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

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 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

antitoxin.

Subsequently, the exact mechanism of action of the toxin was shown, and the

toxin has become a classic model of a bacterial exotoxin.

Human Disease

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

pseudomembrane.

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.

Pathogenicity

The pathogenicity of Corynebacterium diphtheriae includes two distinct

phenomena:

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.

Toxigenicity

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

metabolism.

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

virtually extinct.

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-

ADP-Ribose

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).