Diphtheria (Corynebacterium Diphtheriae) Essay, Research Paper
Diphtheria (Corynebacterium diphtheriae)
Corynebacteria are Gram-positive, aerobic, nonmotile, rod-shaped bacteria
related to the Actinomycetes. They do not form spores or branch as do the
actinomycetes, but they have the characteristic of forming irregular shaped,
club-shaped or V-shaped arrangements in normal growth. They undergo snapping
movements just after cell division which brings them into characteristic
arrangements resembling Chinese letters.
The genus Corynebacterium consists of a diverse group of bacteria including
animal and plant pathogens, as well as saprophytes. Some corynebacteria are part
of the normal flora of humans, finding a suitable niche in virtually every
anatomic site. The best known and most widely studied species is Corynebacterium
diphtheriae, the causal agent of the disease diphtheria.
History and Background
No bacterial disease of humans has been as successfully studied as diphtheria.
The etiology, mode of transmission, pathogenic mechanism and molecular basis of
exotoxin structure, function, and action have been clearly established.
Consequently, highly effective methods of treatment and prevention of diphtheria
have been developed.
The study of Corynebacterium diphtheriae traces closely the development of
medical microbiology, immunology and molecular biology. Many contributions to
these fields, as well as to our understanding of host-bacterial interactions,
have been made studying diphtheria and the diphtheria toxin.
Hippocrates provided the first clinical description of diphtheria in the 4th
century B.C. There are also references to the disease in ancient Syria and Egypt.
In the 17th century, murderous epidemics of diphtheria swept Europe; in Spain
“El garatillo” (the strangler”), in Italy and Sicily, “the gullet disease”.
In the 18th century, the disease reached the American colonies and reached
epidemic proportions in 1735. Often, whole families died of the disease in a few
The bacterium that caused diphtheria was first described by Klebs in 1883, and
was cultivated by Loeffler in 1884, who applied Koch’s postulates and properly
identified Corynebacterium diphtheriae as the agent of the disease.
In 1884, Loeffler concluded that C. diphtheriae produced a soluble toxin, and
thereby provided the first description of a bacterial exotoxin.
In 1888, Roux and Yersin demonstrated the presence of the toxin in the cell-free
culture fluid of C. diphtheriae which, when injected into suitable lab animals,
caused the systemic manifestation of diphtheria.
Two years later, von Behring and Kitasato succeeded in immunizing guinea pigs
with a heat-attenuated form of the toxin and demonstrated that the sera of
immunized animals contained an antitoxin capable of protecting other susceptible
animals against the disease. This modified toxin was suitable for immunizing
animals to obtain antitoxin but was found to cause severe local reactions in
humans and could not be used as a vaccine.
In 1909, Theobald Smith, in the U.S., demonstrated that diphtheria toxin
neutralized by antitoxin (forming a Toxin-Anti-Toxin complex, TAT) remained
immunogenic and eliminated local reactions seen in the modified toxin. For some
years, beginning about 1910, TAT was used for active immunization against
diphtheria. TAT had two undesirable characteristics as a vaccine. First, the
toxin used was highly toxic, and the quantity injected could result in a fatal
toxemia unless the toxin was fully neutralized by antitoxin. Second, the
antitoxin mixture was horse serum, the components of which tended to be
allergenic and to sensitize individuals to the serum.
In 1913, Schick designed a skin test as a means of determining susceptibility or
immunity to diphtheria in humans. Diphtheria toxin will cause an inflammatory
reaction when very small amounts are injected intracutaneously. The Schick Test
involves injecting a very small dose of the toxin under the skin of the forearm
and evaluating the injection site after 48 hours. A positive test (inflammatory
reaction) indicates susceptibility (nonimmunity). A negative test (no reaction)
indicates immunity (antibody neutralizes toxin).
In 1929, Ramon demonstrated the conversion of diphtheria toxin to its nontoxic,
but antigenic, equivalent (toxoid) by using formaldehyde. He provided humanity
with one of the safest and surest vaccines of all time-the diphtheria toxoid.
In 1951, Freeman made the remarkable discovery that pathogenic (toxigenic)
strains of C. diphtheriae are lysogenic, (i.e., are infected by a temperate B
phage), while non lysogenized strains are avirulent. Subsequently, it was shown
that the gene for toxin production is located on the DNA of the B phage.
In the early 1960s, Pappenheimer and his group at Harvard conducted experiments
on the mechanism of a action of the diphtheria toxin. They studied the effects
of the toxin in HeLa cell cultures and in cell-free systems, and concluded that
the toxin inhibited protein synthesis by blocking the transfer of amino acids
from tRNA to the growing polypeptide chain on the ribosome. They found that this
action of the toxin could be neutralized by prior treatment with diphtheria
Subsequently, the exact mechanism of action of the toxin was shown, and the
toxin has become a classic model of a bacterial exotoxin.
Diphtheria is a rapidly developing, acute, febrile infection which involves both
local and systemic pathology. A local lesion develops in the upper respiratory
tract and involves necrotic injury to epithelial cells. As a result of this
injury, blood plasma leaks into the area and a fibrin network forms which is
interlaced with with rapidly-growing C. diphtheriae cells. This membranous
network covers over the site of the local lesion and is referred to as the
The diphtheria bacilli do not tend to invade tissues below or away from the
surface epithelial cells at the site of the local lesion. At this site they
produce the toxin that is absorbed and disseminated through lymph channels and
blood to the susceptible tissues of the body. Degenerative changes in these
tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys,
liver and spleen, result in the systemic pathology of the disease.
In parts of the world where diphtheria still occurs, it is primarily a disease
of children, and most individuals who survive infancy and childhood have
acquired immunity to diphtheria. In earlier times, when nonimmune populations
(i.e., Native Americans) were exposed to the disease, people of all ages were
infected and killed.
The pathogenicity of Corynebacterium diphtheriae includes two distinct
1.Invasion of the local tissues of the throat, which requires colonization
and subsequent bacterial proliferation. Nothing is known about the adherence
mechanisms of this pathogen.
2.Toxigenesis: bacterial production of the diphtheria toxin. The virulence of
C. diphtheriae cannot be attributed to toxigenicity alone, since a distinct
invasive phase apparently precedes toxigenesis. However, it cannot be ruled out
that the diphtheria toxin plays a (essential?) role in the colonization process
due to its short-range effects at the colonization site.
Three strains of Corynebacterium diphtheriae are recognized, gravis, intermedius
and mitis. They are listed here by falling order of the severity of the disease
that they produce in humans. All strains produce the identical toxin and are
capable of colonizing the throat. The differences in virulence between the three
strains can be explained by their differing abilities to produce the toxin in
rate and quantity, and by their differing growth rates.
The gravis strain has a generation time (in vitro) of 60 minutes; the
intermedius strain has a generation time of about 100 minutes; and the mitis
stain has a generation time of about 180 minutes. The faster growing strains
typically produce a larger colony on most growth media. In the throat (in vivo),
a faster growth rate may allow the organism to deplete the local iron supply
more rapidly in the invaded tissues, thereby allowing earlier or greater
production of the diphtheria toxin. Also, if the kinetics of toxin production
follow the kinetics of bacterial growth, the faster growing variety would
achieve an effective level of toxin before the slow growing varieties.
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 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, since lysogeny 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 bacterial chromosome and toxin production depends upon bacterial iron
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 eukaryotes (and archaebacteria)? 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
Mode of Action of the Diphtheria Toxin
The diphtheria toxin 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 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 a
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 eukaryotic 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 ribosyyl group occurs at an unusual derivative
of histadine called diphthamide.
NAD ATox EF-2-
Nicotinamide ATox-ADP-Ribose EF-2
Mode of Action of the Diphtheria Toxin
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.
Immunity to Diphtheria
Acquired immunity to diphtheria is due primarily to toxin-neutralizing antibody
(antitoxin). Passive immunity in utero is acquired transplacentally and can last
at most 1 or 2 years after birth. In areas where diphtheria is endemic and mass
immunization is not practiced, most young children are highly susceptible to
infection. Probably active immunity can be produced by a mild or inapparent
infection in infants who retain some maternal immunity, and in adults infected
with strains of low virulence (inapparent infections).
Individuals that have fully recovered from diphtheria may continue to harbor the
organisms in the throat or nose for weeks or even months. In the past, it was
mainly through such healthy carriers that the disease was spread, and toxigenic
bacteria were maintained in the population. Before mass immunization of children,
carrier rates of C. diphtheriae of 5% or higher were observed.
Because of the high degree of susceptibility of children, artificial
immunization at an early age is universally advocated. Toxoid is given in 2 or 3
doses (1 month apart) for primary immunization at an age of 3 – 4 months. A
booster injection should be given about a year later, and it is advisable to
administer several booster injections during childhood. Usually, infants in the
United States are immunized with a trivalent vaccine containing diphtheria
toxoid, pertussis vaccine, and tetanus toxoid (DPT or DTP vaccine).