Diphtheria (page 3)
This chapter has 4 pages
© Kenneth Todar, PhD
Two factors have great influence on the ability of Corynebacterium
to produce the diphtheria toxin: (1) low extracellular
of iron and (2) the presence of a lysogenic prophage in the
bacterial chromosome. The gene for toxin production occurs on the
of the prophage, but a bacterial repressor protein controls the
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
only by lysogenic bacteria under conditions of iron deficiency.
The role of iron. In artificial culture the most important
controlling yield of the toxin is the concentration of inorganic iron
or Fe+++) present in the culture medium. Toxin is
synthesized in high
only after the exogenous supply of iron has become exhausted (This has
practical importance for the industrial production of toxin to make
Under the appropriate conditions of iron starvation, C. diphtheriae
will synthesize diphtheria toxin as 5% of its total protein).
this phenomenon takes place in vivo as well. The bacterium may not
maximal amounts of toxin until the iron supply in tissues of the upper
respiratory tract has become depleted. It is the regulation of toxin
in the bacterium that is partially controlled by iron. The tox gene is
regulated by a mechanism of negative control wherein a repressor
product of the DtxR gene, is activated by iron. The active repressor
to the tox gene operator and prevents transcription. When iron is
from the repressor (under growth conditions of iron limitation),
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
diphtheria toxin. A phage lytic cycle is not necessary for toxin
or release. The phage contains the structural gene for the toxin
The original proof rested in the demonstration that lysogeny of C.
by various mutated Beta phages leads to production of nontoxic but
material (called CRM for "cross-reacting material"). CRMs have shorter
chain length than the diphtheria toxin molecule but cross react with
antitoxins due to their antigenic similarities to the toxin. The
of CRMs established beyond a doubt that the tox genes resided on the
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
(regulatory) gene is on the bacterial chromosome and toxin production
upon bacterial iron metabolism.
5. The Beta phage that encodes the tox gene for the diphtheria
It is of some interest to speculate on the role of the diphtheria
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
Possibly the toxin assists colonization of the throat (or skin) by
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
against diphtheria has been practiced, the disease has virtually
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
Figure 6. The Diphtheria
A (red) is the catalytic
B (yellow) is the binding domain which displays the receptor for cell
T (blue) is the hydrophobic domain responsible for insertion into the
membrane to secure the release of A. The protein is illustrated in its
The diphtheria toxin (DTx) is a two-component bacterial exotoxin
as a single polypeptide chain containing an A (active) domain and a B
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
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
on susceptible cells and enters by receptor-mediated endocytosis.
of the endosome vesicle results in unfolding of the protein and
of the T segment into the endosomal membrane. Apparently, as a result
on the endosome membrane, the A subunit is cleaved and released from
B subunit as it inserts and passes through the membrane. Once in the
the A fragment regains its conformation and its enzymatic activity.
A catalyzes the transfer of ADP-ribose from NAD to the eucaryotic
Factor 2 which inhibits the function of the latter in protein
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
derivative of histadine called diphthamide.
Mechanism of action of Diphtheria toxin DTxA.
8. Uptake and
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
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
toxin to its cognate receptor and to permit the escape of fragment A
the endosome. The C terminal end of Fragment B contains the peptide
that attaches to the HB-EGF receptor on the sensitive cell membrane,
the N-terminal end is a strongly hydrophobic region which will insert
a membrane lipid bilayer.
The specific membrane receptor,
heparin-binding epidermal growth
(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
the susceptibility of an animal species, and certain cells of an animal
species, to the diphtheria toxin. Normally, the HB-EGF precursor
a peptide hormone that influences normal cell growth and
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
B fragment when the A fragment makes its way through the endosomal
into the cytoplasm.