cloned in pGEM-T vector, for further studying of recombinant vaccine. Conclusion: Our results showed the possibility to prepare a potent recombinant vaccine containing whole DT gene or DT-A against C. diphtheriae or could be used in treatment of cancer as it give high levels of IL-2 and IFN-γ.

Corynebacterium ulcerans from Diseased Wild Boars

Two Corynebacterium strains were isolated from lymph nodes of wild boars showing severe alterations caused by caseous lymphadenitis. The wild boars came from different districts in southern Germany; one was found dead, the other had been shot. The two Corynebacterium strains obtained were both positive for phospholipase D. Further analysis of biochemical profiles did not allow unambiguous differentiation between Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Fourier-transformed infrared spectroscopy as well as partial sequencing of the genes for 16S rRNA and RNA polymerase beta subunit (rpoB) clearly identified both strains as Corynebacterium ulcerans. The tox gene for diphtheria toxin (DT) could be detected in both porcine isolates by PCR. Partial DNA sequencing of this tox gene showed significant differences from sequences described for other Corynebacterium ulcerans strains and a higher degree of similarity to that of Corynebacterium diphtheria. Production of diphtheria toxin could not be detected. These results indicate that wild game could be a reservoir for zoonotic Corynebacterium ulcerans.

PERSPECTIVES: Tethered-toxin debut gets cold reception

Since the discovery of the functional diversity of Na+ currents in primary sensory neurones, the physiological reasons for their heterogeneity have been the subject of investigation and speculation. This is certainly true for the tetrodotoxin-resistant and kinetically slow NaV1.8 current expressed in small diameter nociceptive neurones. The normal expression pattern is confined to neurones in sensory ganglia, and this channel has been the subject of scrutiny over many years as a possible novel pharmacological target for the treatment of pain. Because of its relatively depolarized activation voltage dependence, one possible functional attribute is that the channel may continue to function in small diameter afferents when depolarized, for example in the face of raised external K+. This possibility was recognized by Jim Elliott and colleagues many years ago, but it is in the study of cold reception that this plausibility has become something close to a certainty. In the presence of large and long-lasting receptor potentials, when other Na+ channel subtypes should be substantially inactivated, NaV1.8 still functions and endows excitability to corneal cold receptors (e.g. Carr et al. 2002). The knock-out of NaV1.8 has recently been shown to suppress nociception under cold conditions (Zimmermann et al. 2007), and also to inhibit menthol-induced enhanced cold responses (Zimmermann et al. 2007). A widely held view concerning gene knock-outs is that the functional consequences can be modified or diluted by alterations in the expression of other genes, and this argument was put forward at the time in order to explain the phenotype of the NaV1.8 knock-out mouse, where the (apparently) fairly mild phenotypic changes accompanying channel loss were in part explained by a compensatory up-regulation of NaV1.7. What has been described now in the paper by Sturzebecher et al. (2010) in this issue of The Journal of Physiology is a cell-autonomous tethered-toxin approach for the investigation of NaV1.8 function, where the expression of NaV1.8 is expected to be normal, but where channel function is suppressed (although not abolished) by the provision of tethered conotoxin MrVIa (Daly et al. 2004) on the outside of the nerve membrane. The utilized transgene encoding the toxin included genomic DNA incorporating modified SCN10a, resulting in expression of the toxin in sensory neurones when the SCN10a promoter is activated. The toxin was engineered as the most distal part of a glycophosphatidylinositol anchored extracellular protein, and the evidence presented indicates that in practice this fusion protein is sufficiently flexible to allow the tethered-toxin to exhibit its pharmacological action and block NaV1.8. The partial toxin block of NaV1.8 brings about a reduction in noxious cold sensitivity and a substantially reduced behavioural response to noxious cold (0°C; Fig. 4C in Sturzebecher et al. 2010; highlighted in the authors’ Supplemental movie, available online). These findings are consistent with the abolition of behavioural responses to cold following the ablation of neurochemically distinguished isolectin-IB4+ve neurones, by endogenous production of diphtheria toxin controlled by the SCN10a promotor (Abrahamsen et al. 2008). A contribution made by Snape et al. (2010) was to show that IB4+ve, small diameter sensory neurones, well known to be endowed with more functional NaV1.8 than similarly sized IB4–ve neurones, retain their excitability with applied depolarization approaching –20 mV, potentials at which IB4–ve neurones were rendered inexcitable. The following conclusions seem almost inescapable. Firstly, nociceptive nerve endings in the cold may be undergoing a resting depolarization to some degree, by loss of K+ channels as suggested by Gordon Reid (Reid & Flonta 2001) (whether or not they contain e.g. TRPM8 menthol receptors or other cold-gated channels), or tetrodotoxin-sensitive Na+ channels become functionally unavailable because of the effect of cold on gating (Zimmermann et al. 2007). Secondly, NaV1.8 allows sensory encoding to occur in depolarized endings in the cold (Carr et al. 2002; Zimmermann et al. 2007), and finally, NaV1.8 is the likely channel to encode cold (Carr et al. 2002; Zimmermann et al. 2007; Sturzebecher et al. 2010). It is our expectation that NaV1.8 will play other additional special roles that have yet to be clearly defined.

Development of a biological test to evaluate the bioavailability of iron in culture media

To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium. In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress. The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism. The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.

Ablation of developing podocytes disrupts cellular interactions and nephrogenesis both inside and outside the glomerulus

Podocyte loss in adults leads to glomerulosclerosis. However, the impact of podocyte loss on glomerulogenesis and the development of the kidney as a whole has not been directly studied. Here, we used a podocyte-specific Cre transgene to direct the production of diphtheria toxin (DTA) inside podocytes during nephrogenesis. Affected podocytes underwent translational arrest and apoptosis, leading to oliguria, proteinuria, hematuria, interstitial hemorrhage, and perinatal death. Glomerular cell-cell interactions were disrupted, even before overt podocyte apoptosis. VEGF production by podocytes was greatly decreased, and this was associated with reduced endothelial fenestration and altered glomerular vascular architecture. In addition to these glomerular anomalies, embryonic podocyte ablation also led to structural changes and increased apoptosis in proximal tubules. The collecting ducts, however, only showed molecular changes that are likely an indirect effect of the greatly reduced urine flow. Although podocyte loss significantly impacted the development and maintenance of the vasculature both inside and outside the glomerulus, our results suggest that there is a lack of long-range signaling from deep-seated, mature glomeruli to the differentiating cells in the outer nephrogenic zone. This study illustrates the tight integration of various cell types in the developing kidney and shows that the impact of podocyte loss during development is much greater than that in adults. This study also shows the specificity and effectiveness of a genetically controlled podocyte ablation system in mice where the additional readily available tools can further expand its applications.

Bacteriophage control of bacterial virulence.

In 1930, Felix d’Herelle wrote “. . .the actions and reactions are not solely between these two beings, man and bacterium, for the bacteriophage also intervenes; a third living being and, hence, a third variable is introduced” (19). The contribution of bacteriophages to the pathogenicity of their bacterial hosts began to be uncovered as early as 1927, when Frobisher and Brown discovered that nontoxigenic streptococci exposed to filtered supernatants of toxigenic streptococcal cultures acquired the ability to produce scarlatinal toxin (28). We now know that these supernatants contained a bacteriophage encoding the scarlatinal toxin and that these investigators were describing transduction, i.e., the transfer of genetic material to a bacterial cell via phage infection. Although these early investigators lacked a mechanistic explanation for their observations, they postulated that the bacterium was “within certain limits, perhaps a matter of secondary importance and that toxicogenicity might be a property that could be acquired by different types of organisms.” (28). Their hypothesis that bacteria acquire virulence properties has since gained widespread acceptance, as many virulence genes have been shown to undergo transfer among bacteria by phages (via transduction) and other mobile genetic elements such as plasmids (via conjugation). Over time, a number of toxin genes were found to be phage encoded, and consideration of the role of phages in bacterial pathogenesis emphasized the dissemination of toxin genes among bacterial strains (10). However, it has become increasingly clear that toxin genes are only a subset of the diverse virulence factors encoded by bacteriophages. For example, some phages encode regulatory factors that increase expression of virulence genes not encoded by the phage (84), while others encode enzymes that alter bacterial components related to virulence (31, 58). Furthermore, phages have unique properties that enable them to contribute more directly to bacterial virulence than via transduction. Structural components of virion particles, for example, may be directly pathogenic (5, 6, 99). Additionally, phage-encoded genes frequently undergo replication and transcriptional activation following prophage induction, a process that was speculated to have a role in the production of diphtheria toxin by Corynebacterium diphtheriae as early as 1960 (3). Since d’Herelle’s time, his notion of the phage as a third variable in bacterial pathogenesis has proven correct. However, while d’Herelle emphasized the diminution of bacterial virulence by bacteriophages (19), we have instead come to learn that phages serve as a driving force in bacterial pathogenesis, acting not only in the evolution of bacterial pathogens through gene transfer, but also contributing directly to bacterial pathogenesis at the time of infection. This review provides a discussion of (i) the discovery of phage-encoded virulence factors, (ii) bacterial virulence properties altered by phages, (iii) regulation of phage-encoded virulence factors, and (iv) the role of in situ prophage induction in the control of bacterial virulence.

Process optimization for enhanced production of diphtheria toxin by submerged cultivation

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7–8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.

Process Optimization for Enhanced Production of Diphtheria Toxin by Submerged Cultivation.

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7-8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.

SirR, a novel iron-dependent repressor in Staphylococcus epidermidis.

In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of the sitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within the sitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start of sitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus, S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent metal cation-dependent transcriptional repressor which is widespread among the staphylococci.

Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin.

To assess the performance of the polymerase chain reaction (PCR) when used to screen rapidly large numbers of corynebacteria for toxin production; and to determine the incidence of false positive PCR results with non-toxigenic Corynebacterium diphtheriae isolates. Eighty seven recent British isolates of corynebacteria were assayed by PCR. All isolates were assayed from both blood and tellurite agar within a five day period. Thirty three non-toxigenic isolates of C diphtheriae from six countries were also tested by PCR and by the Elek immunodiffusion assay. There was complete concordance between the results of PCR and traditional methods on the recent British isolates, with one exception: an Elek positive "C ulcerans" isolate, which was PCR positive from tellurite but not from blood agar. One of the thirty three (3%) non-toxigenic isolates of C diphtheriae was PCR positive. These results suggest that PCR compares favourably with traditional methods for the detection of toxigenic corynebacteria and that it represents a powerful new tool in the diagnosis of an old disease.

Transcription analysis and nucleotide sequence of tox promoter/operator mutants of corynebacteriophage β

The production of diphtheria toxin (DT) by Corynebacterium diphtheriae C7(β) is transcriptionally regulated by the iron-dependent diphtheria toxin repressor, DtxR. Transcription of the tox gene was studied in wild-type C. diphtheriae C7(β) and in lysogens carrying mutants of β that determine insensitivity to inhibition of DT production by iron. Under low iron conditions in all strains, tox-specific mRNA appeared and DT production began during late-log phase, and they increased to maximal levels at stationary phase. Under high iron conditions, tox-specific mRNA and DT production were strongly repressed in C7(β) but only partially repressed in C7( β tox-202 ) and C7( β tox-201 ). Under high and low iron conditions, DT production and tox-specific mRNA levels were greater in C7( β tox-201 ) and C7( β tox-202 ) than in wild-type C7(β). Addition of iron or rifampicin to low iron cultures of C. diphtheriae C7(β) repressed tox-mRNA production promptly and with a similar time course. In contrast, repression of tox-mRNA synthesis in C. diphtheriae C7( β tox-201 ) occurred promptly after addition of rifampicin but more slowly after addition of iron. Nucleotide sequence analysis revealed single G to A mutations at positions −47 and −48, within the preferred ‘−10’ sequence of the tox promoter, in β tox-201 and β tox -202, respectively. The single nucleotide substitutions in the tox-201 and tox-202 regulatory alleles, therefore, have pleiotropic effects, causing increased activity of the promoter and partial resistance of the operator to iron-dependent repression.

Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae

The production of diphtheria toxin and siderophore by the Corynebacterium diphtheriae regulatory mutant C7(beta)hm723 is resistant to the inhibitory effects of iron, and the mutant strain is defective for function of the regulatory gene dtxR. A 2.8-kb HindIII fragment carrying the C7(beta)hm723 dtxR allele was cloned and characterized in Escherichia coli. The restriction endonuclease maps of the 2.8-kb HindIII fragment from C7(beta)hm723 and the corresponding fragment from wild-type C. diphtheriae C7 were identical. RNA dot blot analysis with total RNA isolated from wild-type C. diphtheriae C7 and C7(beta)hm723 indicated that the dtxR gene was transcribed at very low but equivalent levels in both strains and was not regulated by iron. beta-Galactosidase synthesis from a tox-lacZ translational fusion construct in E. coli in high-iron medium was not repressed by the C7(beta)hm723dtxR allele, but was strongly repressed by the wild-type dtxR gene. The 28- to 29-kDa polypeptide expressed from the mutant dtxR allele in E. coli had the same electrophoretic mobility as the wild-type dtxR gene product in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nucleotide sequence of the coding region and the 5' upstream region of the C7(beta)hm723 dtxR allele was determined and compared with the wild-type nucleotide sequence. The dtxR allele from C7(beta)hm723 contained a single-base change located 140 nucleotides from the 5' start of the gene, which resulted in replacement of arginine in the wild-type sequence by histidine in the mutant protein. These data demonstrate that C7(beta)hm723 expresses a mutant DtxR repressor protein that is severely defective in repressor activity.

Production of diphtheria toxin by selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis.

We determined the diphtheria toxin phenotype of specially selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (C. ovis). All produced proteins similar in size and immunological structure to diphtheria toxin. As with diphtheria toxin, they exhibited ADP-ribosylating activity, and their synthesis was regulated by iron.

Effect of metal ions on diphtheria toxin production.

The effect of several metal ions on the production of diphtheria toxin was tested. By using the gel immunodiffusion system for detecting toxin, a wide range of metal ion concentrations was conveniently surveyed. Five divalent cations, Fe2+, Cu2+, Co2+, Ni2+, and Mn2+ inhibited toxin production within a range of concentrations that did not inhibit growth of the producing strain. Growth and toxin production were inhibited at identical concentrations by both Cd2+ and Zn2+, whereas Al3+ and Sr2+ affected neither growth nor toxin production over the range of concentrations tested. The data showed that Fe2+ was the most effective inhibitor on an equivalence basis, followed by Cu2+, Co2+, and Ni2+ in descending order. All eight strains of Corynebacterium diphtheriae chosen from diverse ecological origins responded similarly to all metals at similar concentrations. A mutant strain which produces toxin at Fe2+ concentrations 500 times greater than are inhibitory for the parent strain had simultaneously acquired resistance to inhibitory concentrations of Cu2+, Co2+, Ni2+, and Mn2+. This suggests that there is at least one common point in the activity of all these metal ions, and that toxin may respond broadly to changes in metal ion concentrations in the environment.

Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.

It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.