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Phage Philes: Part 1

Introduction to Bacteriophage

 

Bacteriophages: What are they?

Bacteriophage means “bacteria eater.” Referred to simply as phages, these microbes are viruses that infect and kill bacteria. As the most common biological entity in nature, phages have been responsible for constraining bacterial growth since microbial life emerged some 3.5 billion years ago. Remarkably, phages kill up to 40% of all marine bacteria every 24 hours.(1)

Bacteriophages may be the most abundant thing you have never heard of. Estimates suggest something like 4.8 x 10(31) phages sprinkled across the world.(1) That is 4.8 nonillion or 48,000,000,000,000,000,000,000,000,000,000 bacteria-killing machines. This inconceivably large number is the equivalent of about one trillion phages for every grain of sand on Earth.

Several thousands of phage varieties exist, each specialized to infect only one or a few types of bacteria. Like other viruses, phages cannot replicate independently and must hijack bacteria’s cellular machinery. To do this, they attach to the outside of a bacterium and inject genetic material inside with directions to assemble new viral particles, which are all released when the bacterial cell lyses, or bursts open.

Where are all of these bacteria-busting viruses hiding? Phages are found wherever bacteria are present. From the bottom of the ocean to lakes and streams, deep in the soil, and even water treatment plants, phages are just about everywhere. Unpolluted freshwater has about 10 million phages in just one drop.(2) Phages will also hitch a ride on plants and animals, and that’s right, humans, too. But don’t worry, phages are harmless to humans. They are only interested in snacking on bacteria.

Over the past decade, the human microbiome (all the bacteria, viruses, fungi, and archaea in and on the body) has been a growing field of research. And along the way, we have learned about the human “phageome,” the collection of phages that regularly reside on people. Researchers have detected phages in healthy individuals’ gut, mouth, lungs, urinary tract, and skin. In fact, one key to good health may be a healthy phageome.(3) Disruptions of this set of common phages shared by healthy individuals are associated with obesity, bowel disorders, acne, and other diseases.

 

Phage discovery: A brief history

Félix d’Hérelle, a French-Canadian microbiologist, first published reports about the bacteria-killing viruses for which he coined the term ‘bacteriophage’ in 1917. He quickly recognized that phages might be clinically valuable to control bacterial growth and prevent deadly infections. In the first documented use of phage therapeutically, d’Hérelle administered a phage concoction to four sick children at a hospital in Paris, successfully treating them for dysentery in 1919. Throughout the early 20th century, phage therapy centers and commercial phage production plants opened across Europe and India, treating people for dysentery, cholera, bubonic plague, and other bacterial illnesses. Despite early successes, phage therapy fell out of popularity in Western medicine as antibiotics became more readily available. Notably, phage-based research and treatments continued unabated in some regions, including the former Soviet Union and Poland.(4)

 

Rediscovering the power of phage

Today, the popularity of phages is rising once again in Western medicine. With antibiotic-resistant bacteria becoming a global health crisis, doctors are again turning to bacteriophages to handle deadly bacteria that don’t respond to other measures.(5)

Phages became classified as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration (FDA) for food safety in the early 2000s. The food industry now commonly uses phage sprays to prevent bacterial contamination. Phage formulations applied to food packaging and the food itself (including meat, dairy, fruits, and vegetables) control the growth of foodborne pathogens, including Salmonella spp., Listeria monocytogenes, and Escherichia coli, to name a few. The use of these commercial phage preparations keeps food fresh from farm to fork and curbs the occurrence of foodborne illnesses.

Clinically, phages have proven to be life-saving when antibiotics fall short. In 2016, with emergency use authorization from the FDA, Tom Patterson became one of the first patients in the US to receive phage therapy after his deadly infection proved resistant to all known antibiotics. He quickly bounced back from his illness after receiving a series of custom phage cocktails intravenously. While systemic use of phages is still only done on a case-by-case basis in the US, physicians in Russia, Georgia, and Poland regularly administer phages to manage uncontrollable superbugs found in infections. These successes over the past century support bacteriophages as an essential therapeutic agent for controlling bacterial growth moving forward.

Throughout the Phage Philes, we will investigate many topics related to bacteriophages.

  • Examine the details of phage biology: What are phages? How do they infect bacteria? How can we be sure they don’t infect humans?

  • Explore applications for phages: How have phages been used therapeutically in humans? What else can they help us with?

  • Compare phages with other antibacterial measures: What are the benefits of phages? Could they really replace antibiotics?

  • Take a deep dive into the human phageome: Where do people encounter phage? How do phages interact with humans and their microbiome?

Want to learn more about bacteriophages? Check out these sources:

1 Wittebole, X., De Roock, S. & Opal, S. M. A historical overview of bacteriophage therapy as an alternative to         antibiotics for the treatment of bacterial pathogens. Virulence 5, 226-235, doi:10.4161/viru.25991 (2014).

2 Bergh, O., Børsheim, K. Y., Bratbak, G. & Heldal, M. High abundance of viruses found in aquatic environments.   Nature 340, 467-468, doi:10.1038/340467a0 (1989).

3 Zárate, S., Taboada, B., Yocupicio-Monroy, M. & Arias, C. F. Human Virome. Archives of Medical Research 48,  701-716, doi:https://doi.org/10.1016/j.arcmed.2018.01.005 (2017).

4 Sulakvelidze, A., Alavidze, Z. & Morris, J. G., Jr. Bacteriophage therapy. Antimicrob Agents Chemother 45, 649-659, doi:10.1128/aac.45.3.649-659.2001 (2001).

5 Kortright, K. E., Chan, B. K., Koff, J. L. & Turner, P. E. Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria. Cell Host & Microbe 25, 219-232, doi:10.1016/j.chom.2019.01.014 (2019).

Phage Philes: Part 2

Phage Biology: The Life of Phage

 

Living in a phage-filled world

Bacteriophages, viruses that infect and kill bacteria, are the most numerous entities on the planet. Yet, despite how abundant and prevalent phages are, these tiny bacteria-parasitizing virions weren’t discovered until the early 1900s. Since then, we have learned a lot about the fundamentals of phages, like what they are and how they work. To this day, scientists continue to explore the immense diversity of phages.

Mysterious antibacterial activity

At the turn of the 20th century, scientists initially noticed bacteriophages for their ability to kill bacteria, but it wasn’t immediately clear what they had found. They recorded cases where certain water or fecal filtrates could inhibit the growth of bacteria, which is now referred to as the “bacteriophage phenomenon.” Early hypotheses debated whether it was a protein, a chemical, a virus, or some other substance responsible for the observed bacterial death. Without knowing exactly what he was working with, Félix d’Hérelle, a French-Canadian microbiologist, called the mysterious bacteria-eating microbes “bacteriophages,” a name that stuck.

Over the following decades, researchers continued to characterize phage behavior and applications. Finally, in 1940, German scientists had a powerful enough microscope to see what was eliminating bacteria—these tiny, peculiar bacteriophage particles. With time, the phage photos circulated among scientific communities across the globe.

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Taking a closer look at phages
Like other viruses, phages are not cellular. Instead, they have genetic material inside a protein shell called a capsid head.

 

Phages have immense biodiversity. They are typically categorized based on the type of genetic material they have and their morphology (how they appear under a microscope). 

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Depending on the phage type, the genetic information can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), either single or double-stranded. To date, a majority of characterized phages have DNA genomes.

 

Over 96% of known phages are Caudovirales, or “tailed viruses.”(1) Giving phages the look of a lunar lander, the tail is an appendage stemming off the capsid head capped with little leg-like projections called tail fibers.(2) The most studied families of tailed phages include Myo-, Sipho-, and Podoviridae types with varying tail morphology. Myoviridae have a long contractile tail, while Siphoviridae and Podoviridae have noncontractile tails that are long and short, respectively. (What type of phage is shown under the microscope in the image above? Keep reading to find out!)

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The capsid head of tailed phages usually appears spherical at a glance, but many are actually icosahedral shaped with 20 triangular faces. Notably, capsid structures are often symmetrical, with repeating protein subunits on each side. These highly ordered protein shells house and protect the phage’s genetic information. In fact, DNA packed tightly inside can actually expand the capsid a bit.

Other phages like Corticoviridae and Microviridae have semi-spherical capsids without any tail. Filamentous phages (Inoviridae) are also tailless but with a long wormy-shaped capsid. Interestingly, fifteen types of non-tailed phages are known, yet they make up only 4% of inspected phages.1 It is still unclear to scientists why this might be, but they wonder if tailed phages are most easily recognized under the microscope or if the tail has some major benefits for the phages (more below on what the tail is for). 

Over the past 80 years, scientists have primarily relied on morphological traits to classify phages. However, in recent years, advancements in DNA sequencing technology have allowed for an even closer inspection of phages and more detailed characterization. Genetic analysis has revealed a rather complicated web of evolutionary relationships among phages. Interestingly, even phages that appear similar at a glance can have wildly different genetic markers, suggesting they are not as closely related as initially thought. Scientists continue to refine the classification system of phages as more are discovered and investigated. 

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Enter phage: how bacteria get infected
Like other viruses, phages are non-living entities that cannot reproduce independently; they depend on bacterial hosts to replicate and survive. To expand their populations, phages must infect bacteria.(3)


Phages are very particular about what bacterium they will parasitize. Each phage type has a narrow host preference, usually only select species or even strains of bacteria. For example, one of the most well-characterized phages, called T4, exclusively infects Escherichia coli. 


To achieve this remarkable specificity, bacteriophages use an elaborate “lock and key” recognition system to identify bacterial cells of interest. That is, a phage recognizes and binds the target microbe through specific molecules that coat the bacterium. 


Various proteins and other molecules on the outside of the bacterium make it recognizable to its phage predator including polysaccharides, outer membrane proteins, and components of flagella and pili structures, to name just a few. 


To detect and discern their prey, phages use so-called receptor binding proteins (RBPs) at the phage’s tail-end. Some tails have intricate structures at the tip, including baseplates, spikes, and fibers to guide specific binding to the bacterial host.


In fact, host recognition is a complex process where many tail proteins interact with multiple different surface molecules on the bacteria. For example, Escherichia coli is targeted through lipopolysaccharide (LPS) and OmpC porin proteins by the T4 phage.(4) On the other hand, a siphophage called iEPS5 attaches to Salmonella by recognizing FliC and FljB proteins of the bacterium’s flagellum propeller.(5) These redundant mechanisms ensure the correct bacterium is selected and infected to replicate the phage successfully. 


Once a phage finds and attaches to a suitable bacterial cell, the infection process continues. Enzymes on the tip of the phage’s tail eat away at the exterior of the bacteria. These same types of enzymes allow phages to degrade and infiltrate biofilms, the tough tight-knit bacterial colonies that can contribute to antibiotic resistance. 


After digging through the outside layer, phages inject their viral genetic material into a bacterial cell. In addition to facilitating recognition, the tail is also a conduit connecting the virus to the target microbe. The phage’s genetic material passes through the tubular tail and enters the bacterium. 

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To kill or not to kill: lytic or lysogenic phagesWhat happens once a phage has injected its DNA (or RNA) into a bacterium? Very rapidly, the bacterial cell turns into a phage manufacturing plant. The cellular machinery is hijacked to produce new phage components that quickly assemble to form new phages. When they get the signal, the fresh phages burst the bacterium. Phage multiplication happens in minutes to hours, and over a thousand viral particles spew from the dead bacterium. Other bacteria in the immediate area are now at risk of being infected next. 

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Virulent phages reproduce through this deadly process that is known as the “lytic cycle.” Along with generating many new phage particles, the lytic cycle also breaks open (or lyses) the cell, resulting in the death of that bacterium. Notably, therapeutic applications of phages, commonly referred to as “phage therapy,” depend on the lytic cycle to rapidly kill infected bacteria.

For this reason, so-called temperate phages that follow the “lysogenic cycle” are not typically useful for antibacterial applications. Instead of killing microbes, temperate phages will infect bacteria and enter a dormant state. The genetic material quietly integrates into the bacterial genome instead of starting phage replication. In most cases, the bacterial cell is not affected by the infection of a temperate phage and will continue to grow as usual. The dormant phage genes may eventually reactivate in certain circumstances to produce phage particles.

Key points:

  • Phages have immense biodiversity. Scientists continue to discover and investigate new phages.

  • Phages are highly specific with what cells they will parasitize. They don’t recognize or infect human cells and are very selective in the species or strain of bacteria they will choose.

  • Virulent phages reproduce through the lytic cycle that rapidly kills the bacteria. In the lysogenic cycle, temperate phages go dormant and don’t usually kill the bacteria.

Still wondering? The phages shown under the microscope are Siphoviridae! These are the long, non-contractile-tailed bacteriophages found in DermaPhage® CA .

To learn more about the history and possibilities of phage therapy using lytic phages, see the next post.

 

References

1 Ackermann, H. W. 5500 Phages examined in the electron microscope. Archives of Virology 152, 227-243, doi:10.1007/s00705-006-0849-1 (2007).

2 Dion, M. B., Oechslin, F. & Moineau, S. Phage diversity, genomics and phylogeny. Nature Reviews Microbiology 18, 125-138, doi:10.1038/s41579-019-0311-5 (2020).

3 Stone, E., Campbell, K., Grant, I. & McAuliffe, O. Understanding and Exploiting Phage-Host Interactions. Viruses 11, doi:10.3390/v11060567 (2019).

4 Washizaki, A., Yonesaki, T. & Otsuka, Y. Characterization of the interactions between Escherichia coli receptors, LPS and OmpC, and bacteriophage T4 long tail fibers. Microbiologyopen 5, 1003-1015, doi:10.1002/mbo3.384 (2016).

5 Choi, Y., Shin, H., Lee, J. H. & Ryu, S. Identification and characterization of a novel flagellum-dependent Salmonella-infecting bacteriophage, iEPS5. Appl Environ Microbiol 79, 4829-4837, doi:10.1128/aem.00706-13 (2013).

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