Corynebacterium diphtheriae infects the nasopharynx or skin. Toxigenic strains secrete a potent exotoxin which may cause diphtheria. The symptoms of diphtheria include pharyngitis, fever, swelling of the neck or area surrounding the skin lesion. Diphtheritic lesions are covered by a pseudomembrane. The toxin is distributed to distant organs by the circulatory system and may cause paralysis and congestive heart failure.
Structure, Classification, and Antigenic Types
Corynebacterium diphtheriae is a nonmotile, noncapsulated, club-shaped, Gram-positive bacillus. Toxigenic strains are lysogenic for one of a family of corynebacteriophages that carry the structural gene for diphtheria toxin, tox. Corynebacterium diphtheriae is classified into biotypes (mitis, intermedius, and gravis) according to colony morphology, as well as into lysotypes based upon corynebacteriophage sensitivity. Most strains require nicotinic and pantothenic acids for growth; some also require thiamine, biotin, or pimelic acid. For optimal production of diphtheria toxin, the medium should be supplemented with amino acids and must be deferrated.
Asymptomatic nasopharyngeal carriage is common in regions where diphtheria is endemic. In susceptible individuals, toxigenic strains cause disease by multiplying and secreting diphtheria toxin in either nasopharyngeal or skin lesions. The diphtheritic lesion is often covered by a pseudomembrane composed of fibrin, bacteria, and inflammatory cells. Diphtheria toxin can be proteolytically cleaved into two fragments: an N-terminal fragment A (catalytic domain), and fragment B (transmembrane and receptor binding domains). Fragment A catalyzes the NAD+-dependent ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis in eukaryotic cells. Fragment B binds to the cell surface receptor and facilitates the delivery of fragment A to the cytosol.
Protective immunity involves an antibody response to diphtheria toxin following clinical disease or to diphtheria toxoid (formaldehyde-inactivated toxin) following immunization.
Corynebacterium diphtheriae is spread by droplets, secretions, or direct contact. In situ lysogenic conversion of nontoxigenic strains to a toxigenic phenotype has been documented. Infection is spread solely among humans, although toxigenic strains have been isolated from horses. In regions where immunization programs are maintained, isolated outbreaks of disease are often associated with a carrier who has recently visited a subtropical region where diphtheria is endemic. Large-scale outbreaks of disease may occur in populations where active immunization programs are not maintained.
Clinical diagnosis depends upon culture-proven toxigenic C diphtheriae infection of the skin, nose, or throat combined with clinical signs of nasopharyngeal diphtheria (e.g., sore throat, dysphagia, bloody nasal discharge, pseudomembrane). Toxigenicity is identified by a variety of in vitro (e.g., gel immunodiffusion, tissue culture) or in vivo (e.g., rabbit skin test, guinea pig challenge) methods.
Immunization with diphtheria toxoid is extraordinarily effective. Diphtheria patients must be promptly treated with antitoxin to neutralize circulating diphtheria toxin.
Diphtheria is a paradigm of the toxigenic infectious diseases. In 1883, Klebs demonstrated that Corynebacterium diphtheriae was the agent of diphtheria. One year later, Loeffler found that the organism could only be cultured from the nasopharyngeal cavity, and postulated that the damage to internal organs resulted from a soluble toxin. By 1888, Roux and Yersin showed that animals injected with sterile filtrates of C diphtheriae developed organ pathology indistinguishable from that of human diphtheria; this demonstrated that a potent exotoxin was the major virulence factor.
Diphtheria is most commonly an infection of the upper respiratory tract and causes fever, sore throat, and malaise. A thick, gray-green fibrin membrane, the pseudomembrane, often forms over the site(s) of infection as a result of the combined effects of bacterial growth, toxin production, necrosis of underlying tissue, and the host immune response. Recognition that the systemic organ damage was due to the action of diphtheria toxin led to the development of both an effective antitoxin-based therapy for acute infection and a highly successful toxoid vaccine.
Although toxoid immunization has made diphtheria a rare disease in those regions where public health standards mandate vaccination, outbreaks of diphtheria still occur in nonimmunized and immunocompromised groups. In marked contrast, widespread outbreaks of diphtheria reaching epidemic proportions have been observed in those regions where active immunization programs have been halted.
There are two types of clinical diphtheria: nasopharyngeal and cutaneous. Symptoms of pharyngeal diphtheria vary from mild pharyngitis to hypoxia due to airway obstruction by the pseudomembrane (Fig. 32-1). The involvement of cervical lymph nodes may cause profound swelling of the neck (bull neck diphtheria), and the patient may have a fever (≥ 103 °F). The skin lesions in cutaneous diphtheria are usually covered by a gray-brown pseudomembrane. Life-threatening systemic complications, principally loss of motor function (e.g., difficulty in swallowing) and congestive heart failure, may develop as a result of the action of diphtheria toxin on peripheral motor neurons and the myocardium.
Structure, Classification, and Antigenic Types
Corynebacterium diphtheriae is a Gram-positive nonmotile, club-shaped bacillus. Strains growing in tissue, or older cultures in vitro, contain thin spots in their cell walls that allow decolorization during the Gram stain and result in a Gram-variable reaction. Older cultures often contain metachromatic granules (polymetaphosphate) which stain bluish-purple with methylene blue. The cell wall sugars include arabinose, galactose, and mannose. In addition, a toxic 6,6′-diester of trehalose containing corynemycolic and corynemycolenic acids in equimolar concentrations may be isolated. Three distinct cultural types, mitis, intermedius, and gravis have been recognized (Table 1).
Most strains require nicotinic and pantothenic acids for growth; some also require thiamine, biotin, or pimelic acid. For the optimal production of diphtheria toxin the medium should be supplemented with amino acids and must be deferrated.
As early as 1887, Loeffler described the isolation from healthy individuals of avirulent (nontoxigenic) C diphtheriae that were indistinguishable from the virulent (toxigenic) strains isolated from patients. It is now recognized that avirulent strains of C diphtheriae may be converted to the virulent phenotype following infection and lysogenization by one of a number of distinct corynebacteriophages that carry the structural gene for diphtheria toxin, tox. Lysogenic conversion from the avirulent to virulent phenotype may occur in situ, as well as in vitro. The diphtheria toxin structural gene is not essential for either corynebacteriophage or C diphtheriae. Despite this observation, genetic drift of diphtheria toxin has not been observed.
The pathogenesis of diphtheria is based upon two primary determinants: (1) the ability of a given strain of C diphtheriae to colonize in the nasopharyngeal cavity and/or on the skin, and (2) its ability to produce diphtheria toxin. Since those determinants involved in colonization of the host are encoded by the bacteria, and the toxin is encoded by the corynebacteriophage, the molecular basis of virulence in C diphtheriae results from the combined effects of determinants carried on two genomes. Nontoxigenic strains of C diphtheriae are rarely associated with clinical disease; however, they may become highly virulent following lysogenic conversion to toxigenicity.
Little is known of the colonization factors of C diphtheriae. However, it is apparent that factors other than the production of diphtheria toxin contribute to virulence. Epidemiologic studies have demonstrated that a given lysotype may persist in the population for extended periods. It may later be supplanted by another lysotype. The emergence and subsequent predominance of a new lysotype in the population are presumably due to its ability to colonize and effectively compete in their segment of the nasopharyngeal ecologic niche. Corynebacterium diphtheriae may produce a neuraminidase that cleaves sialic acid from the cell surface into its pyruvate and N-acetylneuraminic acid components. Cord factor (6,6′-di-O-mycoloyl-α,α'-D-trehalose) is a surface component of C diphtheriae, but its role in colonization of the human host is unclear.
Diphtheria Toxin Production
The structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages of which the β-phage is the most extensively studied (Fig. 32-2). The regulation of diphtheria tox expression is mediated by an iron-activated repressor, DtxR, which is encoded on the C diphtheriae genome. The expression of tox depends on the physiologic state of C diphtheriae. Under conditions in which iron becomes the growth-rate limiting substrate, iron dissociates from DtxR, the tox gene becomes derepressed, and diphtheria toxin is synthesized and secreted into the culture medium at maximal rates (Fig. 32-3).
Diphtheria toxin is extraordinarily potent; in sensitive species (e.g., humans, monkeys, rabbits, guinea pigs) as little as 100 to 150 ng/kg of body weight is lethal. Diphtheria toxin is composed of a single polypeptide chain of 535 amino acids. Biochemical genetic and X-ray crystallographic analysis show that the toxin is composed of three structural/functional domains: an N-terminal ADP-ribosyltransferase (catalytic domain); (2) a region which facilitates the delivery of the catalytic domain across the cell membrane (transmembrane domain); and (3) the eukaryotic cell receptor binding domain (Fig. 32-4). Following mild digestion with trypsin and reduction under denaturing conditions, diphtheria toxin may be specifically cleaved in its protease-sensitive loop into two polypeptide fragments (A and B). Fragment A is the N-terminal 21 kDa component of the toxin and contains the catalytic center for the ADP-ribosylation of elongation factor 2 (EF-2) according to the following reaction:
The C-terminal fragment, fragment B, carries the transmembrane and receptor binding domains of the toxin.
The intoxication of a single eukaryotic cell by diphtheria toxin involves at least four distinct steps (Fig. 32-5): (1) the binding of the toxin to its cell surface receptor; (2) clustering of charged receptors into coated pits and internalization of the toxin by receptor-mediated endocytosis; following acidification of the endocytic vesicle by a membrane-associated, ATP-driven proton pump, (3) the insertion of the transmembrane domain into the membrane and the facilitated delivery of the catalytic domain to the cytosol, and (4) the ADP-ribosylation of EF-2, which results in the irreversible inhibition of protein synthesis. It has been shown that a single molecule of the catalytic domain delivered to the cytosol is sufficient to be lethal for the cell.
Immunity to diphtheria involves an antibody response to diphtheria toxin following clinical disease or immunization with diphtheria toxoid.
Before mass immunization of the U.S. population with diphtheria toxoid, diphtheria was typically a disease of children. A remarkable aspect of mass immunization with diphtheria toxoid is that as the percentage of the population with protective levels of antitoxin immunity (≥ 0.01 IU/ml) increases, the frequency of isolation of toxigenic strains from the population decreases. Today in the United States where there is an almost complete disappearance of clinical diphtheria, the isolation of toxigenic strains of C diphtheriae is rare. Since subclinical infection is no longer a source of diphtherial antigen exposure and, if not boosted, antitoxin immunity wanes, a large percentage of the adults (30 to 60%) have antitoxin levels that are below the protective level and are at risk. In the United States, Europe, and Eastern Europe recent outbreaks of diphtheria have occurred largely among alcohol and/or drug abusers. Within this group, carriers of toxigenic C diphtheriae have moderately high levels of antitoxic immunity. The recent breakdown of public health measures in Russia has resulted in diphtheria becoming epidemic. By the end of 1994, Russia recorded more than 80,000 cases and greater than 2,000 deaths.
Focal outbreaks of diphtheria are almost always associated with an immune carrier who has returned from a region where diphtheria is endemic. Indeed, recent outbreaks of clinical diphtheria in the United States and Europe have been associated with travelers returning from Russia and Eastern Europe. Toxigenic strains of C diphtheriae spread directly from person to person by droplet infection. It is known that toxigenic strains may directly colonize the nasopharyngeal cavity. In addition, the tox gene may be spread indirectly by release of toxigenic corynebacteriophage and lysogenic conversion of nontoxigenic, autochthonous C diphtheriae in situ.
In addition to the determination of biotype and lysotype of C diphtheriae isolates, it is now possible to use molecular biologic techniques in the study of diphtheria outbreaks. Restriction endonuclease digestion patterns of C diphtheriae chromosomal DNA, as well as the use of cloned corynebacterial insertion sequences as a genetic probe have been used in the study of clinical outbreaks of disease.
The Schick test has been used for many years to assess immunity to diphtheria toxin, although today it has been replaced in many regions by a serologic test for specific antibodies to diphtheria toxin. In the Schick test, a small amount of diphtheria toxin (ca. 0.8 ng in 0.2 ml) is injected intradermally into the forearm (test site) and 0.0124 μg of diphtheria toxoid in 0.2 ml is injected intradermally at a control site. After 48 and 96 hours, readings are made. Nonspecific skin reactions generally peak by 48 hours. At 96 hours, an erythematous reaction with some possible necrosis at the test site indicates that there is insufficient antitoxic immunity to neutralize the toxin (≤ 0.03 IU/ml). Inflammation at both the test and control sites at 48 hours indicates a hypersensitivity reaction to the antigen preparation. In many instances, diphtheria toxin is only partially purified prior to inactivation with formaldehyde, and as a result preparations of toxoid may contain other corynebacterial products, which may elicit a hypersensitivity reaction in some individuals.
The clinical diagnosis of diphtheria requires bacteriologic laboratory confirmation of toxigenic C diphtheriae in throat or lesion cultures. For primary isolation, a variety of media may be used: Loeffler agar, Mueller-Miller tellurite agar, or Tinsdale tellurite agar. Sterile cotton-tipped applicators are used to swab the pharyngeal tonsils or their beds. Calcium alginate swabs may be inserted through both nares to collect nasopharyngeal samples for culture. Since diphtheritic lesions are often covered with a pseudomembrane, the surface of the lesion may have to be carefully exposed before swabbing with the applicator.
Following initial isolation, C diphtheriae may be identified as mitis, intermedius, or gravis biotype on the basis of carbohydrate fermentation patterns and hemolysis on sheep blood agar plates (Table 1). The toxigenicity of C diphtheriae strains is determined by a variety of in vitro and in vivo tests. The most common in vitro assay for toxigenicity is the Elek immunodiffusion test (Fig. 32-6). This test is based on the double diffusion of diphtheria toxin and antitoxin in an agar medium. A sterile, antitoxin-saturated filter paper strip is embedded in the culture medium, and C diphtheriae isolates are streak-inoculated at a 90° angle to the filter paper. The production of diphtheria toxin can be detected within 18 to 48 hours by the formation of a toxin-antitoxin precipitin band in the agar. Alternatively, many eukaryotic cell lines (e.g., African green monkey kidney, Chinese hamster ovary) are sensitive to diphtheria toxin, enabling in vitro tissue culture tests to be used for detection of toxin. Several sensitive in vivo tests for diphtheria toxin have also been described (e.g., guinea pig challenge test, rabbit skin test).
The control of diphtheria depends upon adequate immunization with diphtheria toxoid: formaldehyde-inactivated diphtheria toxin that remains antigenically intact. The toxoid is prepared by incubating diphtheria toxin with formaldehyde at 37° C under alkaline conditions. Immunization against diphtheria should begin in the second month of life with a series of three primary doses spaced 4 to 8 weeks apart, followed by a fourth dose approximately 1 year after the last primary inoculation. Diphtheria toxoid is widely used as a component in the DPT (diphtheria, pertussis, tetanus) vaccine. Epidemiologic surveys have shown that immunization against diphtheria is approximately 97% effective. Although mass immunization against diphtheria is practiced in the United States and Europe and there is an adequate immunization rate in children, a large proportion of the adult population may have antibody titers that are below the protective level. The adult population should be reimmunized with diphtheria toxoid every 10 years. Indeed, booster immunization with diphtheria-tetanus toxoids should be administered to persons traveling to regions with high rates of endemic diphtheria (Central and South America, Africa, Asia, Russia and Eastern Europe). In recent years, the use of highly purified toxoid preparations for immunization has minimized the occasional severe hypersensitivity reaction.
Although antibiotics (e.g., penicillin and erythromycin) are used as part of the treatment of patients who present with diphtheria, prompt passive immunization with diphtherial antitoxin is most effective in reducing the fatality rate. The long half-life of specific antitoxin in the circulation is an important factor in ensuring effective neutralization of diphtheria toxin; however, to be effective, the antitoxin must react with the toxin before it becomes internalized into the cell.
Other Corynebacterium Species
In addition to C diphtheriae, C ulcerans and C pseudotuberculosis, C pseudodiphtheriticum and C xerosis may occasionally cause infection of the nasopharnyx and skin. The last two strains are recognized by their ability to produce pyrazinamidase. In veterinary medicine, C renale and C kutscheri are important pathogens and cause pyelonephritis in cattle and latent infections in mice, respectively.
Redesigning of Diphtheria Toxin for the Development of Eukaryotic Cell-Receptor Specific Cytotoxins
Protein engineering is a new and rapidly developing area within the field of molecular biology; it brings together recombinant DNA methodologies and solid phase DNA synthesis in the design and construction of chimeric genes whose products have unique properties. The study of diphtheria toxin structure/function relationships has clearly shown this toxin to be a three-domain protein: catalytic, transmembrane, and receptor binding (Fig. 32-4). It has been possible to genetically substitute the native diphtheria toxin receptor-binding domain with a variety of polypeptide hormones and cytokines (e.g., α-melanocyte-stimulating hormone [α-MSH], interleukin (IL) 2, IL-4, IL-6, IL-7, epidermal growth factor). The resulting chimeric proteins, or fusion toxins, combine the receptor-binding specificity of the cytokine with the transmembrane and catalytic domains of the toxin. In each instance, the fusion toxins have been shown to selectively intoxicate only those cells which bear the appropriate targeted receptor. The first of these genetically engineered fusion toxins, DAB389 IL-2, is currently being evaluated in human clinical trials for the treatment of refractory lymphomas and autoimmune diseases, in which cells with high affinity IL-2 receptors play a major role in pathogenesis. Administration of DAB389 IL-2 has been shown to be safe, well tolerated, and capable of inducing durable remission from disease in the absence of severe adverse effects. It is likely that the diphtheria toxin-based fusion toxins will be important new biological agents for the treatment of specific tumors or disorders in which specific cell surface receptors may be targeted.
- Bishai WR, Murphy JR: Bacteriophage gene products that cause human disease. In Calendar R (ed): The Bacteriophages. Plenum, New York, 1988.
- Hesketh P, Caguioa P, Koh H. et al. Clinical activity of a cytotoxic fusion protein in the treatment of cutaneous T cell lymphoma. J Clin Oncol. 1993;11:1682. [PubMed: 8355035]
- LeMaistre CF, Craig FE, Meneghetti C. et al. Phase I trial of a 90-minute infusion of the fusion toxin DAB486 IL-2 in hematological cancers. Cancer Res. 1992;53:3930. [PubMed: 8358720]
- Pappenheimer AM, Jr: Diphtheria. In Germanier R (ed) Bacterial Vaccines. Academic Press, San Diego, 1984 .
- Rappuoli R, Perugini M, Falsen E. Molecular epidemiology of the 1984–1986 outbreak of diphtheria in Sweden. N Engl J Med. 1988;318:12. [PubMed: 2827023]
- Tao X, Schiering N, Zeng H-Y. et al. DtxR, iron and the regulation of diphtheria toxin expression. Mol Microbiol. 1994;14:191. [PubMed: 7830565]
- VanderSpek J, Cosenza L, Woodworth T. et al. Diphtheria toxin-related cytokine fusion proteins: elongation factor 2 as a target for the treatment of neoplastic disease. Molec Cell Biochem. 1994;138:151. [PubMed: 7898459]
John R. Murphy.
University of Texas Medical Branch at Galveston, Galveston (TX)
Murphy JR. Corynebacterium Diphtheriae. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 32.