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An Interview with Professor Manuel Varela: Who Found Those Bacteriophages? Frederick Twort Did!

Oct 4, 2018 by

Frederick Twort

Michael F. Shaughnessy

1) Professor Varela- it has been years since my college science days- but I still remember a short discussion about bacteriophages. Refresh my memory, and enlighten our readers- what exactly is a bacteriophage?

Bacteriophages, or simply “phages,” are viruses that primarily infect bacterial microbes as their main hosts. The phages have a propensity for infecting bacteria, and often a particular phage is quite specific for a certain bacterial species, a restriction that quite often is narrowed down even to the strain level.

As with many other of the known viruses, the phages have, in general, a center that contains a nucleic acid-based genomic component, like DNA or RNA, of which a protective protein coat, called a capsid, usually encases. The protein coat encasement keeps the phage genome from degradation, as nucleic acids can be delicate when exposed in an unprotected environment and easily damaged. Thus, the external capsid protects the internal phage genome.

Like many animal viruses, the phage genome may consist of RNA or DNA, and these genomes can be single- or double-stranded nucleic acids. Furthermore, phage genomes may be circular or linear in their global structures.

Very often the inside container of the phage protein capsid has extremely limited room for large genome molecules. Thus, the genomes of the phages will be correspondingly limited in their sizes. Therefore, the phage genome, depending on the nature of the particular phage, may encode only a few specialized functions needed by a phage, such as the capsid proteins and specialized nucleic acid replication machinery.

Phages cannot infect animals, including humans, because such higher organisms lack the bacterial receptors for phages on their animal and human cells. Humans lack, for instance, the porins and lipopolysaccharide molecules, on their eukaryotic-based cells. Because of a lack of phage receptors in humans, and in virtually all other non-bacterial organisms for that matter, the phages cannot infect any such living beings. Phages are limited, therefore, to infecting only the bacteria.

2) I was fortunate enough to once experience an electron microscope (after it was ” warmed up”). Nevertheless, how did Frederick Twort “discover” these bacteriophages?

In 1915, Dr. Frederick Twort published his historic paper detailing the phage discovery in the scientific journal called Lancet. Being the first person in recorded history to deal with these types of viruses that infected bacteria, Dr. Twort referred to these microbes initially as parts of “transparent filterable material” and then later on in the paper as an “ultra-microscopic virus” that gave his Micrococcus bacteria a disease of its own. It was not until 1917, with the studies of Dr. Felix d’Herelle, that these microbes would later be named bacteriophages, for their ability to eat their bacterial hosts.

Dr. Twort had been familiar with the scientific literature regarding bacterial microbes, noting that both pathogenic and non-pathogenic bacteria had been discovered living in nature. At first, Dr. Twort was interested in discovering viruses that were non-pathogenic, as up to that time, only seemingly pathogenic viruses had been found, such as in the cases with vaccinia and tobacco mosaic viruses. Vaccinia virus was pathogenic to humans, causing the notorious small pox, a scourge that had wreaked havoc upon humans for multiple centuries; and the tobacco mosaic virus, the first virus to be discovered, causes the terrible mosaic disease in such plants.

Thus, Dr. Twort set out to find putative non-pathogenic viruses by incubating soils, grasses, hay, animal fecal material, straw, and pond water, all mixed in with over a hundred different culture media from the lab. These laboratory-based media contained agar, and in some cases, eggs, blood serum, and yeast extract. He then filtered the incubated materials and attempted to purify viruses by incubating the filtrates on fresh culture media. The amount of work involved with the hundreds of different culture media was incredible.  Yet, this initial effort showed no purified viruses. 

Dr. Twort even tried to inoculate laboratory animals, such as guinea pigs and rabbits with his filtrates and still found no viruses.  This effort was enormous, too.  Interestingly, he followed up with this work by attempting to find a bacterium as a candidate for dog distemper but was unsuccessful in this endeavor, as well.

However, from a follow up experiment originating from this latter work emerged a new finding.  In this new experiment Dr. Twort first subjected some material that he had isolated from the dog distemper work mentioned above, a preparation called glycerinated calf vaccinia material, to a fresh culture medium with agar and incubated the mixture for a while.

Out of this new experiment came a novel finding. Dr. Twort noticed that in certain areas on the agar surface, the mixture turned watery. Out of these watery parts, Dr. Twort had been able to demonstrate two types of behaviors in cultures of bacteria called Micrococcus. The first type grew the Micrococcus readily, and the second type could grow at first, but then would not grow anymore by any conventional means on various agar media available at the time. Dr. Twort then found that if he further incubated these previously non-culturable Micrococcus bacteria for longer periods, the watery bacterial colonies turned into a glassy and transparent appearance. Left in the wake of these disappearing bacterial colonies were merely the presence of so-called fine granules. This finding is strongly indicative of bacterial host cell lysis occurring as a result of the phage infection. He took elements of this glassy transparent granule-filled non-culturable Micrococcus and added them to the culturable Micrococcus, turning them into glassy transparent granules, too. The younger the glassy Micrococcus, the more potent the conversion had been.  He also observed that heating killed this conversion process. 

When he filtered the glassy transparent Micrococcus, it readily passed through the tiniest of the holes in the porcelain filters. The glassy filtrate potently converted the regular culturable Micrococcus into more of the non-culturable glassy type, complete with its granules. Dr. Twort interpreted these findings as a sickness of the Micrococcus, and that the bacterial disease was conferred upon the bacteria by some ultra-microscopic filterable virus.

Further analysis by Dr. Twort showed that this glassy transparent granular filtrate harboring the ultra-microscopic filterable virus could not grow by itself on agar media.  He found that the ultra-microscopic virus needed the Micrococcus bacteria in order to grow. The older the host bacteria, the more difficult it was for the virus to perform the conversion of the bacteria into the glassy transparent type. Interestingly, the virus did not affect laboratory test animals, only the Micrococcus bacteria. These studies, therefore, had been the first written record to demonstrate the presence of the bacteriophages and in their propensity to infect bacteria, killing them by lysis.

Disappointingly, Dr. Twort made a mention in his 1915 paper that due to lack of funding he could no longer pursue this line of work to learn more about how the virus caused an infectious disease in the Micrococcus to make them sick.  Thus, advancement in the field was delayed two more years, at which time Dr. d’Herelle formulated his theory that an invisible microbe killed Shigella bacteria which he had isolated from his dysentery patients. Dr. d’Herelle named these microbes bacteriophages. The bacteriophage infection of bacteria and its lytic effect upon the host became known as the so-called Twort-d’Herelle phenomenon. It spurred many future groundbreaking studies in genetics, molecular biology, microbiology, and biomedical science.

3) How would you describe bacteriophages, and what function do they serve?

In short, a bacteriophage functions to serve its own interests, by making more copies of itself. However, the phages cannot multiply by themselves without a bacterial host. Thus, phages function by infecting bacteria, producing more phage progeny.

These phage infections of bacteria, in general, proceed in the following manner.

The infection of a host bacterium starts first by the binding of the phage to the outside portion of the bacterial cell wall, using a phage attachment protein to connect to a receptor that’s embedded in the bacterium host. Next, the phage often inserts much of its genomic material into the cytoplasm of the bacterium, frequently leaving out the empty phage coats to remain on the outside milieu of the bacterium.

Next, the phage genome will confiscate the internal cellular machinery of the host bacterium in order to undergo a process called biosynthesis, which consists of phage genome replication, RNA production, and protein synthesis. Here, the biosynthesis of bacterial structures is often compromised, focusing primarily on the production of new phage structures. Eventually, the bacterium will lose its life at the expense of making phage progeny.

Then, once the productions of the phage protein coats and the genome molecules are completed, the phage then proceeds to assemble together the various viral components (e.g., capsid and genome).  The phage assembly process will necessitate that the genomic material be packaged inside the capsid containers.

The last step in the phage infection mechanism involves a degradation of the bacterial host, a process called lysis, which is a lytic procedure that kills the bacterium and releases the newly made and quite infectious phage. 

The newly released phage starts the infection process all over again by finding a new bacterial host with which to infect. This viral phage infection mechanism is referred to as the lytic cycle.

Sometimes the phage infection process leaves the bacterium alive, converting the host into a lysogen. Here, after phage binding and entry, the nucleic acid genome will integrate in to the bacterial genome, making the phage exist as a so-called prophage stage. In this stage, the prophage can be propagated in the bacterial lysogen virtually indefinitely, growing like a normal bacterium would grow. 

Sometimes, the lysogen-prophage combination will be induced to undergo the lytic cycle again, depending on the environmental conditions.  If conditions of the bacterial lysogen are dire, the cell will go lytic, producing new phage progeny for the purpose of finding a new bacterial host that has a better chance of growing and allowing phage lysogeny to occur.

4) Why are bacteriophages important in the big scheme of things?

Interestingly, phages are important for several reasons. First, because the phages were so easy to propagate in the laboratory—one merely needed only the bacteria hosts, the phages, and some culture media in test tubes or Petri dishes, in order to grow the phages—the phages made great microbes for the study of genetics. In fact, much of the early advances in the field of genetics was due to the ease of the phage culturing. Mutants of both phages and bacteria could easily be made, and, importantly, they could be readily counted. Mapping of the locations of both phage and bacteria genes could be performed, allowing the further development of new systems for regulating genes. The famous lac operon comes to mind.  Here, a genetic element could be used to turn on genes, or turn off, if so desired, to induce expression of RNA and of proteins encoded in the regulated genes.

Another reason phages became important is that such viruses could be easily exploited for cloning genes. Early pioneers of gene cloning readily used phages for this purpose. Such gene cloning, using phages as cloning vehicles, led to early advancements in DNA sequencing technologies invented to elucidate the human genome, mapping the human genes and determining the actual nucleotide sequences for all of the genes.

Soon after the discovery of the phages by Dr. Twort, the idea arose about using phages to destroy bacterial pathogens, a discovery called phage therapy. While early studies were met with variable degrees of success, the enthusiasm for phage therapy declined after the discovery of the antibiotics, such as penicillin.  However, with the emergence of multiple antibiotic resistance in pathogenic bacteria, the notion of phage therapy has once again become an important avenue for treatment of infectious bacterial diseases.  In fact, as of this writing phage therapy is available for treatment against listeriosis by the serious bacteria pathogen called Listeria monocytogenes.  Recently, phage therapy for treatment against infection by certain strains of the so-called methicillin-resistant Staphylococcus aureus (MRSA) has become available.  It is anticipated that phage therapy will become a common mode of infectious disease therapy.

In 2018, the Nobel Prize in Chemistry was awarded to scientific investigators Francis Arnold, Gregory Winter and George P. Smith. Both Drs. Winter and Smith used phages to invent a new technology called phage display. They attached phages with new fittings to a molecular platform and used them as receptors to select better chemotherapeutics and antibody molecules, in order to treat certain diseases like, for example, cancer, autoimmune disorders and bacterial infections. It is said that a new type of cancer treatment emerging from the phage display invention was successfully used to treat President Jimmy Carter’s melanoma, in 2015.

5) What was Frederick Twort also known for? What other discoveries did he come up with?

Frederick William Twort, born in Camberley, England, on the 22nd day of October in the year 1877, earned his medical degree in 1894 from St. Thomas Hospital, housed in London, England. With an interest in learning pathology but needing living expenses, he took the first paying post he could find where he supervised a clinical diagnostic laboratory at St. Thomas. Nevertheless, he managed to find ways to satisfy his interest in the field of pathology during his tenure. Next, Dr. Twort moved to London University at the Brown Institute, becoming professor of bacteriology and successfully conducting research there until a dramatic end came to his career, in 1944.

In addition to discovering the bacteriophage viruses that infected bacterial microbes as their receptive hosts, Dr. Twort had additional scientific discoveries of importance. For instance, in 1907, he published work dealing with bacterial sugar fermentation. He found that certain bacteria could alter their glucoside sugar fermentative behaviors on various indicator culture media, strongly suggesting that bacteria could undergo mutation to produce new variants. Some of these novel bacterial mutants had acquired new abilities to ferment sugars that they had not had before. He noticed that bacteria could adapt to new environments, such as the lack or the presence of abilities to utilize new sugars as nutrients. He reasoned that the acquisition of such novel fermentative behaviors could make harmless bacteria become potentially pathogenic.

In 1908, Dr. Twort also studied a glucoside sugar called ericolin, which inhibited the growth of many types of bacteria, but did not affect the growth of the Mycobacterium tuberculosis, a bacterium referred to at the time as the tubercle bacillus and known to be the cause of the consumption or tuberculosis, a severe type of pneumonia. He used the ericolin compound in culture media to enrich the growth of his tubercle bacilli.

Interestingly, in 1910, Dr. Twort also used the ericolin sugar to purify a clinical isolate of the so-called leprosy bacillus, a bacterium known now as Mycobacterium leprae, the causative agent of the serious disease leprosy. Next, Dr. Twort hypothesized that because the bacilli of tuberculosis and of leprosy were related, then the two distinctive microbes underwent similar metabolic activities.  If such was the case then in Dr. Twort’s mind it predicted that cellular material extracted from one of the bacteria could be used as food for the growth of the other. Thus, he tested this prediction by killing the Mycobacterium tuberculosis and using the dead bacteria as part of a new concoction of culture media to grow the Mycobacterium leprae. The experiment worked.

In another project, Dr. Twort studied the causative bacterial agent of an intestinal disease in cattle, called Johne’s disease, or paratuberculosis. The so-called Johne’s bacillus is known today as Mycobacterium avium. He was the first investigator to culture successfully the Johne’s microbe by also incorporating dead Mycobacterium tuberculosis lysate as a suitable component in laboratory culture media. Using their newly cultured and purified Mycobacterium avium, they inoculated test animals, producing lesions.  From the induced lesions, they recovered the same microbe.  This work was published in a series of papers, starting in 1912. The culturing method for the Mycobacterium avium was then used to develop an effective diagnostic test for the Johne’s disease in cattle.

After the start of the Great War, World War I, Dr. Twort became the director of a medical laboratory, in 1915, while stationed at Salonika, a theatre of war in the Macedonian or Salonica Front.  While there he was refused permission by the ruling Medical Advisory Committee to put his ideas to the test regarding putative causes of dysentery transmission.  Dr. Twort had believed, correctly, that the dysentery problem that was plaguing the allies during the war was due to a bacterium, a form of the so-called bacillary dysentery. Afterwards, in 1921, he published a sort of “tell-all” paper outlining his various ideas regarding the nature of the dysenteric problems. Dysentery had been a dreadful and appalling disaster during the Great War.

During the intervening years between wars, Dr. Twort had formulated the notion that bacteria had evolved from viruses, and that the viruses, in turn, had evolved from even more primitive types of life forms, developing the idea of a pre-phage. Apparently, this line of work did not progress further.

Another area of study by Dr. Twort focused on clay materials as potential candidates for the development of new types of culture media. This particular area of investigation is still an active field of study in modern times.

In 1944, during World War II, Dr. Twort’s research laboratory was destroyed by a German air raid bombing attack, essentially ending his scientific progress. Dr. Twort died on the 20th day of March in the year 1950, at the age of 72.

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