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An Interview with Professor Manuel Varela: Diphtheria and Friedrich Loeffler

Sep 21, 2018 by

Michael F. Shaughnessy –

1) Professor Varela- we have all heard the word “diphtheria” but I would surmise that unless we were in the medical field, we would not know EXACTLY what it is. Can you enlighten us?

Diphtheria is an infectious disease that has brought about misery and death for countless centuries, perhaps even for millennia, and it has accordingly had many profound consequences in numerous aspects of our human history. Furthermore, human intervention in the form of effective preventative measures that have been developed within the 20th century has changed the way we address the illness today.

The causative agent of diphtheria is a microbe, a bacterium referred to by many according to its scientific name Corynebacterium diphtheriae.  This microbe-caused diphtheria involves pathology at the respiratory tracts or the skin of human patients.

Human patients of diphtheria acquire the Corynebacterium diphtheriae bacteria by inhalation into the lungs or by physical contact of their skin with other individuals who have the disease. Humans are apparently the only suitable hosts for the diphtheria-causing microbes. Once the bacteria gain entrance into the patient or access to the skin, the microorganisms synthesize a toxin, a protein which in turn mediates the clinical signs and symptoms of the diphtheria.

With the help of an infecting virus, a bacteriophage called β-phage, the Corynebacterium diphtheriae bacteria readily produce the diphtheria toxin, a virulence factor known as an exotoxin, which is in somewhat of an inactive state. A leader peptide portion of the larger toxin protein signals it for secretion from the bacterial cell.

The remaining toxin protein is broken again into of two main parts. The first part is called exotoxin A, or the A subunit, and it contains the so-called catalytic domain, an active enzyme. Likewise, the second part, called exotoxin B, or the B subunit, harbors a cell receptor-binding portion and a transmembrane or translocation portion.

Once the bacteria secrete the diphtheria toxin, the pathology upon the cell is initiated. The resulting pathogenesis in the human patient goes through several stages.

First in the disease process is the binding of the diphtheria toxin to receptors that are situated within the membrane of the human cell.  The diphtheria toxin receptor is normally made for the purpose of binding human epidermal growth factor, but in the case of diphtheria disease, the bacterial toxin binds the receptor, instead.

The second stage in the pathological process involves a migration of several toxin-receptor complexes to an indented area of the human cell membrane, a structure called a coated pit. The toxin-receptors are clustered together within the coated pit structure.

The third stage of the diphtheria pathology involves the entrance into the insides of the human cells of the clustered diphtheria-toxin-cell receptor complexes. This process is often referred to as internalization. The bacterial toxin is now officially inside the human cell and contained within a sub-cellular structure called an endosome or a vesicle—a tiny membrane-enclosed packet harboring the diphtheria toxin.

In the fourth stage, a physiological process occurs, called acidification.  In this stage, the endosome that contains the toxin allows protons to enter, effectively lowering the pH values of the sub-cellular toxin-containing packet.

The fifth stage permits the insertion of a transporter part of the toxin into the membrane of the acidified toxin-harboring endosome. This transporter part is referred to as a transmembrane or translocation domain of the B subunit. The insertion of this part of the toxin permits the next stage of the pathogenesis to occur.

The six stage is called delivery, and it entails the transport of the active part of the toxin across the endosome membrane in order to accomplish the deliverance of the toxin into the cytoplasm of the human host cell. The toxic part of the toxin, the catalytic domain, is an enzyme called ADP-ribosyltransferase.

The last stage involves the killing of the human cell that has been invaded by the diphtheria toxin. The bacterially made toxin, now inside the cytoplasm of the human cell, accomplishes its dastardly deed by performing an enzymatic reaction, called ADP-ribosylation. The enzyme attaches a nucleotide-sugar complex, called ADP-ribose, to the protein-making machinery of the cell, called the ribosome.  The region on the human ribosome to which the ADP-ribose moiety attaches is referred to as elongation factor 2 (EF-2), and this AD-ribosylation inactivates the EF-2 and the ribosome along with it.  With the EF-2 component of the ribosome now prevented from making protein, the human host cell will die. This situation will then lead to the formation of the pseudomembrane in the patient and is quite characteristic of the diphtheria.

2) How common is diphtheria and is it only in certain parts of the world?

The diphtheria aliment can be found in many parts of the globe, but it is especially common in regions of the world where poverty exists, crowded conditions are prevalent, and where vaccination levels are poor.

In particular, diphtheria is continually present in southeastern Asia and certain parts of India and the African continent, where it may be endemic. Periodically, there are outbreaks of diphtheria in various regions of the planet, with thousands of individuals sometimes affected. The diphtheria is considered extremely rare in the U.S., but reliable case numbers of the contagion may be unattainable because it is no longer deemed a reportable disease by the Center for Disease Control and Prevention.

The main victims of the diphtheria are children, and the incidence numbers of cases are known to be enhanced in adults in regions where vaccination measures are invoked. The Corynebacterium diphtheriae bacterium can live in the throat of the upper airway in human beings who show no clinical signs or symptoms and are themselves immune to the microbe. These individuals may be considered carriers of the microbe to others who may not be immune to the diphtheria bacterium. This carriage phenomenon may serve to move the infectious microbe through human populations.

3) How is it treated?

The treatment of choice is the diphtheria antitoxin, an antibody protein which is known to specifically target the bacterial toxin itself, resulting in its neutralization. It is a form of passive immunization. The bacterial derived toxin targets the human, and the human derived antitoxin targets the bacterial toxin.

The antitoxin treatment, however, must be provided quickly, prior to the entry of the toxin into the human host cell. Otherwise, the affected cell can die with certainty.

Antimicrobial chemotherapy may be indicated, but it is a second treatment choice. The two main antimicrobial agents are penicillin or erythromycin. The penicillin can target the cell wall of the bacterium to prevent the construction its protective barrier.  The bacterium will then explode into pieces and die.

The erythromycin, on the other hand, will target the bacterial protein making machinery, while leaving the human ribosomes largely unaffected. It is interesting that both the erythromycin and the bacterial diphtheria toxin will target the protein synthetic machineries. The bacterial toxin targets the human while the erythromycin antibiotic targets the bacterium. It is fortuitous that the ribosomes of humans and of bacteria are sufficiently different such that specific poisons (bacterial toxin versus erythromycin) will affect the protein-making machinery of the pathogenic microbe or of the human cell, but not both.

4) Now, the human element- Friedrich Loeffler- what does he have to do with diphtheria?

In the year 1883, Dr. Friedrich Loeffler, a German investigator in the burgeoning field of bacteriology, was the first investigator to successfully isolate and grow the bacterium that causes diphtheria in pure laboratory culture. Approximately a year earlier, prior to the bacteriological work of Dr. Loeffler, a one Prof. Edwin Theodore Klebs had described essentially the same bacterium, as he had found it in his diphtheria patients.  Dr. Klebs, however, had not purified the microbe as Dr. Loeffler had done. Thus, it has historically been Dr. Loeffler who is credited with the discovery of the bacterium that causes diphtheria.

In any case, the bacterium had then become known as the Klebs-Loeffler bacillus. In 1888, investigators Emile Roux and Alexandre Yersin had then been able to inject laboratory test animals with filtrates of the bacterial cultures, demonstrating that the bacterium produced a smaller agent that passed through the filters, strongly suggesting the involvement of a toxin in conferring diphtheria. In 1883, Dr. Loeffler coined the term exotoxin, a name still used in the scientific literature today to denote the fact that the bacteria make and release the virulence to the outside milieu of the cell. The pioneering bacteriological work of Dr. Loeffler had set the stage for the eventual discovery of the diphtherial antitoxin by Emil Von Behring, leading to an effective serum therapy and prevention by active immunization. 

5) Loeffler- where was he born, raised, educated and what other things did he study or investigate?

Friedrich August Johannes Loeffler (Löffler) was born in Frankfurt am-Oder, Brandenburg, Prussia, now Germany, on the 24th day of June, in 1852.  Loeffler’s father, Gottfried, was a high-ranking army surgeon and a university professor at the Kaiser Wilhelm Academy for military medicine. 

Friedrich Loeffler attended the University of Würzburg focusing on the study of medicine and providing military service during the Franco-Prussian war.  In 1874, he took his M.D. degree from the Institute of Military Medicine, at the University of Berlin, Germany. In 1879, Dr. Loeffler became an army physician as one of the first assistants to the famous Dr. Robert Koch, while working at the Imperial Health Office, in Berlin.  There Dr. Loeffler collaborated with others in Koch’s laboratory to assess various sterilization protocols, comparing hot air and steam conditions for improvement of autoclaving methodology.

In 1888, Dr. Loeffler moved to the University of Greifswald to take on a post as professor of hygiene. In 1913, he became the director of the Robert Koch Institute for Infectious Diseases, at Berlin, in Germany. Shortly thereafter, in 1915, Dr. Loeffler died on the ninth of April, at the age of 62.  

In addition to purifying and cultivating the Corynebacterium diphtheriae bacteria in the laboratory, Dr. Loeffler made several other significant discoveries. For instance, in 1882, Dr. Loeffler and collaborator Wilhelm Schütz discovered the bacterial agent that causes the disease called glanders in horses. They had called the microbe Bacillus mallei, but it is known today as Burkholderia mallei. In 1884, Dr. Loeffler developed a new culture medium composed of thickened serum in order to culture the diphtherial bacteria. Today, it is known as Loeffler’s serum. In 1898, Dr. Loeffler, working with Dr. Paul Frosch, discovered the microbial agent that caused the so-called foot-and-mouth disease; they had found that the agent was filterable, strongly supporting the notion that it was a virus. Some sources say that this discovery was the first report of a virus that was known to cause infection in non-human animals.

6) What have I neglected to ask about Loeffler or diphtheria?

In the early 1880s, Dr. Loeffler became involved in a dispute between his mentor, Dr. Robert Koch, and the famous Prof. Louis Pasteur.  Dr. Loeffler, along with other colleagues of Prof. Koch, such as Georg Gaffky, had a disagreement with Pasteur’s method of attenuation for the bacterial agent of anthrax. Apparently, Koch and Pasteur held long lasting disputes in other areas, such as the ability of the Bacillus anthracis bacterium to form endospores at specific temperatures, the nature of the cholera-causing microbial agent, and the efficacy of Pasteur’s rabies vaccine, among others. 

Several sources credit Dr. Loeffler with defining the experiments necessary to invoke and experimentally test Koch’s famous postulates and with better explaining the postulates than Koch had been. With a clearer understanding of the steps necessary to claim that a microbe causes a disease, many investigators applied the postulates with staggering successes.

I mentioned above that a virus, a bacteriophage called β-phage, helps the Corynebacterium diphtheriae become pathogenic. Without the phage, the bacterium is harmless. With the β-phage, however, the bacterium becomes dangerous.  When the β-phage virus infects the Corynebacterium diphtheriae, it becomes lysogenized, meaning that the phage inserts its viral genome into the bacterial genome, keeping the bacterium alive and pathogenic.

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