Virus World

Mammalian cells outpace bacteriophages in the microbial food chain by devouring phages to fuel their growth. The human gut is a bustling highway for a highly diverse microbial community, including an abundance of bacteriophages that modulate the gut microbiome. It’s a phage-infect-bacteria world, and while bacteriophages cannot infect mammalian cells, their paths still intersect. Mammalian cells can engulf phages within the gut. Researchers have observed that different bacteriophages induce opposing reactions such as anti- or proinflammatory responses in mammalian cells. However, it is unclear how bacteriophages interact with cells and modulate these cellular and immune responses. Jeremy Barr, a bacteriophage biologist at Monash University, and his team set out to clarify whether or not phages activate inflammatory pathways. Their findings, published in PLOS Biology, demonstrated that mammalian cells engulfed bacteriophages to fuel cellular growth without inducing inflammation....

No one had ever seen one virus latching onto another virus, until anomalous sequencing results sent a UMBC team down a rabbit hole leading to a first-of-its-kind discovery. It's known that some viruses, called satellites, depend not only on their host organism to complete their life cycle, but also on another virus, known as a "helper," explains Ivan Erill, professor of biological sciences. The satellite virus needs the helper either to build its capsid, a protective shell that encloses the virus's genetic material, or to help it replicate its DNA. These viral relationships require the satellite and the helper to be in proximity to each other at least temporarily, but there were no known cases of a satellite actually attaching itself to a helper—until now. In a paper published in The ISME Journal, a UMBC team and colleagues from Washington University in St. Louis (WashU) describe the first observation of a satellite bacteriophage (a virus that infects bacterial cells) consistently attaching to a helper bacteriophage at its "neck"—where the capsid joins the tail of the virus. In detailed electron microscopy images taken by Tagide deCarvalho, assistant director of the College of Natural and Mathematical Sciences Core Facilities and first author on the new paper, 80 percent (40 out of 50) helpers had a satellite bound at the neck. Some of those that did not had remnant satellite tendrils present at the neck. Erill, senior author on the paper, describes them as appearing like "bite marks." "When I saw it, I was like, I can't believe this," deCarvalho says. "No one has ever seen a bacteriophage—or any other virus—attach to another virus."

A long-term virus relationship

After the initial observations, Elia Mascolo, a graduate student in Erill 's research group and co-first author on the paper, analyzed the genomes of the satellite, helper, and host, which revealed further clues about this never-before-seen viral relationship. Most satellite viruses contain a gene that allows them to integrate into the host cell's genetic material after they enter the cell. This allows the satellite to reproduce whenever a helper happens to enter the cell from then on. The host cell also copies the satellite's DNA along with its own when it divides. A bacteriophage sample from WashU also contained a helper and a satellite. The WashU satellite has a gene for integration and does not directly attach to its helper, similar to previously observed satellite-helper systems. However, the satellite in UMBC's sample, named MiniFlayer by the students who isolated it, is the first known case of a satellite with no gene for integration. Because it can't integrate into the host cell's DNA, it must be near its helper—named MindFlayer—every time it enters a host cell if it is going to survive. Given that, although the team did not directly prove this explanation, "attaching now made total sense," Erill says, "because otherwise, how are you going to guarantee that you are going to enter into the cell at the same time?" Additional bioinformatics analysis by Mascolo and Julia López-Pérez, another Ph.D. student working with Erill, revealed that MindFlayer and MiniFlayer have been co-evolving for a long time. "This satellite has been tuning in and optimizing its genome to be associated with the helper for, I would say, at least 100 million years," Erill says, which suggests there may be many more cases of this kind of relationship waiting to be discovered.

Original research published in ISME (October 31, 2023):

https://doi.org/10.1038/s41396-023-01548-0 

The Danish creeks, Odense Å and Lindved Å, have surprised researchers and students at SDU by containing previously unknown virus species. "We have found five new species that we believe are unknown to science," said associate professor Clare Kirkpatrick, who studies bacterial stress-response at the Department of Biochemistry and Molecular Biology at University of Southern Denmark. The somewhat surprising discovery was made during the coronavirus pandemic, when some of Kirkpatrick's students could not carry out their normal microbe studies in the laboratory and therefore went on field trips to local creeks to see if they had any interesting microbes to offer. The fact that viruses exist in nature is not surprising, as they are the world's most widespread organism. They are everywhere and part of all kinds of microbial cycles and ecosystems, but the fact that five potentially new species have appeared in local creeks, did surprise Clare Kirkpatrick. While four of the five have not yet had their genome mapped in a genome sequencing, one species has now been fully sequenced, scientifically described, named and published in Microbiology Resource Announcements. The name is Fyn8. Many viruses are so-called bacteriophages (or phages), meaning that they kill bacteria, and Fyn8 is no exception. It can attack and kill the bacteria Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a bacterium found naturally in soil and water. It is normally harmless towards healthy people, but like many other bacteria it has developed resistance to antibiotics and is found in hospitals. For example, patients with wounds (like burn patients) and ventilator patients are at risk of getting an infection that cannot be fought with antibiotics. The researchers have no doubt that Fyn8 can effectively kill Pseudomonas aeruginosa: "We could see it with the naked eye: Clear holes appeared in the layer of Pseudomonas aeruginosa bacteria in our petri dishes, where Fyn8 had infected the bacterial cells, killed them, multiplied and proceeded to attack the next." Considering that the world is facing a resistance crisis, where more people will die from an infection with resistant bacteria than from cancer, the new finding is of course interesting and raises an significant question: Can phages help us in the fight against resistant bacteria? Research in this field has been uncommon until recently, both in academic research institutions and in pharmaceutical companies. In the past and in other parts of the world however, there has been some research, and phages have also been used to treat infections in Eastern European countries in particular.

The phages were discovered at the beginning of the 20th century by researchers who had their bacterial cultures destroyed by virus infections. The benefits of that discovery were obvious, but antibiotics, not phages, became the most widespread cure against bacterial infections. One reason was perhaps that antibiotics were easy to produce and easy to use, while the phages were difficult to isolate and give to patients. Another reason was probably also that an antibiotic dose could kill many different bacteria, while a phage only matches with a single bacterial species. "But today it is relatively easy to make precision medicine for the individual patient. First you find out what exact bacteria a patient is infected with—and then you can treat the patient with exactly the phage that will kill the bacteria," explained Clare Kirkpatrick. She adds that this strategy works even on bacteria which are resistant to all known antibiotics. Time will tell whether there are more new virus species in the local creeks near University of Southern Denmark campus, but it is quite probable, Clare Kirkpatrick believes that "many, many more are waiting to be discovered."

Research published in Bacteriophages (Feb. 13, 2023):

Phages probably picked up DNA-cutting systems from microbial hosts, and might use them to fight other viruses. A systematic sweep of viral genomes has revealed a trove of potential CRISPR-based genome-editing tools. CRISPR–Cas systems are common in the microbial world of bacteria and archaea, where they often help cells to fend off viruses. But an analysis1 published on 23 November in Cell finds CRISPR–Cas systems in 0.4% of publicly available genome sequences from viruses that can infect these microbes. Researchers think that the viruses use CRISPR–Cas to compete with one another — and potentially also to manipulate gene activity in their host to their advantage. Some of these viral systems were capable of editing plant and mammalian genomes, and possess features — such as a compact structure and efficient editing — that could make them useful in the laboratory. “This is a significant step forward in the discovery of the enormous diversity of CRISPR–Cas systems,” says computational biologist Kira Makarova at the US National Center for Biotechnology Information in Bethesda, Maryland. “There is a lot of novelty discovered here.”

DNA-cutting defences

Although best known as a tool used to alter genomes in the laboratory, CRISPR–Cas can function in nature as a rudimentary immune system. About 40% of sampled bacteria and 85% of sampled archaea have CRISPR–Cas systems. Often, these microbes can capture pieces of an invading virus’s genome, and store the sequences in a region of their own genome, called a CRISPR array. CRISPR arrays then serve as templates to generate RNAs that direct CRISPR-associated (Cas) enzymes to cut the corresponding DNA. This can allow microbes carrying the array to slice up the viral genome and potentially stop viral infections. Viruses sometimes pick up snippets of their hosts’ genomes, and researchers had previously found isolated examples of CRISPR–Cas in viral genomes. If those stolen bits of DNA give the virus a competitive advantage, they could be retained and gradually modified to better serve the viral lifestyle. For example, a virus that infects the bacterium Vibrio cholera uses CRISPR–Cas to slice up and disable DNA in the bacterium that encodes antiviral defences2. Molecular biologist Jennifer Doudna and microbiologist Jillian Banfield at the University of California, Berkeley, and their colleagues decided to do a more comprehensive search for CRISPR–Cas systems in viruses that infect bacteria and archaea, known as phages. To their surprise, they found about 6,000 of them, including representatives of every known type of CRISPR–Cas system. “Evidence would suggest that these are systems that are useful to phages,” says Doudna. The team found a wide range of variations on the usual CRISPR–Cas structure, with some systems missing components and others unusually compact. “Even if phage-encoded CRISPR–Cas systems are rare, they are highly diverse and widely distributed,” says Anne Chevallereau, who studies phage ecology and evolution at the French National Centre for Scientific Research in Paris. “Nature is full of surprises.”

Small but efficient

Viral genomes tend to be compact, and some of the viral Cas enzymes were remarkably small. This could offer a particular advantage for genome-editing applications, because smaller enzymes are easier to shuttle into cells. Doudna and her colleagues focused on a particular cluster of small Cas enzymes called Casλ, and found that some of them could be used to edit the genomes of lab-grown cells from thale cress (Arabidopsis thaliana), wheat, as well as human kidney cells. The results suggest that viral Cas enzymes could join a growing collection of gene-editing tools discovered in microbes. Although researchers have uncovered other small Cas enzymes in nature, many of those have so far been relatively inefficient for genome-editing applications, says Doudna. By contrast, some of the viral Casλ enzymes combine both small size and high efficiency. In the meantime, researchers will continue to search microbes for potential improvements to known CRISPR–Cas systems. Makarova anticipates that scientists will also be looking for CRISPR–Cas systems that have been picked up by plasmids — bits of DNA that can be transferred from microbe to microbe. “Each year we have thousands of new genomes becoming available, and some of them are from very distinct environments,” she says. “So it’s really going to be interesting.”

Published in Nature (Nov. 23, 2022): https://doi.org/10.1038/d41586-022-03837-8

For the first time, bacteriophages — viruses that kill bacteria — eliminated an antibiotic-resistant mycobacterial lung infection in a man with advanced cystic fibrosis (CF) lung disease, a study reported. As a result, the patient was able to undergo a successful lung transplant after on.  “I am so grateful for the effort, persistence and creativity of all the people who were involved in my treatment,” Jarrod Johnson, the recipient of the lung transplant, said in a press release. “I thought I was going to die. They have literally saved my life.” Added Jerry Nick, MD, study lead and director of the adult CF program at National Jewish Health in Colorado: “This research can serve as a road map for future use of phages to treat patients with severe Mycobacterium abscessus lung infection and to save lives.” The study, “Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection,” was published in Cell. The buildup of thick mucus in the lungs is a hallmark of CF, which can lead to recurrent and damaging bacterial infections and eventually lung failure, requiring transplant. Mycobacteria are a common type of bacteria that can cause tuberculosis, leprosy, and non-tuberculous mycobacterial (NTM) infections. They can be aggressive and difficult to treat, even with combinations of multiple antibiotics over long periods. Johnson is a 26-year-old man with CF who experienced repeated lung infections throughout childhood and, as a result, was admitted to hospitals several times a year. As an adult, his lung function rapidly declined due to persistent infection by Mycobacterium abscessus, which did not respond to antibiotic treatment. It recently dropped below 30% and he needed a transplant. However, due to the ongoing mycobacteria infection, which had spread to his skin and other tissues, and the lack of effective treatments, he was unable to undergo the procedure.

Bacteriophages, or phages, are viruses that selectively target and kill bacteria. There has been a growing interest in using phages to treat antibiotic-resistant bacterial infections in recent years. To date, phages have not been used to treat a mycobacterial infection of the lungs. In this report, Nick and colleagues describe how Johnson’s mycobacteria infection was eradicated by phage therapy, which allowed him to undergo a lung transplant. “We had tried unsuccessfully for years to clear the mycobacterial infection with a variety of antibiotics,” Nick said. “When we used the bacteria’s own natural enemies, we were able to clear the infection which resulted in a successful lung transplant.” Sputum samples were collected from Johnson, M. abscessus was grown in culture, then screened against a panel of phages, which were genetically engineered to improve their bacteria-killing potential. Two specific phages were identified that efficiently killed the bacteria, either alone or in combination, over a wide range of phage and bacterial levels. While he was in the hospital for an acute worsening of his symptoms, the therapeutic phage cocktail was infused into Johnson’s bloodstream twice daily. It was well tolerated, with no reported side effects. He was discharged five days later and continued on two phage treatments a day. Johnson was readmitted to the hospital four times in the year after starting phage therapy because of pulmonary exacerbations. Sputum samples were collected for up to a year, which were largely positive for M. abscessus to day 96 (about three months). However, most of the following cultures were negative for up to one year. At around three months, he began CF treatment with Trikafta.

Although his lung function ranged from 23% to 31% over the year following phage therapy, similar to before treatment, his time in the hospital decreased. Based on the absence of M. abscessus infection, he was listed for a lung transplant one year after phage therapy and underwent a successful transplant soon after, in October 2021. Phage therapy continued throughout his recovery for up to 500 days (about 16 months). CT scans of his lung during phage therapy showed some improvements over time, along with markers for M. abscessus death. There was no evidence of resistance to phage therapy. All M. abscessus samples isolated over the course of his infection were tested against 20 antibiotics, most of which were consistently resistant or demonstrated little variability, “suggesting the combination of phage and antibiotics did not result in greater antibiotic resistance,” the researchers wrote. Lastly, because phage treatment can fail due to the emergence of anti-phage antibodies, his blood was collected and tested. Surprisingly, Johnson had a substantial antibody response to both phages even before treatment, suggesting that “this individual had naturally encountered phages,” the researchers noted. Antibody levels increased modestly in post-treatment blood samples, but he did not mount a substantial additional antibody response to one of the phages until late in the treatment regimen. At that point, sputum cultures were primarily negative for M. abscessus. “The use of phages in very advanced lung disease with the objective to sufficiently control M. abscessus as a bridge to lung transplant may be considered based on this report,” the researchers wrote. The Cystic Fibrosis Foundation and the National Institutes of Health provided funding for the treatment and study.

Cited research published in Cell (May 13, 2022):

https://doi.org/10.1016/j.cell.2022.04.024 

The achievement promises to unlock the potential for making new classes of chemicals — for medicine, food production, and manufacturing. Two years ago, scientists in Britain swapped out the DNA of the bacteria Escerichia coli for a genetic coding program that was entirely human-made. At the time, it was the largest and most complex synthetic genome ever created. On Thursday, that same group of researchers at the Medical Research Council Laboratory of Molecular Biology reported in Science that with continued tinkering, they’ve made their artificial life form virtually invincible to viral infection. Other adjustments to the bacteria’s designer genome endowed the bug with the ability to string together non-natural amino acids to produce proteins never before seen inside a living cell. In an accompanying editorial, scientists not involved in the work said the achievement promises to unlock the potential for making all sorts of new classes of chemicals — for medicine, food production, and industrial manufacturing.  “It’s brilliant work,” Tom Ellis, director of the Center for Synthetic Biology at Imperial College London, who was not involved in the new study, told STAT via email. “It shows two really exciting applications that can be achieved by having cells with synthetic genomes that have been genetically recoded.” 

DNA, fundamentally, is an information storage system; your DNA stores all the information required to build the molecules and cells and tissues that make you, you. But to actually build those things, you also need a storage-retrieval and decoding system. And on Earth, all of life — from pond scum to peregrine falcons — evolved to use a single system. Every organism decodes DNA using the same 64 codons (three-letter combinations of the DNA bases known as A’s, T’s, C’s, and G’s)  to specify which of the 20 amino acids are going into the proteins it makes. Which means there are redundancies.  So, TCA, for example, tells the cell to grab an amino acid called serine and stick it next in the chain of whatever protein it’s building. But so does TCT. And AGT. In fact, there are six codons that all specify serine.  For years, Jason Chin, a molecular biologist at the MRC lab who led the new study, worked to whittle down some of those redundant codons and assign them new functions. Previously he and his team had systematically eliminated three codons across their E. coli’s genome, creating a bacteria, dubbed Syn61, that lived and squirmed and formed colonies despite using 61 codons rather than nature’s 64. But Syn61 still possessed the genetic program for creating all the molecules that actually read the codons and shuttle the correct amino acid to the site of protein production. These critical decoding molecules are called transfer ribonucleic acids, or tRNAs. In their latest work, Chin and his colleagues went through and deleted the tRNAs that recognize the codons they had already removed. The exercise wasn’t just about further streamlining their synthetic genome. Viruses don’t have any of their own decoding or protein production machinery. They rely on a host cell to do that. And in theory, if a virus invaded a bacteria that didn’t have the ability to read all of the codons in the viral genome, it would stymie the would-be hijacker’s plans for self-replication.  “The hypothesis was that the cells should be completely resistant to viruses,” said Chin. And indeed, that’s exactly what his team found. When mixed together with a cocktail of five different bacteriophages — viruses that prey on bacteria — their genetically reformulated E. coli rebuffed the viral onslaught. Eliminating the tRNAs was crucial. Colonies of their original synthetic strain, Syn61, were blasted apart by the phage invaders.

The National Institute of Allergy and Infectious Diseases awarded $2.5 million in grants to 12 institutions to study using bacteriophage therapy to combat antimicrobial-resistant bacteria. Bacteriophages, or phages, are viruses that target and consume bacteria. They are being used in some cases as a last resort to treat resistant bacterial infections that have not responded to traditional antibiotics.  “In recent decades, multidrug-resistant bacteria, particularly those that cause potentially deadly diseases like tuberculosis, have become a serious and growing global public health concern,” NIAID Director Anthony S. Fauci, MD, said in a press release. “With these awards, NIAID is supporting research needed to determine if phage therapy might be used in combination with antibiotics — or replace them altogether — in treating evolving antibiotic-resistant bacterial diseases.” The NIH said the funding will address knowledge gaps related to phages, including a study characterizing various types of phages, research investigating how phages combat biofilms, and research to determine how to identify new phages. Grant recipients include researchers from the Albert Einstein College of Medicine, University of Alabama at Birmingham, University of Connecticut, Georgia Institute of Technology, Harvard School of Public Health, University of Pittsburgh, Queens College, Texas A&M Agrilife Research, University of Wisconsin-Madison, Geneve Foundation, Guild Associates, and PhagePro.

Large bacteriophages that dwarf the smallest bacteria also carry bacterial genes, including CRISPR and ribosomal proteins, blurring the line between living microbes and viral machines.  These phages — short for bacteriophages, so-called because they “eat” bacteria — are of a size and complexity considered typical of life, carry numerous genes normally found in bacteria and use these genes against their bacterial hosts.  University of California, Berkeley, researchers and their collaborators found these huge phages by scouring a large database of DNA that they generated from nearly 30 different Earth environments, ranging from the guts of premature infants and pregnant women to a Tibetan hot spring, a South African bioreactor, hospital rooms, oceans, lakes and deep underground. Altogether they identified 351 different huge phages, all with genomes four or more times larger than the average genomes of viruses that prey on single-celled bacteria.

Among these is the largest bacteriophage discovered to date: Its genome, 735,000 base-pairs long, is nearly 15 times larger than the average phage. This largest known phage genome is much larger than the genomes of many bacteria. “We are exploring Earth’s microbiomes, and sometimes unexpected things turn up. These viruses of bacteria are a part of biology, of replicating entities, that we know very little about,” said Jill Banfield, a UC Berkeley professor of earth and planetary science and of environmental science, policy and management, and senior author of a paper about the findings appearing Feb 12 in the journal Nature. “These huge phages bridge the gap between non-living bacteriophages, on the one hand, and bacteria and Archaea. There definitely seem to be successful strategies of existence that are hybrids between what we think of as traditional viruses and traditional living organisms.”

Ironically, within the DNA that these huge phages lug around are parts of the CRISPR system that bacteria use to fight viruses. It’s likely that once these phages inject their DNA into bacteria, the viral CRISPR system augments the CRISPR system of the host bacteria, probably mostly to target other viruses. “It is fascinating how these phages have repurposed this system we thought of as bacterial or archaeal to use for their own benefit against their competition, to fuel warfare between these viruses,” said UC Berkeley graduate student Basem Al-Shayeb. Al-Shayeb and research associate Rohan Sachdeva are co-first authors of the Nature paper....

Published in Nature (Feb. 12, 2020):

https://doi.org/10.1038/s41586-020-2007-4 

Researchers from the Utrecht University, in collaboration with colleagues at the University of York (UK) and the Nanjing Agricultural University (China) have developed a new technology to selectively destroy the pathogen that causes the devastating bacterial wilt disease without side effects on other beneficial microorganisms. Bacterial wilt disease caused by Ralstonia solanacearum infects several plants including tomatoes, potatoes, banana and roses. It causes huge economic losses around the world, and to date, no control method is available. 

"Current management of plant diseases rely on aggressive chemicals. These treatments are, however, extremely damaging to beneficial microorganisms that naturally protect plants. As a result, fumigation only provides a brief respite, but pathogens outbreaks become progressively worse after every treatment," plant biologist Alexandre Jousset from the Institute of Environmental Biology at Utrecht University explains.

Precision medicine

"Here, we developed a novel approach using bacteriophages: special viruses that only attack pathogenic bacteria. This precision medicine is fully natural and highly efficient," Jousset says. This new technology provides a long-lasting protection by destroying the pathogen while allowing soil life to recover. Even if pathogens survive the treatments, they are so weakened that they can no longer compete with natural microbes and are thus eradicated. The researchers have published their results in Nature Biotechnology. Bacterial plant diseases such as Ralstonia, Clavibacter, Xyllela, Xanthomonas and Erwinia are major issues for the Dutch agrifood and horticulture sectors. By providing the first solution to these scourges, the study forms the basis of highly efficient and sustainable plant protection methods that make pesticides redundant.

Published in Nat. Biotechnology (2 December 2019):

https://doi.org/10.1038/s41587-019-0328-3

ETH researchers are using synthetic biology to reprogram bacterial viruses—commonly known as bacteriophages—to expand their natural host range. This technology paves the way for the therapeutic use of standardized, synthetic bacteriophages to treat bacterial infections.

Bacteriophages ("phages" for short) are viruses that infect bacteria. Phages are highly host-specific and will typically only infect and kill an individual species or even subspecies of bacteria. Compared to conventional antibiotics, phages do not indiscriminately kill bacteria. Therefore when used as a therapeutic, phages do not cause collateral damage to beneficial "good" bacteria living in the gut. This ability to target only disease-causing bacteria has led to phages being seen as potential "magic bullets" in the fight against bacterial infections, especially against bacteria that have developed antibiotic resistance. However, the high specificity of phages is also a disadvantage: Clinicians have to administer different combinations of phages to be sure the right phage is present to target a single bacterial infection. Not only does this approach limit the chances of phage therapy becoming a standardized treatment option, but also finding a phage, or combination of phages, for every infection becomes a time-consuming and labor-intensive task. Until now, phages had to be first isolated from their natural environment, tested against the bacterial strain(s) in question, and—most importantly—have their genomes sequenced to ensure they are safe for use in humans.

Genetically modified phages

Under the direction of Samuel Kilcher, an "Ambizione" fellow funded by the Swiss National Science Foundation, researchers from the Institute of Food, Nutrition and Health (IFNH) at ETH Zurich have genetically reprogrammed phages to produce synthetic phages that recognize and attack a broader range of bacterial strains beyond their natural host. The researchers reported their findings in the journal Cell Reports.

On the bottoms of phage tails are specialized receptor binding proteins that recognize specific receptors on the exposed cell walls of a target bacterium. "Using X-ray crystallography, we cracked the atomic structure of the first receptor binding protein from a Listeria phage, providing the structural blueprint for re-engineering our phages," says lead author Matthew Dunne.

Akin to building with Lego blocks, the researchers assembled new receptor binding proteins by fitting together protein components derived from different phages to provide different host specificities. Finally, the researchers genetically modified Listeria phages with their designer receptor binding proteins, resulting in phages that recognize and kill new strains of the target bacterium. Although these designer phages attack different new hosts, they all share the same genome, except for the gene encoding their receptor binding proteins.

Phage cocktail as a form of therapy

A mixture of such phage variants could now be used to treat patients. "We could cover a broad range of hosts by administering several synthetically produced phages in a single cocktail," Kilcher explains. The difference to a wild-type phage cocktail is that the synthetic ones could be developed, produced and adapted in a much more targeted fashion. Cultivating artificial phages in pure culture is neither expensive nor labor-intensive. "We can program them accordingly for almost every specific purpose," he adds. Alongside therapeutic applications, the researchers could also use the synthetic phages as diagnostic markers of specific molecular structures, such as for detecting pathogenic strains among a mixed bacterial population...

Studies published in Cell Reports (October 29, 2019):

https://doi.org/10.1016/j.celrep.2019.09.062

Research into bacteriophages adds to evidence that gut and brain interactions influence our behaviour. Viruses are widely regarded as harmful to our health, but a subset of viruses living in the gut could play a crucial role in regulating stress, research suggests. The discovery adds to mounting evidence that interactions between the gut and brain influence people’s behaviours, and could eventually lead to new treatments for stress-related conditions that target the vast community of viruses living inside us. While previous studies have suggested that the composition of microbes living in the gut changes in response to stress, these have largely focused on bacteria, rather than on this “virome”. “The way the virome interacts with bacteria, and how they affect stress-related health and disease status is largely unexplored,” said Dr Nathaniel Ritz, of the APC Microbiome Ireland research centre at University College Cork. “Our research opens up the potential to target the virome to treat and reduce the effects of stress.”

Ritz and his colleagues focused on a subset of viruses called bacteriophages, which infect bacteria and replicate alongside them. They studied what happened to these viruses when the mice they inhabited were exposed to chronic social stresses, such as being housed alone or in overcrowded conditions, and found that stress exposure led to changes in the composition of the viruses and the bacteria in the animals’ guts. Next, they harvested viruses from the droppings of unstressed healthy animals, and transplanted some of them back in, once the mice had been exposed to chronic social stress. The research, published in Nature Microbiology, suggested these transplants reduced levels of stress hormones and curbed depression- and anxiety-like behaviour in the mice. While further studies are needed to assess whether virus transplants are beneficial to humans suffering from stress-related conditions, the research provides some of the first evidence that gut viruses are involved in the response to stress, and that manipulating them could have therapeutic benefits. “Given that the virome composition varies greatly among individuals, it may open the door for personalised medicine approaches for stress-related disorders in the future,” said Prof John Cryan at APC Microbiome Ireland, who led the research. “One thing for certain, we must acknowledge that not all viruses are bad and they can play a key role in keeping the bad bacteria in our gut at bay especially in times of stress.”

Cited research published in Nature Microbiology (Feb.5, 2024):

https://doi.org/10.1038/s41564-023-01564-y 

Researchers create virus-resistant, safely restrained E. coli for medical and industrial applications.  Engineering bacterial genomes with beneficial traits is the goal of many synthetic biology researchers. Now, work from the lab of George Church, PhD, at Harvard Medical School (HMS), reports the construction of an Escherichia coli strain that is not only immune to viral infections but has reduced potential of escaping into the wild. The work may reduce the threat of viral contamination when harnessing bacteria to produce medicines such as insulin as well as other useful substances, such as biofuels. Currently, viruses that infect vats of bacteria can halt production, compromise drug safety, and cost millions of dollars.

Cryo-electron microscopy by University of Alabama at Birmingham researchers has exposed the structure of a bacterial virus with unprecedented detail. This is the first structure of a virus able to infect Staphylococcus epidermidis, and high-resolution knowledge of structure is a key link between viral biology and potential therapeutic use of the virus to quell bacterial infections. Bacteriophages or "phages" is the terms used for viruses that infect bacteria. The UAB researchers, led by Terje Dokland, Ph.D., in collaboration with Asma Hatoum-Aslan, Ph.D., at the University of Illinois Urbana-Champaign, have described atomic models for all or part of 11 different structural proteins in phage Andhra. The study is published in Science Advances. Andhra is a member of the picovirus group. Its host range is limited to S. epidermidis. This skin bacterium is mostly benign but also is a leading cause of infections of indwelling medical devices. "Picoviruses are rarely found in phage collections and remain understudied and underused for therapeutic applications," said Hatoum-Aslan, a phage biologist at the University of Illinois. With emergence of antibiotic resistance in S. epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in potentially using bacteriophages to treat bacterial infections. Picoviruses always kill the cells they infect, after binding to the bacterial cell wall, enzymatically breaking through that wall, penetrating the cell membrane and injecting viral DNA into the cell. They also have other traits that make them attractive candidates for therapeutic use, including a small genome and an inability to transfer bacterial genes between bacteria.

Knowledge of protein structure in Andhra and understanding of how those structures allow the virus to infect a bacterium will make it possible to produce custom-made phages tailored to a specific purpose, using genetic manipulation. "The structural basis for host specificity between phages that infect S. aureus and S. epidermidis is still poorly understood," said Dokland, a professor of microbiology at UAB and director of the UAB Cryo-Electron Microscopy Core. "With the present study, we have gained a better understanding of the structures and functions of the Andhra gene products and the determinants of host specificity, paving the way for a more rational design of custom phages for therapeutic applications. Our findings elucidate critical features for virion assembly, host recognition and penetration." Staphylococcal phages typically have a narrow range of bacteria they can infect, depending on the variable polymers of wall teichoic acid on the surface of different bacterial strains. "This narrow host range is a double-edged sword: On one hand, it allows the phages to target only the specific pathogen causing the disease; on the other hand, it means that the phage may need to be tailored to the patient in each specific case," Dokland said. The general structure of Andhra is a 20-faced, roundish icosahedral capsid head that contains the viral genome. The capsid is attached to a short tail. The tail is largely responsible for binding to S. epidermidis and enzymatically breaking the cell wall. The viral DNA is injected into the bacterium through the tail. Segments of the tail include the portal from the capsid to the tail, and the stem, appendages, knob and tail tip. The 11 different proteins that make up each virus particle are found in multiple copies that assemble together. For instance, the capsid is made of 235 copies each of two proteins, and the other nine virion proteins have copy numbers from two to 72. In total, the virion is made up of 645 protein pieces that include two copies of a 12th protein, whose structure was predicted using the protein structure prediction program AlphaFold.

The atomic models described by Dokland, Hatoum-Aslan, and co-first authors N'Toia C. Hawkins, Ph.D., and James L. Kizziah, Ph.D., UAB Department of Microbiology, show the structures for each protein—as described in molecular language like alpha-helix, beta-helix, beta-strand, beta-barrel or beta-prism. The researchers have described how each protein binds to other copies of that same protein type, such as to make up the hexameric and pentameric faces of the capsid, as well as how each protein interacts with adjacent different protein types. Electron microscopes use a beam of accelerated electrons to illuminate an object, providing much higher resolution than a light microscope. Cryo-electron microscopy adds the element of super-cold temperatures, making it particularly useful for near-atomic structure resolution of larger proteins, membrane proteins or lipid-containing samples like membrane-bound receptors, and complexes of several biomolecules together. In the past eight years, new electron detectors have created a tremendous jump in resolution for cryo-electron microscopy over normal electron microscopy. Key elements of this so-called "resolution revolution" for cryo-electron microscopy are:

• Flash-freezing aqueous samples in liquid ethane cooled to below -256 degrees F. Instead of ice crystals that disrupt samples and scatter the electron beam, the water freezes to a window-like "vitreous ice."
• The sample is kept at super-cold temperatures in the microscope, and a low dose of electrons is used to avoid damage to the proteins.
• Extremely fast direct electron detectors are able to count individual atoms at hundreds of frames per second, allowing sample movement to be corrected on the fly.
• Advanced computing merges thousands of images to generate three-dimensional structures at high resolution. Graphics processing units are used to churn through terabytes of data.
• The microscope stage that holds the sample can also be tilted as images are taken, allowing construction of a three-dimensional tomographic image, similar to a CT scan at the hospital.

The analysis of Andhra virion structure by the UAB researchers started with 230,714 particle images. Molecular reconstruction of the capsid, tail, distal tail and tail tip started with 186,542, 159,489, 159,489 and 159,489 images, respectively. Resolution ranged from 3.50 to 4.90 angstroms.

Cited research published (Nov. 30, 2022) in Science Advances:

https://doi.org/10.1126/sciadv.ade0459 

The number of reported cases using viruses to treat deadly Mycobacterium infections just went up by a factor of five. In a new study, a team led by researchers from the University of Pittsburgh and the University of California San Diego report 20 new case studies on the use of the experimental treatment, showing the therapy’s success in more than half of the patients. The number of reported cases using viruses to treat deadly Mycobacterium infections just went up by a factor of five. In a new study, a team led by researchers from the University of Pittsburgh and the University of California San Diego report 20 new case studies on the use of the experimental treatment, showing the therapy’s success in more than half of the patients.  It’s the largest ever set of published case studies for therapy using bacteria-killing viruses known as bacteriophages, providing unprecedented detail on their use to treat dire infections while laying the groundwork for a future clinical trial. “Some of those are spectacular outcomes, and others are complicated,” said Graham Hatfull, the Eberly Family Professor of Biotechnology in the Kenneth P. Dietrich School of Arts and Sciences at the University of Pittsburgh. “But when we do 20 cases, it becomes much more compelling that the phages are contributing to favorable outcomes — and in patients who have no other alternatives.” Each patient treated in the study was infected with one or more strains of Mycobacterium, a group of bacteria that can cause deadly, treatment-resistant infections in those with compromised immune systems or with the lung disorder cystic fibrosis.

In 2019, Hatfull led a team showing the first successful use of phages to treat one of these infections. “For clinicians, these are really a nightmare: They’re not as common as some other types of infections, but they’re amongst some of the most difficult to treat with antibiotics,” said Hatfull. “And especially when you take these antibiotics over extended periods of time, they’re toxic or not very well-tolerated.”  Last month, the University of Pittsburgh researchers were involved in two new case studies successfully treating patients in collaboration with colleagues at National Jewish Health and Harvard University. But those reports represent only a fraction of the cases the team has been involved in behind the scenes. Since 2019, Hatfull and his lab have fielded requests from more than 200 clinicians looking for treatments for their patients, working with them to find phages that could be effective against the particular strain of bacteria infecting each patient. The newest paper, published today in the journal Clinical Infectious Diseases alongside collaborators from 20 institutions, dramatically scales up the body of published evidence on the effectiveness of the therapy.  “These are incredibly brave physicians, jumping off the ledge to do an experimental therapy to try to help patients who have no other options,” said Hatfull. “And each of these collaborations represents a marker that can move the field forward.” Looking at measures of patient health and whether samples from the patient still showed signs of Mycobacterium infections, the team found that the therapy was successful in 11 out of 20 cases. No patients showed any adverse reactions to the treatment. In another five patients the results of the therapy were inconclusive, and four patients showed no improvement. According to Hatfull, even these apparent failures are key to making the therapy available to more patients. “In some ways, those are the most interesting cases,” he said. “Understanding why they didn’t work is going to be important.” Several unexpected patterns emerged from the case studies. In 11 cases, researchers were unable to find more than one kind of phage that could kill the patient’s infection, even though standard practice would be to inject a cocktail of different viruses so the bacteria would be less likely to evolve resistance.

“If you’d asked me whether that was a good idea three years ago, I would have had a fit,” Hatfull said. “But we just didn’t observe resistance, and we didn’t see a failure of treatment from resistance even when using only a single phage.” In addition, the team saw that some patients’ immune systems attacked the viruses, but only in a few cases did their immune systems render the virus ineffective. And in some instances, the treatment was still successful despite such an immune reaction. The study paints an encouraging picture for the therapy, said Hatfull, and one that opens the possibility for new phage regimens that clinicians could use to maximize the treatment’s chance of success.  Along with the study’s significance to patients facing Mycobacterium infections, it also represents a substantial advance for the wider field of phage therapy. A concern in some corners is that researchers may be only publishing case studies in which phage therapy is successful. “A series of consecutive case studies, where we’re not cherry-picking, is a much more transparent way of looking to see what works and what doesn’t,” said Hatfull. “This adds considerable weight to the sense that the therapy is safe.” The lab continues to provide phages for more patients — while at the same time conducting research to widen the funnel that narrowed an initial group of 200 patients down to 20, in the hopes of providing treatment to more people with no other options. “We’ve not yet figured out how to find or engineer phages that will get every strain of bacteria in these patients,” said Hatfull. “That represents one of the major challenges ahead.”

Research cited published in Clinical Infectious Diseases (June 9, 2022):

https://doi.org/10.1093/cid/ciac453 

Scientists who study glacier ice have found viruses nearly 15,000 years old in two ice samples taken from the Tibetan Plateau in China. Most of those viruses, which survived because they had remained frozen, are unlike any viruses that have been cataloged to date. The findings, published today in the journal Microbiome, could help scientist understand how viruses have evolved over centuries. For this study, the scientists also created a new, ultra-clean method of analyzing microbes and viruses in ice without contaminating it.  “These glaciers were formed gradually, and along with dust and gases, many, many viruses were also deposited in that ice,” said Zhi-Ping Zhong, lead author of the study and a researcher at The Ohio State University Byrd Polar and Climate Research Center who also focuses on microbiology. “The glaciers in western China are not well-studied, and our goal is to use this information to reflect past environments. And viruses are a part of those environments.” The researchers analyzed ice cores taken in 2015 from the Guliya ice cap in western China. The cores are collected at high altitudes – the summit of Guliya, where this ice originated, is 22,000 feet above sea level. The ice cores contain layers of ice that accumulate year after year, trapping whatever was in the atmosphere around them at the time each layer froze. Those layers create a timeline of sorts, which scientists have used to understand more about climate change, microbes, viruses and gases throughout history. Researchers determined that the ice was nearly 15,000 years old using a combination of traditional and new, novel techniques to date this ice core. When they analyzed the ice, they found genetic codes for 33 viruses. Four of those viruses have already been identified by the scientific community. But at least 28 of them are novel. About half of them seemed to have survived at the time they were frozen not in spite of the ice, but because of it.

“These are viruses that would have thrived in extreme environments,” said Matthew Sullivan, co-author of the study, professor of microbiology at Ohio State and director of Ohio State’s Center of Microbiome Science. “These viruses have signatures of genes that help them infect cells in cold environments – just surreal genetic signatures for how a virus is able to survive in extreme conditions. These are not easy signatures to pull out, and the method that Zhi-Ping developed to decontaminate the cores and to study microbes and viruses in ice could help us search for these genetic sequences in other extreme icy environments – Mars, for example, the moon, or closer to home in Earth’s Atacama Desert.”  Viruses do not share a common, universal gene, so naming a new virus – and attempting to figure out where it fits into the landscape of known viruses – involves multiple steps. To compare unidentified viruses with known viruses, scientists compare gene sets. Gene sets from known viruses are cataloged in scientific databases. Those database comparisons showed that four of the viruses in the Guliya ice cap cores had previously been identified and were from virus families that typically infect bacteria. The researchers found the viruses in concentrations much lower than have been found to exist in oceans or soil. The researchers’ analysis showed that the viruses likely originated with soil or plants, not with animals or humans, based on both the environment and the databases of known viruses.

The study of viruses in glaciers is relatively new: Just two previous studies have identified viruses in ancient glacier ice. But it is an area of science that is becoming more important as the climate changes, said Lonnie Thompson, senior author of the study, distinguished university professor of earth sciences at Ohio State and senior research scientist at the Byrd Center. “We know very little about viruses and microbes in these extreme environments, and what is actually there,” Thompson said. “The documentation and understanding of that is extremely important: How do bacteria and viruses respond to climate change? What happens when we go from an ice age to a warm period like we’re in now?” This study was an interdisciplinary effort between Ohio State’s Byrd Center and its Center for Microbiome Science. The 2015 Guliya ice cores were collected and analyzed as part of a collaborative program between the Byrd Polar and Climate Research Center and the Institute of Tibetan Plateau Research of the Chinese Academy of Sciences, funded by the U.S. National Science Foundation and the Chinese Academy of Sciences. Funding also came from the Gordon and Betty Moore Foundation and the U.S. Department of Energy.

Published in Microbiome (July 20, 2021):

https://doi.org/10.1186/s40168-021-01106-w

Some bacteria-killing viruses spell out their genetic instructions in a different DNA alphabet. More than 40 years ago, scientists in Russia reported that a type of bacteriophage called cyanophage S-2L replaces the DNA building block adenine, commonly known as A, with 2-aminoadenine, designated Z. But no one knew how the phage went from A to Z, or why. After decades of wondering, two independent groups of scientists have discovered how the viruses make and build Z into their genetic instructions, and one reason they do it, the teams report in three studies in the April 30 Science.

The findings have implications for the origins of life on Earth, the search for life on other planets and multiple potential applications in biomedicine, synthetic biology, material sciences and computing, says Farren Isaacs, a molecular and synthetic biologist at Yale University who coauthored a commentary in the same issue of Science. “It’s a really fundamental discovery.” In the 1990s, Philippe Marlière, a xenobiologist then at the Pasteur Institute in Paris, was “looking for examples divergent from life as we know it,” when he came across the 1977 Russian study describing the cyanophage with the unusual DNA. After getting a sample of the virus, Marlière and colleagues deciphered the phage’s complete set of genetic instructions, or genome.

In the virus’s genome, the researchers found instructions for building an enzyme, called PurZ, that could carry out the first step in making Z — also known as diaminopurine. The Pasteur Institute filed a patent on the enzyme in Marlière’s name in 2003.

With the enzyme in hand, “it became crystal clear how Z was made, but we didn’t [do] any experiments to prove that we were right,” says Marlière, now president of the European Syndicate of Synthetic Scientists and Industrialists in Berlin. The project was halted for a variety of reasons. The researchers didn’t publish their findings until now, in part, because PurZ wasn’t the enzyme Marlière was looking for. Instead, he says he had hoped to find a different enzyme, a polymerase that would reject adenine and instead build DNA with Z in its place. “I was very, very disappointed,” he says, “because the polymerase I was craving could not be detected in that phage.”   Indeed, this phage’s polymerase isn’t what he was looking for. Marlière’s collaborator Pierre Alexandre Kaminski and colleagues found that cyanophage S-2L’s polymerase isn’t picky about using A or Z. Instead, another viral enzyme called DatZ degrades adenine building blocks, leaving the polymerase no choice but to use Z, Kaminski, a biochemist at the Pasteur Institute, and colleagues report April 23 in Nature Communications. Periodically, Marlière searched genetic databases for other phages that have PurZ and might contain the elusive picky polymerase. Then about four years ago, he says, “I got results. Ding, ding, ding! And I didn’t get just one. I got 12. And bingo, right next door to this PurZ gene was, guess what, a polymerase gene. Aha!”

The Siphoviridae bacteriophages that infect a wide variety of bacteria all have versions of the polymerase, called DpoZ, that preferentially insert Z instead of A into the viruses’ DNA, the researchers report. Marlière has filed a patent on the enzyme. The alternative alphabet may be used far more widely than previously thought, says Huimin Zhao, a synthetic biologist at the University of Illinois at Urbana-Champaign. He first heard about the bacteriophage that use Z-containing DNA at a dinner party a few years ago, he recalls. Not knowing that the French scientists were still working on the puzzle, he also searched databases and found 60 bacteriophages that contain PurZ, including phages from both the Siphoviridae and Podoviridae families. His team also worked out the biochemical pathway the phages use to make and incorporate Z, and found enzymes that degrade A. Just because the phages have the enzymes, they don’t necessarily use Z in their DNA. So Zhao and colleagues in China chose a phage called SH-Ab 15497 that infects Acinetobacter bacteria, and confirmed that its DNA alphabet also has Z in place of A, his team reports.

Replacing As with Zs

Why phages would bother with the unconventional DNA was still unknown. One hypothesis is that replacing A with Z is a countermeasure against bacterial defense enzymes, known as restriction enzymes, that chop up DNA from invading phages. Such enzymes have a hard time recognizing and cutting DNA containing Z bases, Zhao and colleagues found. “The phage is trying to avoid being destroyed by the host,” he says. “This is really a protection mechanism for the phage.” It’s also part of a never-ending arms race between phages and bacteria, says Steven Benner, a chemist and astrobiologist at the Foundation for Applied Molecular Evolution in Alachua, Fla. It’s possible that other phages that use Z or other alternative DNA bases may still be out there. “We have overlooked this life-form on Earth because our molecular tools didn’t allow us to look for it,” he says. “What these guys have done is discover an entire biosphere that was missing from our inventory.” It’s debatable whether Z-containing phages are new forms of life (not to mention the ongoing debate about whether viruses are alive), says Floyd Romesberg, a synthetic biologist at the global pharmaceutical and biotechnology company Sanofi’s site in La Jolla, Calif. But it does open up new possibilities, he says, for what life is, was, and could become. “Life isn’t exactly what we thought it was. Life doesn’t have to be GTAC,” he says, referring to the four letters of the standard DNA alphabet. “What it says is that life can be more diverse.” That realization could influence the search for life on other planets (SN: 4/18/16). Scientists often assume that they should search for guanine, thymine, adenine and cytosine, the bases of DNA as we’ve known it until now. But maybe researchers should be looking for 2-aminoadenine, the Z base, instead, Benner says. After all, Z forms three hydrogen bonds with thymine, instead of the two hydrogen bonds that hold A–T base pairs together. That makes Z–T paired DNA more stable and potentially able to stand up to hotter or harsher conditions than conventional DNA can, he says.

With the extra stability, one might wonder why all organisms on Earth don’t use Z. Stability isn’t everything, Romesberg says. DNA has to be unwound and split apart to be copied. That may be harder to do with Z–T base pairs. Z also changes how DNA curves and bends, perhaps making it harder to pack into tight spaces the way A-containing genetic material can. That might make A more attractive for other organisms. Or perhaps it was just an accident that A came first. Once cells started using that base, too many things would have to change to completely switch to another base, says Romesberg, who has been working for years to get bacteria to incorporate exotic DNA bases (SN: 5/7/14). Cells have a hard time swapping because there are so many different parts that would have to be changed to accommodate a new DNA base. Viruses’ stripped-down genomes are more flexible, Romesberg says: They carry around less machinery because they make the host do most of the work. Even the Z-phage do just the first step in making Z and rely on several host enzymes to finish the recipe. It’s still not known whether cellular organisms can write Z into their DNA, too.

The U.S. Food and Drug Administration (FDA) cleared Armata Pharmaceuticals‘ investigational new drug (IND) application for a Phase 1b/2a clinical trial of AP-PA02 for the treatment of the Pseudomonas aeruginosa bacterial infections that are a hallmark of cystic fibrosis (CF).  “We are very pleased that the FDA has cleared our IND, and we plan to initiate clinical development of AP-PA02 by the end of this year,” Todd R. Patrick, Armata’s CEO, said in a press release. P. aeruginosa can colonize the lungs, causing difficult-to-treat infections, which can severely impact both the quality of life and survival rates among people with CF. Treatment for P. aeruginosa infections typically consists of antibiotics. AP-PAo2, conversely, utilizes a cocktail of viruses called bacteriophages to attack the infecting bacteria. Because phages are specific to bacteria — they cannot infect the cells of the patients’ organs — they cause fewer side effects than antibiotics and can target even antibiotic-resistant microbes. AP-PA02 delivers phages specific to P. aeruginosa directly to the lungs via inhalation.

The new trial — called SWARM-P.a. — is a multi-center, double-blind, randomized, placebo-controlled, single ascending dose (SAD) and multiple ascending dose trial that will assess the safety and tolerability of AP-PA02 in CF patients with chronic pulmonary P. aeruginosa infections. Armata expects to begin testing in the SAD group later this year. Of note, in a double-blind study, neither the participants nor the researchers know who is receiving the investigational therapy and who is given a placebo. “Results from this study, which we are calling SWARM-P.a. to reflect the manner in which phage attack dangerous pathogens, will be our company’s first clinical trial to evaluate a phage-based therapy as a potential treatment for Pseudomonas aeruginosa airway infections,” Patrick said. “This clinical trial will contribute to the evaluation of the potential of phage to combat multi-drug resistant infections, and potentially usher in a new era in the fight to develop alternatives to antibiotics,” he added. The Cystic Fibrosis Foundation is supporting Armata’s research through a $5 million grant, awarded earlier this year.. 

Bacteria and the viruses that infect them are engaged in a molecular arms race as ancient as life itself. Evolution has equipped bacteria with an arsenal of immune enzymes, including CRISPR-Cas systems, that target and destroy viral DNA. But bacteria-killing viruses, also known as phages, have devised their own tools to help them outmaneuver even the most formidable of these bacterial defenses. Now, scientists at UC San Francisco and UC San Diego have discovered a remarkable new strategy that some phages employ to avoid becoming the next casualty of these DNA-dicing enzymes: after they infect bacteria, these phages construct an impenetrable "safe room" inside of their host, which protects vulnerable phage DNA from antiviral enzymes. This compartment, which resembles a cell nucleus, is the most effective CRISPR shield ever discovered in viruses. "In our experiments, these phages didn't succumb to any of the DNA-targeting CRISPR systems they were challenged with. This is the first time that anyone has found phages that exhibit this level of pan-CRISPR resistance," said Joseph Bondy-Denomy, PhD, assistant professor in the Department of Microbiology and Immunology at UCSF. Bondy-Denomy led the research team that made the discovery, which is detailed in a paper published Dec. 9, 2019 in the journal Nature.

The Hunt for DNA That CRISPR Can't Cut

To find CRISPR-resistant phages, the researchers selected viruses from five different phage families and used them to infect a common bacteria that had been genetically engineered to deploy four different Cas enzymes, the DNA-cutting component of CRISPR systems. These CRISPR-fortified bacteria emerged victorious against most of the phages they faced off against. But two jumbo phages -- so named because their genomes are five to 10 times larger than the genomes of the most well-studied phages -- were found to be impervious to all four CRISPR systems. The researchers decided to put these jumbo phages to the test and probe the limits of their CRISPR-resistance. They exposed them to bacteria outfitted with a completely different type of CRISPR, as well as bacteria equipped with restriction-modification systems -- a DNA-cleaving enzyme that's more common than CRISPR (restriction systems are found in about 90 percent of bacterial species, whereas as CRISPR is present in only about 40 percent) -- but which can only target a limited number of DNA sequences. The results were the same as before: petri dishes littered with the exploded remains of phage-infected bacteria. "It was really surprising because we engineered the bacteria to massively overproduce components of the immune system, but none of them could cut the phage DNA. These phages were resistant to all six bacterial immune systems tested. No other phage even comes close," said Bondy-Denomy.

Jumbo phages, it seemed, were virtually indestructible. But test tube experiments suggested otherwise -- jumbo phage DNA was, in fact, as vulnerable to CRISPR and restriction enzymes as any other DNA. The CRISPR resistance that was observed in phage-infected cells had to be the result of something the viruses were producing that interfered with CRISPR. But what?

Found: An Impenetrable CRISPR Shield

Microscope-based experiments finally revealed what was happening. When these jumbo phages infect bacteria, they build a spherical compartment in the middle of the host cell, which keeps antiviral enzymes at bay and provides a "safe room" for the viral genome to replicate. This compartment, it turns out, was identical to one first discovered in 2017 by UCSD Professor Joe Pogliano, PhD, and UCSF Professor David Agard, PhD, both of whom are co-authors of the new study. Though these researchers previously demonstrated that the phage genome replicated in this nucleus-like shell, nobody knew until now that the shell also serves as an impenetrable shield against CRISPR and other DNA-dicers....

Publisjed in Nature (09 December 2019):

https://doi.org/10.1038/s41586-019-1786-y

UC San Diego researchers linked a gut bacteria toxin to worse clinical outcomes in patients with alcoholic liver disease, and discovered that treatment with bacteriophages clears the bacteria and eliminates the disease in mice. 

Bacteriophages (phages) are viruses that specifically destroy bacteria. In the early 20th century, researchers experimented with phages as a potential method for treating bacterial infections. But then antibiotics emerged and phages fell out of favor. With the rise of antibiotic-resistant infections, however, researchers have renewed their interest in phage therapy. In limited cases, patients with life-threatening multidrug-resistant bacterial infections have been successfully treated with experimental phage therapy after all other alternatives were exhausted. Researchers at University of California San Diego School of Medicine and their collaborators have now for the first time successfully applied phage therapy in mice for a condition that’s not considered a classic bacterial infection: alcoholic liver disease. The study publishes November 13, 2019 in Nature.

“We not only linked a specific bacterial toxin to worse clinical outcomes in patients with alcoholic liver disease, we found a way to break that link by precisely editing gut microbiota with phages,” said senior author Bernd Schnabl, MD, professor of medicine and gastroenterology at UC San Diego School of Medicine and director of the National Institutes of Health-funded San Diego Digestive Diseases Research Center. Up to 75 percent of patients with severe alcoholic hepatitis, the most serious form of alcohol-related liver disease, die within 90 days of diagnosis. The condition is most commonly treated with corticosteroids, but they aren’t highly effective. Early liver transplantation is the only cure, but is only offered at select medical centers to a limited number of patients. In fact, there are only approximately 8,000 liver transplants for all reasons in the United States each year, according to the American Liver Foundation, with a waiting list of roughly 14,000 people.  

Alcohol itself can directly damage liver cells. But Schnabl and team had previously discovered that alcohol is also harmful to the liver for a second reason: It diminishes natural gut antibiotics, leaving mice more prone to bacterial growth in the liver and exacerbating alcohol-induced liver disease. In the current study, Schnabl’s team — including many collaborators around the world — addressed two primary questions: How do gut bacteria contribute to liver damage? And can phages be used to reduce the bacteria and thus alleviate alcoholic liver disease?  The researchers discovered that liver cells are injured by cytolysin, a toxin secreted by Enterococcus faecalis, a type of bacteria typically found in low numbers in the healthy human gut. They also found that people with alcoholic hepatitis have more cytolysin-producing E. faecalis in their guts than healthy people. The more E. faecalis present, the more severe their liver disease. For people with alcoholic liver disease, more than five percent of their fecal bacteria were Enterococcus, compared to almost none in healthy people or people with alcoholic use disorder. Approximately 80 percent of alcoholic hepatitis patients have E. faecalis living in their feces, and 30 percent are positive for cytolysin. Moreover, the researchers found that nearly 90 percent of cytolysin-positive patients with alcoholic hepatitis died within 180 days of hospital admission, compared to approximately 4 percent of cytolysin-negative patients.

Next, the team transferred feces from cytolysin-positive and cytolysin-negative people with alcoholic hepatitis to mice. Mice with cytolysin-positive humanized gut microbiomes developed more severe alcohol-induced liver disease and survived less than mice without cytolysin. To investigate the potential for phage therapy, the researchers isolated from sewage water four different phages that specifically target cytolysin-producing E. faecalis. When they treated the mice with the targeting phages, the bacteria were eradicated and alcohol-induced liver disease was abolished. Control phages that target other bacteria or non-cytolytic E. faecalis had no effect. “This phage therapy has only so far been tested in mice, and a clinical trial will be required to test the safety of this approach, and validate our findings in patients with alcoholic hepatitis,” Schnabl said.

Published in Nature (November 13, 2019):

https://doi.org/10.1038/s41586-019-1742-x

Most of the viruses present in people’s guts are bacteriophages, but how they interact with resident bacteria is still an open question. There’s a lot that scientists don’t know about the gut microbiota, and when it comes to the viruses present there they know even less. To learn more, researchers have monitored the gut viromes of nine people for a full year and that of one person for more than two years. They find that many types of bacteriophages are present and that each individual’s virome is stable over time and different from that of the other subjects.

This study “generates an important database for phages in the gut,” says Corrine Maurice, a microbiologist at McGill University who did not participate in the work. “That’s a database that we just didn’t have, and so that data is going to allow us to formulate some really cool hypotheses going forward. It’s really providing us with tools to . . . look further into what these phages may be doing for our health. “It confirms recent reports that there is no such thing as a core gut virome shared between adult individuals, which is in contrast with the bacterial component of our microbiota where there are more members shared between humans,” Evelien Adriaenssens, who studies gut viruses at the Quadram Institute in the UK and was not involved in the work, writes in an email to The Scientist. “We need more studies on the gut virome like these to establish a baseline about what a healthy human gut virome looks like, taking into account differences in for example geography, ethnicity and lifestyle. After we know what is healthy, we can start looking at complex disease syndromes . . . and identify what changes in the virome can be used as a marker for disease.”

There’s a lot that scientists don’t know about the gut microbiota, and when it comes to the viruses present there they know even less. To learn more, researchers have monitored the gut viromes of nine people for a full year and that of one person for more than two years. They find that many types of bacteriophages are present and that each individual’s virome is stable over time and different from that of the other subjects.

This study “generates an important database for phages in the gut,” says Corrine Maurice, a microbiologist at McGill University who did not participate in the work. “That’s a database that we just didn’t have, and so that data is going to allow us to formulate some really cool hypotheses going forward. It’s really providing us with tools to . . . look further into what these phages may be doing for our health.”

“It confirms recent reports that there is no such thing as a core gut virome shared between adult individuals, which is in contrast with the bacterial component of our microbiota where there are more members shared between humans,” Evelien Adriaenssens, who studies gut viruses at the Quadram Institute in the UK and was not involved in the work, writes in an email to The Scientist. “We need more studies on the gut virome like these to establish a baseline about what a healthy human gut virome looks like, taking into account differences in for example geography, ethnicity and lifestyle. After we know what is healthy, we can start looking at complex disease syndromes . . . and identify what changes in the virome can be used as a marker for disease.”

Andrey Shkoporov, a microbiologist at University College Cork, and colleagues set out to establish that baseline. “We thought, ‘Okay, before we embark on comparative studies of the virome in different health conditions, why don’t we look at the longitudinal stability and inter-individual variability of the gut virome between healthy human subjects,’” he tells The Scientist. 

The research team collected fecal samples from 10 adults—four men and six women—every month for a year. From one female subject, they collected three additional samples at months 19, 20, and 26. Then they separated viral particles from fecal matter and cells and isolated and sequenced viral nucleic acids. Because 99 percent of gut viruses are unknown to science, Shkoporov says, it was not possible to rely on existing viral sequence databases to figure out what was there. Instead, the authors assembled the reads into overlapping DNA sequences, predicted protein coding genes, and then tried to detect any similarities between proteins in databases with those likely encoded by the long stretches of DNA. “This can help to get a rough idea what kind of viruses we are dealing with,” Shkoporov adds. The researchers reported yesterday (October 9) in Cell Host & Microbe  that the individual viromes were stable over the 12 or 26 months of the study and diverse, meaning there were many types of bacteriophages present. While the subjects’ viral communities stayed consistent over time, each person’s complement of gut viruses looked different from that of the others.....

Published in Cell Host & Microbe on October 9, 2019:

https://doi.org/10.1016/j.chom.2019.09.009