BACTERIOPHAGES

First World War superbug treatment could save NHS millions – but is blocked by red tape

Joe Pinkstone
Tue 29 October 2024\
THE TELEGRAPH

The use of bacteria-killing viruses known as phages to treat patients dates back to the First World War


William Stocking, 81, has spent much of the last four years in and out of hospital as an infection slowly destroys his leg.

He caught a superbug, an antimicrobial-resistant strain of Staphylococcus aureus, after going to the Royal Devon and Exeter Hospital in early 2020 with a chest infection.

Superbugs are notorious for being hard to treat because of their immunity to antibiotics. Around 52,000 people a year catch superbugs in the UK, causing around 2,000 deaths and costing the NHS around £180 million annually.

However, a little-known treatment dating back to the First World War is available, if NHS doctors are prepared to spend hundreds of hours fighting a mountain of government red tape.

Bacteriophages, known as phages, are bacteria-killing viruses that inject their own DNA into a bacteria to seize control of the cell and produce more phages until the bacteria bursts.

It is an effective killing mechanism and phages are the most abundant entity in the world.

They are highly precise with only specific strains of bacteria targeted by a certain phage, and phages can be effective against superbug infections impervious to all known medication.

Doctors are increasingly looking at phage therapy to help patients who are otherwise out of options, and Mr Stocking is a pioneering patient in the UK.

The superbug bacteria was attracted to the metal in his knee replacement which he had in the late 1980s following a career-ending injury suffered in the line of duty while a sergeant in the Hampshire Constabulary.

William Stocking and his wife Lorraine hope the treatment will allow him to walk properly again - Eddie Mulholland

“It works a bit like [the video game] Pac-Man and goes around eating the infection. It’s been a partial success so far, and time will tell,” Mr Stocking told The Telegraph from his hospital bed at the Royal National Orthopaedic Hospital (RNOH) in north-west London after receiving his third and final dose of phage therapy last week.

“I’ve had numerous pills, potions, antibiotics, transfusions and none of them worked. We have exhausted the available options and are down to phage which was raised as a last resort,” Mr Stocking said.

The cost of this bespoke and unique treatment, which was paid for and administered by the NHS, is thought to be similar to a course of the most premium and highly-preserved antibiotics, at a few thousand pounds. The procedure is a last hope for him and his wife, Lorraine, 72, also a retired police officer.

After retiring, the couple moved to a smallholding in Devon and ran a rural B&B for a decade. On Wednesday, they celebrated their 48th wedding anniversary by sharing a Mars bar in hospital.

Mr Stocking now has three sinuses on his left leg from his infection which weep constantly and need regular tending.

“It’s got more and more painful, and it’s got worse and worse to the point where I can’t walk very far and I am very unstable,” Mr Stocking said.

“It has prevented us doing lots of things we would have wanted to have done. Everything has just been put on hold,” adds Lorraine.



The couple were not put off by the therapy’s experimental nature, and hope it could allow Mr Stocking’s leg to heal enough to allow him to walk with greater ease.

“I thought it was brilliant when it was first suggested to me,” Mr Stocking said.

“It was put to me that I would be the first one to have phage for something like this and that it was an experiment that could work for a lot of people and, if it works, also help a lot of people.

“I am 81 and I can’t pioneer much more with my life so whatever I can do to be of use to anybody then let’s give it a go.

“I’m never going to win a Butlins Knobbly Knees competition, but I’d like to see my leg sufficiently well to use it and walk. Walking is the main thing, to get about for the final few years of my life.”

But the path to this point, the couple say, has been exhausting. Endless red tape has delayed treatment and made access to phage a multi-year struggle.


Phage therapy is not a licensed medicine in the UK and a phage from the UK can not be provided to an NHS patient unless it reaches Good Manufacturing Practice (GMP) standard. There is no GMP facility for phages in the UK.

However, a “GMP-like” phage from abroad can be used for compassionate use if it is approved by the Medicines and Health Products Regulatory Agency (MHRA) and imported.

Proving this, and sourcing an importer to bring an unlicensed, non-GMP medicine from a laboratory in Brussels required more than 200 hours of work from Mr Stocking’s clinical team and caused most of the delays.
‘Get on with it’

The Stocking family and the doctors both urged politicians and regulators to alter the current legislation to make it easier for other patients to access phage therapy through the NHS.

“While all the faceless bureaucracy goes on we are left 200 miles away with no answers,” said Mr Stocking.

“It’s not been a very pleasant time. Phage could be useful to a lot of people but red tape is holding it all up. It has been months and months of hanging around.

“Give it a chance, expedite it,” he urged politicians. “Money is money but lives are lives and limbs are limbs. Get on with it.”

A medic treats Mr Stocking - RNOH Images

Lorraine said phage therapy could save the NHS millions of pounds a year and help treat thousands of different people around the country who have run out of options.

“Behind the delays and red tape are mental, emotional and physical impacts,” she said.

“His condition three-and-a-half years ago was not so bad and maybe phage would be more effective had we not been this far down the line before getting it.”

Tariq Azamgarhi, the principal antimicrobial pharmacist at RNOH, and Dr Antonia Scobie, research lead for the Bone Infection Unit at RNOH, were central in securing phage therapy for Mr Stocking.

Mr Azamgarhi said phages “fall between the cracks” of much of the existing UK regulation, and urged politicians to make changes to help doctors better access phages for compassionate use in patients with no other option.
‘Huge potential’

Dr Scobie, who is also lead for the UK Clinical Working Group for bacteriophage therapy, told The Telegraph: “We are in the camp of strongly supporting phage therapy and I think it has a huge amount of potential.

“We’re under no illusion that on its own, phage therapy is never going to be able to replace antibiotics but what it does offer is a safe treatment that can be an adjunct to our existing therapies.

“We would never dream of injecting the phage and just crossing fingers and hoping that would be enough. It needs to be used in the right way, but it’s an extra tool. I think it has huge potential and it just needs to be used in the right way.”

The RNOH team is now beginning the long-winded process again for three other superbug patients with joint infections and is helping other hospitals around the country tackle the paperwork.

“We hope that by opening various doors in the process that it may be easier for them and help the next patient,” Dr Scobie said.

A report by MPs on the science and technology committee said earlier this year that the MHRA should change its rules to allow for compassionate use in last-resort medical cases, like those of Mr Stocking.

They also urged the agency to set out new guidance for how doctors can use non-GMP phages.

The MHRA has missed some deadlines set out by the committee at the start of the year and is currently reviewing proposed guidance. It says this will be published “in due course”.

The MHRA declined to comment.

 OUR FRIEND THE BACTERIOPHAGE

Bacteria-fighting viruses team up to treat drug-resistant superbugs

UChicago Pritzker School of Molecular Engineering researchers screened a library of bacteriophages to find combinations of the viruses that can work together to fight antibiotic-resistant Klebsiella pneumoniae infections

University of Chicago

 

image: 

Researchers at the University of Chicago Pritzker School of Molecular Engineering (PME) and UChicago Medicine, including Asst. Prof. Mark Mimee (left) and research specialist Ella Rotman, have shown that a mixture of collections of bacteriophages can successfully treat antibiotic-resistant infections in mice.

view more 

Credit: UChicago Pritzker School of Molecular Engineering / Jason Smit

Researchers have a new battle tactic to fight drug-resistant bacterial infections. Their strategy involves using collections of bacteriophages, viruses that naturally attack bacteria. In a new study, researchers at the University of Chicago Pritzker School of Molecular Engineering (PME) and UChicago Medicine have shown that a mixture of these phages can successfully treat antibiotic-resistant Klebsiella pneumoniae infections in mice.

At the same time, however, the team’s work revealed just how complex the interactions between phages and bacteria can be; the viruses predicted to be most effective in isolated culture dishes did not always work in animals. Moreover, both phages and bacteria can evolve over time – in some cases, phages evolved to be more efficient in killing bacteria while in other cases, Klebsiella evolved resistance to the phages.

“We still think phages are an incredibly promising approach to treating drug-resistant bacteria such as Klebsiella,” said Mark Mimee, assistant professor of molecular engineering and senior author of the new work, published in Cell Host & Microbe. “But phages are like a living, constantly changing antibiotic which gives them a lot of complexity.”

Klebsiella pneumoniae are common bacteria found in people’s intestines where they cause little harm. However, when the bacteria escape to other body sites, such as open wounds, the lungs, the bloodstream, or the urinary tract, they can cause more severe infections. K. pneumoniae are often spread within hospital settings, and drug-resistant strains have become common.

“In my clinic, I see patients with recurrent urinary tract infections caused by Klebsiella,” says urogynecologist Sandra Valaitis, MD, of UChicago Medicine, a co-author of the new work. “Often these bacterial strains develop resistance to oral antibiotics, leaving patients with fewer options to clear the infection. We urgently need new ways of treating these bacteria.”

Phages, for more than a century, have been known as a natural enemy of bacteria and studied for their potential to treat infections. However, phages are usually very specific for one type of bacteria and predicting these matches has been difficult.

In the new research, Ella Rotman – a scientist in the Mimee Lab – screened wastewater to isolate phages that could effectively kill 27 different Klebsiella strains, including 14 that were newly isolated from University of Chicago patients. The team identified several dozen phages with the capability of killing at least some Klebsiella strains, Then, the researchers analyzed what genetic factors in the bacteria made them most prone to being killed or weakened by each of those phages.

Based on that analysis, Rotman and her colleagues developed a mixture of five phages that each targeted different components of the bacteria. In culture dishes as well as mice, this phage cocktail made antibiotic-resistant Klebsiella bacteria more likely to be attacked by the immune system and, in some cases, more susceptible to treatment with antibiotics. However, in other cases, the bacteria became more antibiotic resistant after treatment.

“It’s one of those things where biology often doesn’t work the way you want it to,” says Mimee. “But it gives us an opportunity to study the detailed dynamics between the phages and the bacteria.”

By exposing the phage mixture to a series of isolated Klebsiella bacteria, the researchers gave the phage the opportunity to evolve. This improved the ability of the cocktail to kill Klebsiella. In mice, the mixture effectively killed or weakened Klebsiella. The researchers observed co-evolution between the bacteria and phage in the mouse intestines, where the Klebsiella evolved to evade phage attack and the phage countered to better infect the altered bacteria.

Mimee’s lab group is continuing experiments to better understand how different phage and bacteria pairs interact with each other and how the presence of other phages and bacteria – naturally found in the human body—influences that. At the same time, in collaboration with Valaitis, they are seeking approval from the Food and Drug Administration (FDA) for a small clinical trial testing the phage mixture in patients with urinary tract infections.

“This research is a positive step forward in trying to sort out the complexities of phages and move them closer to the clinic,” says Mimee.

Citation: “Rapid design of bacteriophage cocktails to suppress the burden and virulence of gutresident carbapenem-resistant Klebsiella pneumoniae,” Rotman et al, Cell Host & Microbe, October 4, 2024. DOI: https://doi.org/10.1016/j.chom.2024.09.004

Funding: National Science Foundation, National Institutes of Health, Arnold and Mabel Beckman Foundation, BRaVE Phage Foundry at Lawrence Berkeley National Laboratory.

---

Journal

Cell Host & Microbe

DOI

10.1016/j.chom.2024.09.004 

Article Title

Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

Article Publication Date

4-Oct-2024

 Harnessing bacteriophages as targeted treatments for drug-resistant bacteria

Sep 5 2024

As antibiotic resistance becomes an increasingly serious threat to our health, the scientific and medical communities are searching for new medicines to fight infections. Researchers at Gladstone Institutes have just moved closer to that goal with a novel technique for harnessing the power of bacteriophages.

Bacteriophages, or phages for short, are viruses that naturally take over and kill bacteria. Thousands of phages exist, but using them as treatments to fight specific bacteria has so far proven to be challenging. To optimize phage therapy and make it scalable to human disease, scientists need ways to engineer phages into efficient bacteria-killing machines. This would also offer an alternative way to treat bacterial infections that are resistant to standard antibiotics.

Now, Gladstone scientists have developed a technology that lets them edit the genomes of phages in a streamlined and highly effective way, giving them the ability to engineer new phages and study how the viruses can be used to target specific bacteria.

"Ultimately, if we want to use phages to save the lives of people with infections that are resistant to multiple drugs, we need a way to make and test lots of phage variants to find the best ones," says Gladstone Associate Investigator Seth Shipman, PhD, the lead author of a study published in Nature Biotechnology. "This new technique lets us successfully and rapidly introduce different edits to the phage genome so we can create numerous variants."

The new approach relies on molecules called retrons, which originate from bacterial immune systems and act like DNA-production factories inside bacterial cells. Shipman's team has found ways to program retrons so they make copies of a desired DNA sequence. When phages infect a bacterial colony containing retrons, using the technique described in the team's new study, the phages integrate the retron-produced DNA sequences into their own genomes.

The enemy of your enemy

Unlike antibiotics, which broadly kill many types of bacteria at once, phages are highly specific for individual strains of bacteria. As rates of antibiotic-resistant bacterial infections rise-;with an estimated 2.8 million such infections in the United States each year-;researchers are increasingly looking at the potential of phage therapy as an alternative to combat these infections.

"They say that the enemy of your enemy is your friend," says Shipman, who is also an associate professor in the Department of Bioengineering and Therapeutic Sciences at UCSF, as well as a Chan Zuckerberg Biohub Investigator. "Our enemies are these pathogenic bacteria, and their enemies are phages."

Already, phages have been successfully used in the clinic to treat a small number of patients with life-threatening antibiotic-resistant infections, but developing the therapies has been complex, time-consuming, and difficult to replicate at scale. Doctors must screen collections of naturally-occurring phages to test whether any could work against the specific bacteria isolated from an individual patient.

Shipman's group wanted to find a way to modify phage genomes to create larger collections of phages that can be screened for therapeutic use, as well as to collect data on what makes some phages more effective or what makes them more or less specific to bacterial targets.

"As the natural predators of bacteria, phages play an important role in shaping microbial communities," says Chloe Fishman, a former research associate at Gladstone and co-first author of the new study, now pursuing her graduate degree at Rockefeller University. "It's important to have tools to modify their genomes in order to better study them. It's also important if we want to engineer them so that we can shape microbial communities to our benefit-;to kill antibiotic-resistant bacteria, for example."

Continuous phage editing

To precisely engineer phage genomes, the scientists turned to retrons. In recent years, Shipman and his group pioneered the development and use of retrons to edit the DNA of human cells, yeast, and other organisms.

Shipman and his colleagues began by creating retrons that produce DNA sequences specifically designed to edit invading phages-;a system the team dubbed "recombitrons." Then, they put those retrons into colonies of bacteria. Finally, they let phages infect the bacterial colonies. As the phages infected bacteria after bacteria, they continuously acquired and integrated the new DNA from the recombitrons, editing their own genome as they went along.

The research team showed that the longer they let phages infect a recombitron-containing bacterial colony, the greater the number of phage genomes were edited. Moreover, the researchers could program different bacteria within the colony with different recombitrons, and the phages would acquire multiple edits as they infected the colony.

As a phage is bouncing from bacterium to bacterium, it picks up different edits. Making multiple edits in phages is something that was previously incredibly hard to do; so much so that, most of the time, scientists simply didn't do it. Now, you basically throw some phages into these cultures, wait a while, and get your multiple-edited phages."

Seth Shipman, PhD, lead author

A platform to screen phages

If scientists already knew exactly what edits they wanted to make to a given phage to optimize its therapeutic potential, the new platform would let them easily and effectively carry out those edits. However, before researchers can predict the consequence of a genetic change, they first need to better understand what makes phages work and how variations to their genomes impact their effectiveness. The recombitron system helps makes progress here, too.

If multiple recombitrons are put into a bacterial colony, and phages are allowed to infect the colony for only a short time, different phages will acquire different combinations of edits. Such diverse collections of phages could then be compared.

"Scientists now have a way to edit multiple genes at once if they want to study how these genes interact or introduce modifications that could make the phage a more potent bacterial killer," says Kate Crawford, a graduate student in the Shipman lab and co-first author of the new study.

Shipman's team is working on increasing the number of different recombitrons that can be put into a single bacterial colony-;and then passed along to phages. They expect that eventually, millions of combinations of edits could be introduced to phages to make huge screening libraries.

"We want to scale this high enough, with enough phage variants, that we can start to predict which phage variants will work against what bacterial infections," says Shipman.

Source:

Gladstone Institutes

Journal reference:

Fishman, C. B., et al. (2024). Continuous multiplexed phage genome editing using recombitrons. Nature Biotechnology. doi.org/10.1038/s41587-024-02370-5.

 

CRISPR is promising to tackle antimicrobial resistance, but remember bacteria can fight back

Experts looking to use the Nobel winning technology to target resistance genes and make bacteria sensitive to first line antibiotics again; but the bacteria have ways to fight back

EUROPEAN SOCIETY OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES

In the second new research review on this subject, Assistant Prof. Ibrahim Bitar, Department of Microbiology, Faculty of Medicine and University Hospital in Plzen, Charles University in Prague, Plzen, Czech Republic, will give an overview of the molecular biology of CRISPR technology in explaining how it can used to tackle antimicrobial resistance.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR associated genes (cas) are widespread in the genome of many bacteria and are a defence mechanism against foreign invaders such as plasmids and viruses.  The CRISPR arrays are composed of a repeated array of short sequences, each originating from and exactly matching a nucleic acid sequence that once invaded the host.

Accompanying CRISPR sequences, there are 4-10 CRISPR-associated genes (cas), which are highly conserved and encode the Cas proteins. Cas proteins conduct adaptive immunity in prokaryotes (bacteria) based on immunological memories stored in the CRISPR array. The CRISPR/Cas system integrates a small piece of foreign DNA from invaders such as plasmids and viruses into their direct repeat sequences and will recognise and degrade the same external DNA elements during future invasions.

As the CRISPR/Cas systems integrate DNA from invading pathogens in chronical order, genotyping can be used to trace the clonality and the origin of the isolates and define them as a population of strains that were subjected to the same environmental conditions including geographic location (region) and community/hospital settings and eventually further extended to track pathogenic bacteria around human society.

CRISPR/Cas systems can also be employed for developing antimicrobial agents: introduction of self-targeting crRNAs will effectively and selectively kill target bacterial populations. Due to the shortage of available effective antimicrobial agents in treating multidrug-resistant (MDR) infections, researchers started to search for alternative methods to fight MDR infections rather than going through the process of developing new antimicrobial agents which can go on for decades. As a result, the concept of CRISPR/Cas-based selective antimicrobials was first developed and demonstrated in 2014. Vectors coding Cas9 and guide RNAs targeting genomic loci of a specific bacterial strain/species can be delivered to the target strain via bacteriophages or conjugative bacterial strains. In theory, delivery of the engineered CRISPR/Cas systems specifically eliminates target strains from the bacterial population, yet it is not that simple.

While these systems can seem a target for manipulation/intervention, all bacteria are regulated by multiple pathways to ensure the bacteria retains control over the process. Therefore, there remain several major challenges in using this system as an antimicrobial agent.

Most methods require delivery of the re-sensitised system by conjugation; the vector is carried by a non-virulent lab strain bacteria that is supposed to go and share the vector/plasmid through conjugation. The conjugation process is a natural process that the bacteria do which results in sharing plasmids among each other (even with other species). The percentage of conjugated (successfully delivered) bacteria in the total bacterial population is critical to the re-sensitised efficiency. This process is governed by several complicated pathways.

Bacteria also possess built-in anti-CRISPR systems, that can repair any damage caused by CRISPR-Cas systems. Defence systems that the bacteria uses to protect itself from foreign DNA often co-localise within defence islands (genomic segments that contain genes with similar function in protecting the host from invaders)  in bacterial genomes; for example: acr (a gene that acts, with other similar variants, as a repressor of plasmid conjugative systems) often cluster with antagonists of other host defence functions (e.g., anti-restriction modification systems) and experts hypothesise that MGEs (mobile genetic elements) organise their counter defence strategies in “anti-defence” islands.

Assistant Professor Bitar concludes: “In summary, this method seems very promising as an alternative way of fighting antimicrobial resistance. The method uses the concept of re-sensitising the bacteria in order to make use of already available antibiotics – in other words, removing their resistance and making them vulnerable again to first-line antibiotics. Nevertheless, the bacterial pathways are always complicated and such systems are always heavily regulated by multiple pathways. These regulated pathways must be studied in depth in order to avoid selective pressure favoring anti-CRISPR systems activation, hence prevalence of resistance in a more aggressive manner.”

 

Experts developing way to harness Nobel Prize winning CRISPR technology to deal with antimicrobial resistance (AMR)

EUROPEAN SOCIETY OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES

Antimicrobial resistance (AMR) is continuing to increase globally, with rates of AMR in most pathogens increasing and threatening a future in which every day medical procedures may no longer be possible and infections thought long dealt with could kill regularly again. As such, new tools to battle AMR are vitally needed.

In a new research review at this year’s ESCMID Global Congress (formerly ECCMID – Barcelona 27-30 April) shows how the latest CRISPR-Cas gene editing technology can be used to help modify and attack AMR bacteria. The presentation is by Dr Rodrigo Ibarra-Chávez, Department of Biology, University of Copenhagen, Denmark.

CRISPR-Cas gene editing technology is a groundbreaking method in molecular biology that allows for precise alterations to the genomes of living organisms. This revolutionary technique, which brought its inventors, Jennifer Doudna and Emmanuelle Charpentier, the Nobel Prize in Chemistry in 2020, enables scientists to accurately target and modify specific segments of an organism's DNA (genetic code). Functioning like molecular ‘scissors’ with the guidance of guide RNA (gRNA), CRISPR-Cas can cut the DNA at designated spots. This action facilitates either the deletion of unwanted genes or the introduction of new genetic material into an organism's cells, paving the way for advanced therapies.

Dr Ibarra-Chávez says: “Fighting fire with fire, we are using CRISPR-Cas systems (a bacterial immunity system) as an innovative strategy to induce bacterial cell-death or interfere with antibiotic resistance expression – both hold promise as novel sequence-specific targeted ‘antimicrobials’.”

One line of their work involves creating guided systems against antimicrobial resistance genes could treat infections and prevent dissemination of resistance genes.

Mobile genetic elements (MGEs) are parts of the bacterial genome that can move about to other host cells or also transfer to another species. These elements drive bacterial evolution via horizontal gene transfer.  Dr Ibarra-Chávez explains how repurposing mobile genetic elements (MGEs) and choosing the delivery mechanism involved in the antimicrobial strategy is important for reaching the target bacterium.

A phage is a virus that infects bacteria, and it is also considered MGE, as some can remain dormant in the host cell and transfer vertically. The MGEs his team is using are phage satellites, which are parasites of phages. He says: “These ‘phage satellites’ hijack parts of the viral particles of phages to ensure their transfer to host cells. In contrast to phages, satellites can infect bacteria without destroying them, offering a step-change over existing methods involving phages and thus developing an arsenal of viral particles that are safe to use for applications such as detection and modification via gene delivery. Phage particles are very stable and easy to transport and apply in medical settings. It is our task to develop safe guidelines for their application and understand the resistance mechanisms that bacteria can develop.”

Bacteria can evolve mechanisms to evade the action of the CRISPR-Cas system and delivery vectors can be vulnerable to anti-MGE defences. Thus Dr Ibarra-Chávez’s team and others are developing the use of anti-CRISPRs and defence inhibitors in the delivery payloads to counter these defences, to enable the CRISPR to arrive and attack the AMR genes in the cell.

Dr Ibarra-Chávez will also discuss how combination strategies employing CRISPR-Cas systems could promote antibiotic susceptibility in a target bacterial population. Phages have a particular selective pressure on AMR cells, which can improve the effect of some antibiotics. Similarly, using CRISPR-Cas in combination with phages and/or antibiotics, it is possible to suppress the mechanisms of resistance that infectious bacteria may develop by targeting such virulence/resistance genes, making these therapies safer.

He explains: “Bacteria are particularly good at adapting and becoming resistance. I believe we need to be cautious and try using combinatorial strategies to avoid the development of resistance, while monitoring and creating guidelines of new technologies.”

Dr Ibarra-Chávez has primarily focused on tackling resistance in Staphylococcus aureus and Escherichia coli. Now, in collaboration with Prof. Martha Clokie and Prof. Thomas Sicheritz-Pontén, his team will treat group A Streptococci necrotising soft tissue infection (flesh eating bacteria) using the combination approaches described above.

 

---

ARTICLE PUBLICATION DATE

26-Apr-2024

---

Study suggests host response needs to be studied along with other bacteriophage research

Bacteriophage (magenta) attack Pseudomonas aeruginosa (teal) biofilms grown in 

association with respiratory epithelial cells (nuclei, yellow). Credit: Paula Zamora, 

A team of micro- and immunobiologists from the Dartmouth Geisel School of Medicine, Yale University, and the University of Pittsburgh has found evidence suggesting that future research teams planning to use bacteriophages to treat patients with multidrug-resistant bacterial infections need to also consider how cells in the host's body respond to such treatment.

In their paper published in the open-access journal PLOS Biology, the group describes experiments they conducted that involved studying the way epithelial cells in the lungs respond to bacteriophages.

Over the past decade, medical scientists have found that many of the antibiotics used to treat bacterial infections are becoming resistant, making them increasingly useless. Because of this, other scientists have been looking for new ways to treat such infections. One possible approach has involved the use of bacteriophages, which are viruses that parasitize bacteria by infecting and reproducing inside of them, leaving them unable to reproduce.

To date, most of the research involving use of bacteriophages to treat infections has taken place in Eastern Europe, where some are currently undergoing clinical trials. But such trials, the researchers involved in this new study note, do not take into consideration how cells in the body respond to such treatment. Instead, they are focused on determining which phages can be used to fight which types of bacteria, and how well they perform once employed.

The reason so little attention is paid to host cell interaction, they note, is that prior research has shown that phages can only replicate inside of the bacterial cells they invade; thus, there is little opportunity for them to elicit a response in human cells.

In this new study, the research team suggests such thinking is misguided because it fails to take into consideration the immune response in the host. To demonstrate their point, the team conducted a series of experiments involving exposing human epithelial cells from the lungs (which are the ones that become infected as part of lung diseases) to bacteriophages meant to eradicate the bacteria causing an infection.

They found that in many cases, the immune system responded by producing proinflammatory cytokines in the epithelial cells. They noted further that different phages elicited different responses, and there exists the possibility that the unique properties of some phages could be used to improve the results obtained from such therapies. They conclude by suggesting that future bacteriophage research involve inclusion of host cell response.

More information: Paula F. Zamora et al, Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells, PLOS Biology (2024). DOI: 10.1371/journal.pbio.3002566

Journal information: PLoS Biology 

© 2024 Science X NetworkMammalian cells may consume bacteria-killing viruses to promote cellular health

Study details a common bacterial defense against viral infection

Complex of 2 proteins enhances blockage of phage replication

Peer-Reviewed Publication

OHIO STATE UNIVERSITY

COLUMBUS, Ohio – One of the many secrets to bacteria’s success is their ability to defend themselves from viruses, called phages, that infect bacteria and use their cellular machinery to make copies of themselves.

Technological advances have enabled recent identification of the proteins involved in these systems, but scientists are still digging deeper into what those proteins do.

In a new study, a team from The Ohio State University has reported on the molecular assembly of one of the most common anti-phage systems – from the family of proteins called Gabija – that is estimated to be used by at least 8.5%, and up to 18%, of all bacteria species on Earth.

Researchers found that one protein appears to have the power to fend off a phage, but when it binds to a partner protein, the resulting complex is highly adept at snipping the genome of an invading phage to render it unable to replicate.

“We think the two proteins need to form the complex to play a role in phage prevention, but we also believe one protein alone does have some anti-phage function,” said Zhangfei Shen, co-lead author of the study and a postdoctoral scholar in biological chemistry and pharmacology at Ohio State’s College of Medicine. “The full role of the second protein needs to be further studied.”

The findings add to scientific understanding of microorganisms’ evolutionary strategies and could one day be translated into biomedical applications, researchers say.

Shen and co-lead author Xiaoyuan Yang, a PhD student, work in the lab of senior author Tianmin Fu, assistant professor of biological chemistry and pharmacology at Ohio State.

The study was published April 16 in Nature Structural & Molecular Biology.

The two proteins that make up this defense system are called Gabija A and Gabija B, or GajA and GajB for short.

Researchers used cryo-electron microscopy to determine the biochemical structures of GajA and GajB individually and of what is called a supramolecular complex, GajAB, created when the two bind to form a cluster consisting of four molecules from each protein.

In experiments using Bacillus cereus bacteria as a model, researchers observed the activity of the complex in the presence of phages to gain insight into how the defense system works.

Though GajA alone showed signs of activity that could disable a phage’s DNA, the complex it formed with GajB was much more effective at ensuring phages would not be able take over the bacterial cell.

“That’s the mysterious part,” Yang said. “GajA alone is sufficient to cleave the phage nucleus, but it also does form the complex with GajB when we incubate them together. Our hypothesis is that GajA recognizes the phage’s genomic sequence, but GajB enhances that recognition and helps to cut the phage DNA.”

The large size and elongated configuration of the complex made it difficult to get the full picture of GajB’s functional contributions when bound to GajA, Shen said, leaving the team to make some assumptions about protein roles that have yet to be confirmed.

“We only know GajB helps enhance GajA activity, but we don’t yet know how it works because we only see about 50% of it on the complex,” Shen said.

One of their hypotheses is that GajB may influence the concentration level of an energy source, the nucleotide ATP (adenosine triphosphate), in the cellular environment – specifically, by driving ATP down upon detection of the phage’s presence. That would have the dual effect of expanding GajA’s phage DNA-disabling activity and stealing energy that a phage would need to start replicating, Yang said.

There is more to learn about bacterial anti-phage defense systems, but this team has already shown that blocking virus replication isn’t the only weapon in the bacterial arsenal. In a previous study, Fu, Shen, Yang and colleagues described a different defense strategy: bacteria programming their own death rather than letting phages take over a community.

This work was supported by the National Institute of General Medical Sciences.

Additional co-authors are Jiale Xie, Jacelyn Greenwald, Ila Marathe, Qingpeng Lin and Vicki Wysocki of Ohio State, and Wenjun Xie of the University of Florida.

#

Contact: Tianmin Fu, Fu.978@osu.edu

Written by Emily Caldwell, Caldwell.151@osu.edu; 614-292-8152

---

JOURNAL

Nature Structural & Molecular Biology

DOI

10.1038/s41594-024-01283-w 

ARTICLE TITLE

Molecular basis of Gabija anti-phage supramolecular assemblies

ARTICLE PUBLICATION DATE

16-Apr-2024

MICROVERSE

Attack and defence in the microverse

How small RNA molecules regulate viral infections of bacteria

Peer-Reviewed Publication

FRIEDRICH-SCHILLER-UNIVERSITAET JENA

 

IMAGE: 

VIEW OF A PETRI DISH WITH CHOLERA BACTERIA (VIBRIO CHOLERAE).

view more 

CREDIT: JENS MEYER/UNIVERSITY OF JENA

Viruses need hosts. Whether it’s measles, the flu or coronavirus, viral pathogens cannot multiply or infect other organisms without the assistance of their hosts’ cellular infrastructure. However, humans are not the only ones affected by viruses: animals, plants and even microorganisms can all serve as hosts. Viruses that use bacteria as host cells are called bacteriophages (or simply “phages” for short) and are thought to be the most abundant biological entities of all. Just as the human immune system springs into action to resist a flu or coronavirus infection, bacteria do not simply allow phages to infiltrate their cellular machinery without a fight.

A research team at the University of Jena and its Cluster of Excellence “Balance of the Microverse” has examined in detail the complex interaction of attack and defence strategies when cholera-causing bacteria (Vibrio cholerae) are infected with a bacteriophage known as VP882—and discovered that tiny RNA molecules play a decisive role. The researchers’ findings have been published in the latest issue of a prestigious journal, Cell Host & Microbe.

From harmless housemate to cunning kidnapper

There are two ways in which phages can multiply after infecting bacteria: either as invisible passengers, hidden in the bacteria’s genetic material, or as cunning kidnappers, multiplying in vast numbers in bacterial cells without regard for potential losses and, ultimately, destroying the cells. Which method a phage adopts depends on whether sufficient numbers of other host cells are available in the immediate environment to provide shelter.

But how do phages determine this? “They rely on a chemical counting mechanism that bacteria use to identify other members of their species,” explains Prof. Dr Kai Papenfort of the University of Jena, who headed up the project. Known as “quorum sensing”, this method uses signal molecules that are produced by bacteria and released into their surroundings. At the same time, the bacteria monitor the concentration of these molecules using specific receptors, thereby gaining information about the size of their current population. “The phages’ trick essentially involves ‘listening in’ to this chemical communication between bacteria,” says Papenfort.

In their experiments, the Jena researchers examined what happens to the phages and bacteria once the bacteria emit their quorum sensing signals. “We have observed that 99% of bacteria are destroyed within 60 minutes, in which time the phages take control,” reports Dr Marcel Sprenger, the lead author of the article. The team discovered that this switchover is controlled by tiny RNA molecules, one of which is called “VpdS” (VP882 phage-derived sRNA). “As soon as the phages receive the chemical signal from the bacteria, this RNA is produced in high quantities,” says Sprenger.

How bacteria fight back against viruses

In order to find out precisely which genes are regulated by VpdS, the team adopted a comprehensive, technological approach and infected bacteria cultures with both VP882 phages and genetically modified phages unable to produce VpdS. Applying a method known as “RNA interaction by ligation and sequencing”, the researchers were able to identify the interactions between all RNA molecules in the bacteria cultures at different times. “This not only gave us insights into which genes are active, it also showed how they interact,” says Papenfort.

This method enabled the researchers to examine the genes of the phages as well as those of the host bacteria. As a result, the researchers gained extensive insights into the changes that occurred both during and after quorum sensing. “We were able to demonstrate that VpdS regulates phage genes as well as genes of the host, which effectively explains the destruction of bacterial cells,” says Papenfort.

However, the researchers have been able to deduce further relationships from the data they collected. For example, bacteria also have genes that, when activated by a chemical signal, fight back against the phages’ propagation and thereby counteract their own destruction. According to Papenfort, this aspect is particularly interesting. “We can see these as the precursors to the immune systems in higher organisms. Bacteria have many genes that protect them against viruses.” Given that these genes are also present in higher organisms, the researchers surmise that RNA molecules could also play an important role in their regulation.

 

Original publication:

Sprenger M. et al.: Small RNAs direct attack and defence mechanisms in a quorum sensing phage and its host. Cell. Host & Microbe (2024), https://doi.org/10.1016/j.chom.2024.03.010

 

Contact:

Prof. Dr Kai Papenfort
Institute of Microbiology at Friedrich Schiller University Jena
Winzerlaer Straße 2, 07745 Jena, Germany
Phone: +49 (0)3641 9 49311
Email: kai.papenfort@uni-jena.de

 

---

JOURNAL

Cell Host & Microbe

DOI

10.1016/j.chom.2024.03.010 

METHOD OF RESEARCH

Experimental study

SUBJECT OF RESEARCH

Cells

ARTICLE TITLE

Small RNAs direct attack and defence mechanisms in a quorum sensing phage and its host.

ARTICLE PUBLICATION DATE

4-Apr-2024

New micromaterial releases nanoparticles that selectively destroy cancer cells

UNIVERSITAT AUTONOMA DE BARCELONA

Researchers at the Universitat Autònoma de Barcelona (UAB), in collaboration with the Sant Pau Research Institute and the CIBER-BBN, have developed micromaterials made up only of proteins, capable of delivering over an extended period of time nanoparticles that attack specific cancer cells and destroy them. The micromaterials mimic natural secretory granules found in the endocrine system and were proven effective in mouse models of colorectal cancer.

A team coordinated by Professor Antonio Villaverde from the Institute of Biotechnology and Biomedicine of the Department of Genetics and Microbiology, UAB, and with the participation of the Sant Pau Research Institute and the CIBER-BBN, has developed self-contained micromaterials made up only of proteins that are capable of delivering over an extended period of time the polypeptide that composes them. The technology used for the fabrication of these granules, patented by the researchers, is relatively simple and mimics the secretory granules of the human endocrine system. With regards to its chemical structure, it involves the coordination of ionic zinc with histidine-rich domain, an amino acid essential for living beings and therefore not toxic.
The new micromaterials developed by researchers are formed by chains of amino acids known as polypeptides, which are functional and bioavailable in the form of nanoparticles that can be released and targeted to specific types of cancer cells, for selective destruction.
The research team analyzed the molecular structure of these materials and the dynamics behind the secretion process, both in vitro and in vivo. In an animal model of CXCR4+ colorectal cancer, the system showed high performance upon subcutaneous administration, and how the released protein nanoparticles accumulated in tumor tissues.
“It is important to highlight that this accumulation is more efficient than when the protein is administered in blood. This fact offers an unexpected new way to ensure high local drug levels and better clinical efficacy, thus avoiding repeated intravenous administration regimens”, explains Professor Antonio Villaverde. "In the clinical context, the use of these materials in the treatment of colorectal cancer should largely enhance drug efficiency and patient’s comfort, while at the same time minimizing undesired side effects."
Participating in the research, conducted principally by UAB researcher Julieta M. Sánchez, were researchers from the UAB Department of Genetics and Microbiology, the UAB Institute of Biomedicine and Biotechnology (IBB-UAB), and the Oncogenesis and Antitumor Drugs team led by Professor Ramón Mangues of the Sant Pau Research Institute. Both Professor Antonio Villaverde and Professor Ramón Mangues form part of the CIBER network of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Also participating in the study were the Protein Production Platform (Unit 1) and the Nanotoxicology Platform (Unit 18) of the Singular Infrastructure NANBIOSIS, and funding was received through several competitive research and technology transfer projects (including PID2019 -105416RB-I00/AEI/10.13039/501100011033, PDC2022-133858-I00, PID2022-136845OB-I00, CPP2021-008946, PI21/400), as well as intramural CIBER-BBN projects (VENOM4CANCER, NANOREMOTE and NANOSCAPE).

 

 

---

JOURNAL

Advanced Science

DOI

10.1002/advs.202309427 

METHOD OF RESEARCH

Experimental study

SUBJECT OF RESEARCH

Animals

ARTICLE TITLE

Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids