University of Toronto researchers have made the first discovery of a virus that infects Legionella pneumophila, the bacteria that causes Legionnaires' disease.
The findings, published in Science Advances , open the door for the use of bacterial viruses - also known as bacteriophages, or phages for short - to treat Legionella infections and uncover a surprising insight into how the bacteria evolved to cause disease.
In addition to isolating the new phage, named LME-1, the researchers also showed that it could infect Legionella pneumophila and inhibit the bacteria's growth in human macrophages, the immune cells where these bacteria typically reside.
LME-1 was identified by a team of researchers led by Alexander Ensminger, an associate professor of biochemistry and molecular genetics in U of T's Temerty Faculty of Medicine, and Beth Nicholson, a senior research associate in his lab.
"The Legionella field has been looking for phages for 50 years," says Ensminger. "We were doing all these things like looking in water samples, but all along there was one sitting in our freezer. We just had to figure out how to reveal itself as a phage."
The researchers became interested in finding Legionella phages when they discovered that many isolates of Legionella contained active CRISPR-Cas systems, which are bacterial immune systems that defend against viruses.
They found these CRISPR-Cas systems contained records of previous encounters with uncharacterized phages along with a mysterious genetic element called LME-1. LME-1 had all the genetic hallmarks of being a phage - it contained genes that resembled the structural components needed to build a phage - but previous attempts by Ensminger's team and other research groups could not induce LME-1 to produce any phages.
"All of the standard tools for either activating a phage or isolating a phage didn't work," says Ensminger. "The 'aha!' moment was figuring out how to activate this thing."
The breakthrough came when Nicholson tried using antibiotic resistance to coax the bacteria to start making what she hoped would be LME-1 phages, an idea inspired by data from Chitong Rao, a former PhD student.
The plan worked.
The researchers could detect the production of phage proteins in the bacteria, and they started to see lightbulb-shaped virus particles under an electron microscope.
"At some points, I felt like we were searching for Bigfoot or the Loch Ness Monster because we just had these blurry images of a phage-like thing," says Ensminger, recounting their earlier efforts to find the phage before they figured out how to induce phage production.
Once they could make large quantities of phages, the researchers collaborated with Susan Lea and Justin Deme at the National Cancer Institute in the U.S. to obtain the first ever high-resolution images of the Legionella LME-1 phage, which showed it to have an icosahedral head decorated with surface proteins and a short tail.
To better understand how Legionella resists LME-1 infection, Nicholson and then-undergraduate researcher José Santé looked for cells that were vulnerable to LME-1 in a strain that is normally resistant. They found that in every case, susceptibility was caused by genetic mutations in the lag1 gene. Together with PhD student Elizabeth Chaney, they went on to show that this gene prevents LME-1 attachment by modifying the bacterial cell surface.
Previous research showed lag1 also helps bacteria evade killing by the immune system. Further, 80 per cent of all cases of Legionnaires' disease are caused by Legionella strains that contain the lag1 gene. Despite its role in disease, the evolutionary forces driving the lag1 adaptation have long been a mystery.
Ensminger believes that over the course of evolution, Legionella picked up the gene to protect itself against phages, with the accidental result of making the bacteria better at surviving and causing disease in humans.
"We have a previously unknown phage to 'thank' for Legionnaires' disease," he says.
Nicholson is now focused on creating a "defenseless" strain of the bacteria to hunt for other Legionella-infecting phages, some of which could be good candidates to use in phage therapy.
"Finding the first phage for Legionella opens the door to one day being able to use phage to control Legionella. It's still a ways down the road but at least it's a possibility now," she says.
"Our study is also a cautionary tale that with phage therapy, we need to understand the relationship between phage and bacteria before we deploy it because in some instances, resistance to the phage might make the bacteria more harmful to humans," says Ensminger.
This study was funded by the Canadian Institutes of Health Research and the New Frontiers in Research Fund.
