Bacteriology at UW-Madison
The disease diphtheria is caused by the bacterium, Corynebacterium diphtheriae. Corynebacteria are Gram-positive, aerobic, nonmotile, rod-shaped bacteria. 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.
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 the disease became known as "El garatillo" (the strangler"), in Italy and Sicily as "the gullet disease".
In the 18th century, the disease reached the American colonies where it reached epidemic proportions about 1735. Often, whole families died of the disease in a few weeks.
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 it 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 was neutralized by antibodies in serum directed against the toxin ("antitoxin"), forming a Toxin-Anti-Toxin complex, or TAT, which could also be used to stimulate active immunity. 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 antitoxin.
Subsequently, the exact mechanism of action of the toxin was shown, and the toxin has become a classic model of a bacterial exotoxin.
At the turn of the century, in the United States, diphtheria was common, occurring primarily in children, and was one of the leading causes of death in infants and children. In the l920's, when data were first gathered, in the United States there were approximately 150,000 cases and 13,000 deaths reported annually. After diphtheria immunization was introduced, the number of cases gradually fell to about 19,000 in 1945. When diphtheria immunization became widespread in the late 1940's, a more rapid decrease in the number of cases and deaths occurred.
From 1970 to 1979, an average of 196 cases per year were reported.
outbreaks of 15 or more cases occurred in the United States between
and 1980, but there have been none since 1980. From 1980-1989, the
of cases in the United States dropped to 24; two cases were fatal and
occurred in persons 20 years of age or older. Most cases have occurred
nonimmunized (or inadequately immunized) individuals.
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 many were killed.
About one person in 10 who gets diphtheria dies of it. Diphtheria is more severe for those under 5 and over 40 years of age.
1. Invasion of the local tissues of the throat, which requires colonization and subsequent bacterial proliferation. Nothing is known about the adherence mechanisms of C. diphtheriae
2. Toxigenesis: bacterial production of the toxin. The diphtheria toxin causes the death eukaryotic cells and tissues by inhibition protein synthesis in the cells. Although the toxin is responsible for the lethal symptoms of the disease, the virulence of C. diphtheriae cannot be attributed to toxigenicity alone, since a distinct invasive phase apparently precedes toxigenesis. However, it has not been ruled out that the diphtheria toxin plays an essential role in the colonization process due to short-range effects at the colonization site.
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 animals. 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. This suggests that the toxin might have played a key role in the colonization of the throat in nonimmune individuals.
The diphtheria toxin is a three component bacterial exotoxin
as a single polypeptide chain containing an A (active) domain, a B
domain and a T (translocation) domain. The toxin binds to a target cell
by means od the B domain. This activates the T domain to insert into
target cell membrane and insert the A component which is responsible
the enzymatic action of the toxin.
Figure 2. The Diphtheria Toxin (DT) Monomer
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.
Fig 3. Mode of Action of the Diphtheria Toxin
Figure 4. Uptake and activity of the diphtheria toxin in Eukaryotic cells
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).
The relative absence of diphtheria in the United States is due primarily to the high level of appropriate immunization in children, and to an apparent reduction in toxin-producing strains of the bacterium. However, the increasing percentage of diphtheria cases in adults suggests that many adults may not be protected against diphtheria, because they have not receive booster immunizations within the past ten years. A similar situation exists with tetanus.