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Tag words: diphtheria, Corynebacterium diphtheriae, C. diphtheriae, diphtheria bacteria, pseudomembrane, diphtheria toxin, dtx, Beta phage, Theobald Smith, Freeman, Pappenheimer, diphtheria toxoid, DPT, DTP, DTaP.

Corynebacterium diphtheriae

Kingdom: Bacteria
Phylum: Actinobacteria
Order: Actinomycetales
Suborder: Croynebacterineae
Family: Corynebacteriaceae
Genus: Corynebacterium
Species: C. diphtheriae

Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Diphtheria (page 3)

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Two factors have great influence on the ability of Corynebacterium diphtheriae to produce the diphtheria toxin: (1) low extracellular concentrations of iron and (2) the presence of a lysogenic prophage in the bacterial chromosome. The gene for toxin production occurs on the chromosome of the prophage, but a bacterial repressor protein controls the expression of this gene. The repressor is activated by iron, and it is in this way that iron influences toxin production. High yields of toxin are synthesized only by lysogenic bacteria under conditions of iron deficiency.

The role of iron. In artificial culture the most important factor controlling yield of the toxin is the concentration of inorganic iron (Fe++ or Fe+++) present in the culture medium. Toxin is synthesized in high yield only after the exogenous supply of iron has become exhausted (This has practical importance for the industrial production of toxin to make toxoid. Under the appropriate conditions of iron starvation, C. diphtheriae will synthesize diphtheria toxin as 5% of its total protein). Presumably, this phenomenon takes place in vivo as well. The bacterium may not produce maximal amounts of toxin until the iron supply in tissues of the upper respiratory tract has become depleted. It is the regulation of toxin production in the bacterium that is partially controlled by iron. The tox gene is regulated by a mechanism of negative control wherein a repressor molecule, product of the DtxR gene, is activated by iron. The active repressor binds to the tox gene operator and prevents transcription. When iron is removed from the repressor (under growth conditions of iron limitation), derepression occurs, the repressor is inactivated and transcription of the tox genes can occur. Iron is referred to as a corepressor since it is required for repression of the toxin gene.

The role of B-phage. Only those strains of Corynebacterium diphtheriae that are lysogenized by a specific Beta phage produce diphtheria toxin. A phage lytic cycle is not necessary for toxin production or release. The phage contains the structural gene for the toxin molecule. The original proof rested in the demonstration that lysogeny of C. diphtheriae by various mutated Beta phages leads to production of nontoxic but antigenically-related material (called CRM for "cross-reacting material"). CRMs have shorter chain length than the diphtheria toxin molecule but cross react with diphtheria antitoxins due to their antigenic similarities to the toxin. The properties of CRMs established beyond a doubt that the tox genes resided on the phage chromosome rather than the bacterial chromosome.

Even though the tox gene is not part of the bacterial chromosome, the regulation of toxin production is under bacterial control since the DtxR (regulatory) gene is on the bacterial chromosome and toxin production depends upon bacterial iron metabolism.

Figure 5. The Beta phage that encodes the tox gene for the diphtheria toxin.

It is of some interest to speculate on the role of the diphtheria toxin in the natural history of the bacterium. Of what value should it be to an organism to synthesize up to 5% of its total protein as a toxin that specifically inhibits protein synthesis in eucaryotes and archaea? Possibly the toxin assists colonization of the throat (or skin) by killing epithelial cells or neutrophils. There is no evidence to suggest a key role of the toxin in the life cycle of the organism. Since mass immunization against diphtheria has been practiced, the disease has virtually disappeared, and C. diphtheriae is no longer a component of the normal flora of the human throat and pharynx. It may be that the toxin played a key role in the colonization of the throat in nonimmune individuals and, as a consequence of exhaustive immunization, toxigenic strains have become virtually extinct.

Figure 6. The Diphtheria Toxin (DTx) Monomer. A (red) is the catalytic domain; B (yellow) is the binding domain which displays the receptor for cell attachment; T (blue) is the hydrophobic domain responsible for insertion into the endosome membrane to secure the release of A. The protein is illustrated in its "closed" configuration.

The diphtheria toxin (DTx) is a two-component bacterial exotoxin synthesized as a single polypeptide chain containing an A (active) domain and a B (binding) domain. Proteolytic nicking of the secreted form of the toxin separates the A chain from the B chain. The B chain contains a hydrophobic T (translocation) region, responsible for insertion into the endosome membrane in order to secure the release of A. The toxin binds to a specific receptor (now known as the HB-EGF receptor) on susceptible cells and enters by receptor-mediated endocytosis. Acidification of the endosome vesicle results in unfolding of the protein and insertion of the T segment into the endosomal membrane. Apparently, as a result of activity on the endosome membrane, the A subunit is cleaved and released from the B subunit as it inserts and passes through the membrane. Once in the cytoplasm, the A fragment regains its conformation and its enzymatic activity. Fragment A catalyzes the transfer of ADP-ribose from NAD to the eucaryotic Elongation Factor 2 which inhibits the function of the latter in protein synthesis. Ultimately, inactivation of all of the host cell EF-2 molecules causes death of the cell. Attachment of the ADP ribosyl group occurs at an unusual derivative of histadine called diphthamide.

Figure 7. The Mechanism of action of Diphtheria toxin DTxA.

Figure 8. Uptake and activity of the diphtheria toxin in eucaryotic cells. The figure is redrawn from the Diphtheria Toxin Homepage at UCLA. A represents the A/B toxin's A (catalytic) domain; B is the B (receptor) domain; T is the hydrophobic domain that inserts into the cell membrane.

In vitro, the native diphtheria toxin is inactive and can be activated by trypsin in the presence of thiol. The enzymatic activity of fragment A is masked in the intact toxin. Fragment B is required to bind the native toxin to its cognate receptor and to permit the escape of fragment A from the endosome. The C terminal end of Fragment B contains the peptide region that attaches to the HB-EGF receptor on the sensitive cell membrane, and the N-terminal end is a strongly hydrophobic region which will insert into a membrane lipid bilayer.

The specific membrane receptor, heparin-binding epidermal growth factor (HB-EGF) precursor is a protein on the surface of many types of cells. The occurrence and distribution of the HB-EGF receptor on cells determines the susceptibility of an animal species, and certain cells of an animal species, to the diphtheria toxin. Normally, the HB-EGF precursor releases a peptide hormone that influences normal cell growth and differentiation. One hypothesis is that the HB-EGF receptor itself is the protease that nicks the A fragment and reduces the disulfide bridge between it and the B fragment when the A fragment makes its way through the endosomal membrane into the cytoplasm.

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Kenneth Todar has taught microbiology to undergraduate students at The University of Texas, University of Alaska and University of Wisconsin since 1969.

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