Diphtheria (Corynebacterium diphtheriae)Corynebacteria are Gram-positive, aerobic, nonmotile, rod-shaped bacteriarelated to the Actinomycetes.

They do not form spores or branch as do theactinomycetes, but they have the characteristic of forming irregular shaped,club-shaped or V-shaped arrangements in normal growth. They undergo snappingmovements just after cell division which brings them into characteristicarrangements resembling Chinese letters.The genus Corynebacterium consists of a diverse group of bacteria includinganimal and plant pathogens, as well as saprophytes.

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Some corynebacteria are partof the normal flora of humans, finding a suitable niche in virtually everyanatomic site. The best known and most widely studied species is Corynebacteriumdiphtheriae, the causal agent of the disease diphtheria.History and BackgroundNo bacterial disease of humans has been as successfully studied as diphtheria.The etiology, mode of transmission, pathogenic mechanism and molecular basis ofexotoxin structure, function, and action have been clearly established.Consequently, highly effective methods of treatment and prevention of diphtheriahave been developed.The study of Corynebacterium diphtheriae traces closely the development ofmedical microbiology, immunology and molecular biology.

Many contributions tothese 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 4thcentury 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 reachedepidemic proportions in 1735. Often, whole families died of the disease in a fewweeks.The bacterium that caused diphtheria was first described by Klebs in 1883, andwas cultivated by Loeffler in 1884, who applied Koch’s postulates and properlyidentified Corynebacterium diphtheriae as the agent of the disease.

In 1884, Loeffler concluded that C. diphtheriae produced a soluble toxin, andthereby provided the first description of a bacterial exotoxin.In 1888, Roux and Yersin demonstrated the presence of the toxin in the cell-freeculture 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 pigswith a heat-attenuated form of the toxin and demonstrated that the sera ofimmunized animals contained an antitoxin capable of protecting other susceptibleanimals against the disease. This modified toxin was suitable for immunizinganimals to obtain antitoxin but was found to cause severe local reactions inhumans and could not be used as a vaccine.In 1909, Theobald Smith, in the U.S.

, demonstrated that diphtheria toxinneutralized by antitoxin (forming a Toxin-Anti-Toxin complex, TAT) remainedimmunogenic and eliminated local reactions seen in the modified toxin. For someyears, beginning about 1910, TAT was used for active immunization againstdiphtheria. TAT had two undesirable characteristics as a vaccine.

First, thetoxin used was highly toxic, and the quantity injected could result in a fataltoxemia unless the toxin was fully neutralized by antitoxin. Second, theantitoxin mixture was horse serum, the components of which tended to beallergenic and to sensitize individuals to the serum.In 1913, Schick designed a skin test as a means of determining susceptibility orimmunity to diphtheria in humans. Diphtheria toxin will cause an inflammatoryreaction when very small amounts are injected intracutaneously. The Schick Testinvolves injecting a very small dose of the toxin under the skin of the forearmand evaluating the injection site after 48 hours. A positive test (inflammatoryreaction) 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 humanitywith 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 Bphage), while non lysogenized strains are avirulent. Subsequently, it was shownthat 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 experimentson the mechanism of a action of the diphtheria toxin. They studied the effectsof the toxin in HeLa cell cultures and in cell-free systems, and concluded thatthe toxin inhibited protein synthesis by blocking the transfer of amino acidsfrom tRNA to the growing polypeptide chain on the ribosome.

They found that thisaction of the toxin could be neutralized by prior treatment with diphtheriaantitoxin.Subsequently, the exact mechanism of action of the toxin was shown, and thetoxin has become a classic model of a bacterial exotoxin.Human DiseaseDiphtheria is a rapidly developing, acute, febrile infection which involves bothlocal and systemic pathology. A local lesion develops in the upper respiratorytract and involves necrotic injury to epithelial cells. As a result of thisinjury, blood plasma leaks into the area and a fibrin network forms which isinterlaced with with rapidly-growing C.

diphtheriae cells. This membranousnetwork covers over the site of the local lesion and is referred to as thepseudomembrane.The diphtheria bacilli do not tend to invade tissues below or away from thesurface epithelial cells at the site of the local lesion. At this site theyproduce the toxin that is absorbed and disseminated through lymph channels andblood to the susceptible tissues of the body.

Degenerative changes in thesetissues, 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 diseaseof children, and most individuals who survive infancy and childhood haveacquired immunity to diphtheria. In earlier times, when nonimmune populations(i.e.

, Native Americans) were exposed to the disease, people of all ages wereinfected and killed.PathogenicityThe pathogenicity of Corynebacterium diphtheriae includes two distinctphenomena: 1.Invasion of the local tissues of the throat, which requires colonizationand subsequent bacterial proliferation.

Nothing is known about the adherencemechanisms of this pathogen. 2.Toxigenesis: bacterial production of the diphtheria toxin. The virulence ofC.

diphtheriae cannot be attributed to toxigenicity alone, since a distinctinvasive phase apparently precedes toxigenesis. However, it cannot be ruled outthat the diphtheria toxin plays a (essential?) role in the colonization processdue to its short-range effects at the colonization site.Three strains of Corynebacterium diphtheriae are recognized, gravis, intermediusand mitis. They are listed here by falling order of the severity of the diseasethat they produce in humans. All strains produce the identical toxin and arecapable of colonizing the throat. The differences in virulence between the threestrains can be explained by their differing abilities to produce the toxin inrate and quantity, and by their differing growth rates.The gravis strain has a generation time (in vitro) of 60 minutes; theintermedius strain has a generation time of about 100 minutes; and the mitisstain has a generation time of about 180 minutes.

The faster growing strainstypically 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 supplymore rapidly in the invaded tissues, thereby allowing earlier or greaterproduction of the diphtheria toxin. Also, if the kinetics of toxin productionfollow the kinetics of bacterial growth, the faster growing variety wouldachieve an effective level of toxin before the slow growing varieties.ToxigenicityTwo factors have great influence on the ability of Corynebacterium diphtheriaeto produce the diphtheria toxin: (1) low extracellular concentrations of ironand (2) the presence of a lysogenic prophage in the bacterial chromosome.

Thegene for toxin production occurs on the chromosome of the prophage, but abacterial repressor protein controls the expression of this gene. The repressoris activated by iron, and it is in this way that iron influences toxinproduction. High yields of toxin are synthesized only by lysogenic bacteriaunder conditions of iron deficiency.The role of iron.

In artificial culture the most important factor controllingyield 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 theexogenous supply of iron has become exhausted (This has practical importance forthe industrial production of toxin to make toxoid. Under the appropriateconditions of iron starvation, C. diphtheriae will synthesize diphtheria toxinas 5% of its total protein!). Presumably, this phenomenon takes place in vivo aswell. The bacterium may not produce maximal amounts of toxin until the ironsupply in tissues of the upper respiratory tract has become depleted. It is theregulation of toxin production in the bacterium that is partially controlled byiron.

The tox gene is regulated by a mechanism of negative control wherein arepressor molecule, product of the DtxR gene, is activated by iron. The activerepressor binds to the tox gene operator and prevents transcription. When ironis removed from the repressor (under growth conditions of iron limitation),derepression occurs, the repressor is inactivated and transcription of the toxgenes can occur. Iron is referred to as a corepressor since it is required forrepression of the toxin gene.The role of B-phage. Only those strains of Corynebacterium diphtheriae that thatare lysogenized by a specific Beta-phage produce diphtheria toxin.

A phage lyticcycle is not necessary for toxin production or release. The phage contains thestructural gene for the toxin molecule, since lysogeny by various mutated Betaphages leads to production of nontoxic but antigenically-related material(called CRM for “cross-reacting material”). CRMs have shorter chain length thanthe diphtheria toxin molecule but cross react with diphtheria antitoxins due totheir antigenic similarities to the toxin. The properties of CRMs establishedbeyond a doubt that the tox genes resided on the phage chromosome rather thanthe bacterial chromosome.Even though the tox gene is not part of the bacterial chromosome the regulationof toxin production is under bacterial control since the DtxR (regulatory) geneis on bacterial chromosome and toxin production depends upon bacterial ironmetabolism.

It is of some interest to speculate on the role of the diphtheria toxin in thenatural history of the bacterium. Of what value should it be to an organism tosynthesize up to 5% of its total protein as a toxin that specifically inhibitsprotein synthesis in eukaryotes (and archaebacteria)? Possibly the toxin assistscolonization 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 theorganism.

Since mass immunization against diphtheria has been practiced, thedisease has virtually disappeared, and C. diphtheriae is no longer a componentof the normal flora of the human throat and pharynx. It may be that the toxinplayed a key role in the colonization of the throat in nonimmune individuals and,as a consequence of exhaustive immunization, toxigenic strains have becomevirtually extinct.Mode of Action of the Diphtheria ToxinThe diphtheria toxin is a two component bacterial exotoxin synthesized as asingle polypeptide chain containing an A (active) domain and a B (binding)domain.

Proteolytic nicking of the secreted form of the toxin separates the Achain from the B chainThe toxin binds to a specific receptor (now known as the HB-EGF receptor) onsusceptible cells and enters by receptor-mediated endocytosis. Acidification ofthe endosome vesicle results in unfolding of the protein and insertion of asegment into the endosomal membrane. Apparently as a result of activity on theendosome membrane, the A subunit is cleaved and released from the B subunit asit inserts and passes through the membrane. Once in the cytoplasm, the Afragment regains its conformation and its enzymatic activity. Fragment Acatalyzes the transfer of ADP-ribose from NAD to the eukaryotic ElongationFactor 2 which inhibits the function of the latter in protein synthesis.

Ultimately, inactivation of all of the host cell EF-2 molecules causes death ofthe cell. Attachment of the ADP ribosyyl group occurs at an unusual derivativeof histadine called diphthamide. NADAToxEF-2-ADP-RiboseNicotinamideATox-ADP-Ribose EF-2Mode of Action of the Diphtheria ToxinIn vitro, the native diphtheria toxin is inactive and can be activated bytrypsin in the presence of thiol. The enzymatic activity of fragment A is maskedin the intact toxin. Fragment B is required to bind the native toxin to itscognate receptor and to permit the escape of fragment A from the endosome.

The Cterminal 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 astrongly 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 anddistribution of the HB-EGF receptor on cells determines the susceptibility of ananimal species, and certain cells of an animal species, to the diphtheria toxin.Normally, the HB-EGF precursor releases a peptide hormone that influencesnormal cell growth and differentiation.

One hypothesis is that the HB-EGFreceptor itself is the protease that nicks the A fragment and reduces thedisulfide bridge between it and the B fragment when the A fragment makes its waythrough the endosomal membrane into the cytoplasm.Immunity to DiphtheriaAcquired immunity to diphtheria is due primarily to toxin-neutralizing antibody(antitoxin). Passive immunity in utero is acquired transplacentally and can lastat most 1 or 2 years after birth. In areas where diphtheria is endemic and massimmunization is not practiced, most young children are highly susceptible toinfection. Probably active immunity can be produced by a mild or inapparentinfection in infants who retain some maternal immunity, and in adults infectedwith strains of low virulence (inapparent infections).

Individuals that have fully recovered from diphtheria may continue to harbor theorganisms in the throat or nose for weeks or even months. In the past, it wasmainly through such healthy carriers that the disease was spread, and toxigenicbacteria 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, artificialimmunization at an early age is universally advocated. Toxoid is given in 2 or 3doses (1 month apart) for primary immunization at an age of 3 – 4 months.

Abooster injection should be given about a year later, and it is advisable toadminister several booster injections during childhood. Usually, infants in theUnited States are immunized with a trivalent vaccine containing diphtheriatoxoid, pertussis vaccine, and tetanus toxoid (DPT or DTP vaccine).

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