Northwestern University researchers have successfully coaxed a deadly pathogen to destroy itself from the inside out.
In the new study, the researchers modified DNA from a bacteriophage, or “phage,” a type of virus that infects and replicates inside bacteria. Then, the research team puts the DNA inside Pseudomonas aeruginosa (P. aeruginosa), a deadly bacterium that is also highly resistant to antibiotics. Once inside the bacterium, the DNA is assembled into virions, bypassing the pathogen’s defenses, which cut through the bacterium’s cell to kill it.
The experimental work, based on growing interest in “phage therapy,” represents an important step toward engineering designer viruses as new therapeutics to kill antibiotic-resistant bacteria. It also reveals important information about the inner workings of phages, a little-studied area of biology.
The research will be published Wednesday (Jan. 24) in the journal Microbiology Spectrum.
“Antimicrobial resistance is sometimes referred to as the ‘silent epidemic,'” said Erica Hartman of Northwestern, who led the work. “The number of infections and deaths due to infections is increasing worldwide. This is a huge problem. Phage therapy has emerged as an unnecessary alternative to our reliance on antimicrobial use. But, in many ways, phages are the ‘final frontier’ of microbiology. We don’t know much about them. The more we can learn about how phages work, the more we can engineer more effective therapeutics. Our project is cutting-edge in that we’re learning about phage biology in real time as we engineer them.”
An indoor microbiologist, Hartman is an associate professor of civil and environmental engineering at Northwestern’s McCormick School of Engineering and a member of the Synthetic Biology Center.
Alternatives to antibiotics are desperately needed
Associated with the increase in antimicrobial use, the rise of antibacterial resistance is an urgent and growing threat to the world population. According to the Centers for Disease Control and Prevention (CDC), approximately 3 million antimicrobial-resistant infections occur each year in the United States alone, resulting in more than 35,000 deaths.
The growing crisis has inspired researchers to look for alternatives to antibiotics, which are steadily losing effectiveness. In recent years, researchers have begun to explore phage therapies. But despite billions of phages, scientists know very little about them.
For every bacterium that exists, there are dozens of phages. So, Earth has an astronomically large number of phases, but we understand only a few of them. We weren’t necessarily motivated to really study them. Now, the motivation is there, and we are increasing the number of tools dedicated to their study.”
Erica Hartman, Northwestern University
Treatment without side effects
To explore potential phage therapies, researchers identify or modify an existing virus to selectively target a bacterial infection without disrupting the rest of the body. Ideally, scientists could one day develop a phage therapeutic to infect a specific bacterium and design “à la carte” therapeutics with specific characteristics and properties to treat individual infections.
“What’s powerful about phage is that it can be very specific in a way that antibiotics aren’t,” Hartman said. “If you take an antibiotic for a sinus infection, for example, it disrupts your entire gastrointestinal tract. A phage therapy can be designed to affect only the infection.”
While other researchers have investigated phage therapy, almost all studied have focused on using phages to infect. Escherichia coli. Hartman, however, decided to focus P. aeruginosa, one of the five deadliest human pathogens. Especially dangerous for those with low immunity. P. aeruginosa It is a major cause of nosocomial infections, often infecting burn or surgical wounds in people with cystic fibrosis, as well as patients with lung disease.
“It’s a high-priority, multi-drug-resistant pathogen that a lot of people are really concerned about,” Hartman said. “It is highly drug resistant, so there is an urgent need to develop alternative therapeutics.”
Simulating infection, bypassing defenses
In the study, Hartman and his team began P. aeruginosa Bacteria and purified DNA from different phages. Then, they use electroporation -; A technique that delivers short, high-voltage pulses of electricity -; To make temporary holes in the outer cell of bacteria. Through these pores, the phage DNA enters the bacteria to mimic the process of infection.
In some cases, bacteria recognize the DNA as a foreign object and destroy the DNA to protect themselves. But after using synthetic biology to optimize the process, Hartmann’s team was able to knock out the bacteria’s antiviral defense system. In this case, the DNA successfully carries the information into the cell, causing the virus to kill the bacteria.
“Where we’ve had success, you’ll see black spots on the bacteria,” Hartman said. “This is where the viruses burst out of the cell and kill all the bacteria.”
After this success, Hartmann’s team introduced DNA from two more phages that were naturally unable to infect their strain. P. aeruginosa. Yet again, the process worked.
Phage production in a cell
Not only did the phage kill the bacteria, the bacteria released billions more phages. These phages can then be used to kill other bacteria, such as those that cause infections.
Next, Hartmann plans to continue modifying phage DNA to optimize potential therapies. For now, his team is studying the expelled phages P. aeruginosa.
“This is an important part of developing phage therapy,” he said. “We can study our phages to see which ones to develop and eventually mass-produce them as therapeutics.”
The research, supported by the Walder Foundation, the National Science Foundation and the National Institutes of Health, is titled “A Synthetic Biology Approach to Assemble and Reboot Clinically Relevant Pseudomonas aeruginosa Tailed Phage.”
Ipaucha, T., etc (2024) A Synthetic Biology Approach to Assemble and Reboot Clinically Relevant Pseudomonas aeruginosa Tailed Phages. Microbiology Spectrum. doi.org/10.1128/spectrum.02897-23.