Posted by Written by: Muskan Gupta, Rohit R. Gokhale
Edited by: Abhishek Chari
Disease-causing bacteria have always been a thorn in humanity’s side. Our helplessness against them in the distant past can be measured in the many millions of human lives that were lost to diseases like plague, syphilis, cholera, tuberculosis and other similar horrors. But over time, we have rallied and fought against them, with only a few but trusted weapons. While vaccines and improved sanitation have proven to be a great help in keeping some of them at bay, their use is limited to prevention rather than cure. For curative ability against bacterial diseases, we haven’t had a better therapeutic weapon than antibiotics.
For more than fifty years, the use of antibiotics has made many of us feel almost invincible against a wide range of such maladies. But, with the emerging problem of antibiotic resistance, it looks like we might soon be facing the horrors of the pre-antibiotic era – again! Bacteria can become resistant to not just one but many different types of antibiotics and, amazingly, can even spread this resistance to other species of bacteria. To overcome the dangers of antibiotic resistant bacteria and the diseases they cause, we may need the help of a lesser known, and entirely different kind of therapeutic weapon: bacteriophages.
About 50% of the drugs currently being used to combat bacteria were discovered in the 1950s and 60s – which can be thought of as the ‘golden era of antibiotics’. Since then, overprescription and lack of patient compliance in completing antibiotic courses, have contributed to the evolution of new strains of bacteria that cannot be killed with these antibiotics. Popularly called ‘superbugs’, these bacterial strains have become a source of great concern for doctors and researchers across the globe. Annually, drug resistant infections lead to at least 700,000 deaths worldwide, and this number could rise to 10 million deaths per year by 2050.
But we do not have to panic just yet! The human race is working on another strategy to counter the effect of pathogens, by focussing on bacteriophages. These intriguing organisms are viruses which specifically phage (derived from the Greek word- phagein, meaning to eat) or devour bacteria: colloquially, we refer to them as phages.
1. Artistic rendering of bacteriophage attached to a bacterial cell
[Image by co-author Muskan Gupta and friends (K.M. Kanika, Pulkit Singh, Sachin Sharma and Siddharth Mehdiratta); CC BY-SA 2.0]Ironically, more than a decade before antibiotics were discovered, scientists already knew about phages. The contrasting development of these two revolutionary healthcare technologies represents an interesting paradigm of how biology was and has been driven by rapid success, expectations, geopolitical tensions and war. When phages first came under scientific scrutiny around the beginning of the 20th century, microbiologists were quick to espouse phage-based therapy to control bacterial diseases. The excessive enthusiasm that followed led to many far-fetched assertions about phages when, in reality, not much was known about them!
In the midst of all the hype came the landmark year 1928, when penicillin – the first antibiotic – was discovered. While some researchers in the West did continue working on phages, the overall focus shifted towards developing more antibiotics, as their potency became apparent (starting in World War II). The brisk developments of antibiotics and their astounding efficacy in curing bacterial infections swiftly displaced the concept of phage therapy from the western world. Meanwhile, the application of phage therapy was widely explored in countries like Georgia and Poland in Eastern Europe, besides the erstwhile USSR. Historically, this proved to be quite significant. As the Cold War continued, and the West cut off access to new technologies (including some required for manufacturing antibiotics), the Soviet Union had access to phages as therapeutic agents and could treat diarrhoea, typhoid and bacterial infections of wounds with some success.
But how exactly do bacteriophages kill bacteria? To understand what phages can do, we need to look closely at what they are. From the viewpoint of molecular biology, phages are natural nano-machines that are quite diverse in terms of their size, shape and genomes. But, the most basic structural features common to all the known phages seem to be the presence of a genome packaged inside a protein coat. The detailed visualization of bacteriophages has been made possible with the use of electron microscopy along with other techniques, like X-ray crystallography and cryo electron microscopy, which have been used to study the overall shapes and structures of phages and the bio-molecules they are made of.
While all viruses, and therefore phages too, are composed of biological molecules, it is a bit difficult to think of them as complete living organisms. This is because viruses show some but not all the properties of life. They cannot even reproduce on their own, requiring the biological machinery of a host system (be it bacteria, plant or animal) to reproduce and increase in number. Bacteriophages have this same requirement, categorically depending on different species of bacteria to host them. Bacteriophages are also highly specific, so particular phages can successfully infect only certain bacterial species. This specific relationship of phage and bacteria is pivotal: without an exact match between viral and bacterial proteins, phages cannot even attach themselves – by adsorption – to their favourite bacterial hosts. But once phages successfully attach themselves, they can drive two very different kinds of infections in their target bacteria.
2. Electron microscopy image of a bacteriophage (in terms of size, it is approximately a hundred thousand times smaller than an average house fly.
[Image by AFADadcADSasd; (CC BY 4.0)]
Phages have two modes of replication: Lysogenic and Lytic. After adsorbing onto the bacterial cell surface, they puncture the bacterial cell membrane to create a hole through which the viral genetic material is injected into the cell. If the phage is lysogenic, then its viral genes will integrate into the bacterial genome and the bacterial cell continues to live and reproduce normally. The phage genome that is successively inherited in this way by each of the bacterial progeny cells can make them resistant to phage superinfection. But, if the phage is lytic, things get more interesting: its genes will seize control of the bacterial cell machinery – to replicate and create more copies of the same phage. Phages also have the ability to switch from the lysogenic to the lytic mode under favourable conditions, thereby ‘getting activated’ and rapidly increasing in number. The ‘new born’ viruses that are formed then burst out of the bacterial cell, killing it in the process. This ability of phages to destroy bacterial cells makes them potential weapons for use against disease causing bacteria.
3. Lytic and lysogenic cycle of bacteriophage
[Image by CNX OpenStax; (CC BY 4.0)]
With the emergence of a growing number of multidrug resistant (MDR) bacterial strains, researchers have been forced to look beyond antibiotics for alternatives to treat such infectious diseases. The early 2000s saw phage therapy being taken up more enthusiastically and with a greater sense of purpose by the research community. Recently, therapeutic and prophylactic applications of bacteriophage therapy for hospital-borne and gastroenterological infections have been tested in which either a specific bacteriophage or a phage cocktail has been used. S. aureus, E. coli, Streptococcus spp., P. aeruginosa, Salmonella spp., and Enterococcus spp. are some of the bacteria on which human clinical trials are going on in Poland – with encouraging results being obtained even for MDR strains. Most recently, a 15 year old cystic fibrosis patient who developed a disseminated Mycobacterium abscessus (MABS) infection – a MABS infection that spread to other organs besides the lungs – was successfully treated with a three-phage cocktail therapy. Breakthroughs like this encourage researchers to believe that phage therapy can be used to target other chronic bacterial diseases like tuberculosis and leprosy in the future. Working to improve phage therapies, researchers are also testing ways to continually future-proof them against bacterial resistance.
Bioengineering is an indispensable part of modern-day biological research and development. Till very recently, researchers used natural phages to kill bacterial cells. However, this may not be enough with the bacteria constantly evolving resistance mechanisms. To get the ball back in our court, genetic engineering is being used to create therapeutically useful modifications in phage genomes. Techniques such as bacteriophage recombineering of electroporated DNA (BRED) can create point mutations that have the potential to match bacterial changes as small as a single base pair of DNA. Such methods could enhance the bacteria-killing abilities of phages in many ways. Increasing the host range of bacteria that phages can target and making them work synergistically with antibiotics are just two of the tantalising possibilities on offer.
4. Artistic rendering of a T4 bacteriophage attached to its bacterial host
[Image by co-author Muskan Gupta and friends (K.M. Kanika, Pulkit Singh, Sachin Sharma and Siddharth Mehdiratta). CC BY-SA 2.0]Is this all that bacteriophages have to offer? Definitely not! Using phages to fight bacterial diseases in humans is just one aspect of phage therapy. These multifaceted entities are being manipulated to act as vehicles for carrying genes, proteins or antimicrobial chemicals of our choice. Modified adequately, they can provide efficient drug delivery, gene therapy and even biocontrol of diseases beyond those that affect just the human body, such as agricultural plant diseases caused by bacteria. Bacteriophages also show promising effects in tests that have used them in bioimaging, biosensing, enzyme display and nanomaterial design.
With so many possibilities on the table, you may be wondering why these amazingly talented organisms called bacteriophages are not being used very actively at present. Well, that is what got us thinking too! Historically, there have been several difficulties in bringing phages to the clinic. A huge advantage that antibiotics enjoyed is that they can target and destroy a large spectrum of bacterial species, while phages are strain-specific. Besides this, many biologically important characteristics of phages were not well known. Inadequate purification, processing and storage protocols resulted in low concentrations of the active phage populations and contamination of their cultures often made matters worse. Even today, there are unanswered questions such as the optimal dosage of phage cocktails, their time of action and safe usage. The development of new medications is a stringent process: potential therapeutics have to be thoroughly tested in different model organisms before they can be tested on humans. At the moment, phage research is in its infancy, but enormous research efforts are underway to bring safe and effective versions of them, in the near future, to your neighbourhood pharmacy.
If bacteria can develop resistance against powerful weapons like antibiotics, do you think they will allow the tiny phages to steal their thunder this easily? Bacteria and phages have continuously been at war for survival and are in an arms race to gain any advantage they can over each other. Bacteria are known to have evolved adsorption-blocking mechanisms that prevent phages from even attaching to them. Over time, bacteria have also developed multiple enzymatic defense mechanisms against phages, including the adaptive immune response known as the CRISPR-Cas system, popularly called CRISPR. This system is currently also used as a genome editing tool in biotechnology applications- inspired by the bacterial defence against phages. Bacteria possess six known kinds of CRISPR systems that can be thought of as molecular scissors which chop up the incoming genetic material from invaders such as phages. However, some phages also have a trick up their sleeve! They use anti-CRISPR (Acr) proteins which interact with distinct mechanisms to counter-attack the bacterial defence system. Both the CRISPR and Acr systems are the result of evolutionary processes and the ‘by-products’ of the ancient war being fought between two of the deadliest creatures on earth – bacteria and phages.
Looking to combine the best abilities of bacteria and phages, the Marraffini and Lu labs at Rockefeller University and Massachusetts Institute of Technology, respectively, are focussing on engineering CRISPR-wielding phages. Soon, bio-engineered phages, besides bacteria of course, will have the ability to use this high-precision genetic ‘machete’. In this way, scientists are planning to hijack the arms race between bacteria and phages to benefit humans. High selectivity and the protection of commensal bacteria that are useful to us are the key ideas behind the development of such futuristic biotechnologies.
The emergence of bacteria that are resistant to multiple classes of antibiotics has given a much-needed push to bacteriophage research and, if expert opinions are to be believed, ‘the ship has just set sail’. The historical image of phages as the ‘heavyweights’ of the biological world is now becoming a reality. The time is near, when people will know about “bacteriophage medicines” as well as they know about antibiotics in 2020! This coming age of bacteriophages is likely to propel many transformational advances in healthcare, agriculture, industry and other aspects of human life in the decades to follow.
References: While the original sources for all the information in this article are present as hyperlinks within the body of the text, the authors would like to acknowledge that Wikipedia was very useful in tracing the sources for some of the points referred to in this article and for background information.
Muskan Gupta and Rohit R. Gokhale are undergraduate students at Acharya Narendra Dev College (ANDC), New Delhi, studying in the Department of Biomedical Sciences (BMS). This article was written as part of online training in science writing provided by IndSciComm. Artworks 1 and 4, contributed by the artists, were generated by them as part of a Sci-Art workshop conducted by Dr. Lipsa Panda, visual science communicator, on September 19, 2019 at ANDC.