New study reveals a minimalist bacterial defense that disrupts viral assembly

University of Toronto researchers have expanded our understanding of bacterial immunity with the discovery of a new protein that can both sense and counteract viral infections.

In the new study, published today in Nature, researchers from U of T’s Temerty Faculty of Medicine describe how a single protein named Rip1 recognizes bacteriophages, the viruses that infect bacteria, and cause infected bacteria to die prematurely, thereby ending the chain of transmission.

“There are a lot of parallels between our immune system and bacterial immune systems,” says Karen Maxwell, the study’s co-senior author and a professor of biochemistry at Temerty Medicine.  

Her research is focused on understanding how bacteria protect themselves against phages and how phages overcome these defences, with the long-term goal of using this information to develop safe and effective phage therapies.

As a PhD student in Maxwell’s lab, Pramalkumar Patel found the Rip1 gene hidden in a prophage, which are bacteriophages that have integrated their genetic information into the host bacteria’s genome. Prophage genes often confer additional survival benefits to their host, such as enhancing its ability to cause disease and protecting it against other phages. 

After their initial finding that Rip1 protected bacteria against some phages, the researchers delved into how the protein works. They found that it caused the bacterial cell to change from an elongated pill-like shape to a round sphere before bursting. They also showed that Rip1 proteins could bind to two different phage proteins, both involved in building new viruses.

“The biological data were all pointing to Rip1 being something that punches holes in the bacterial membrane, but we wanted to look structurally to determine is that really what’s happening?” says Maxwell. 

She and Patel took their data and protein samples down the hall to Michael Norris, an assistant professor of biochemistry and structural biologist who studies how viruses assemble. Together the researchers used a technique called cryogenic electron microscopy (cryo-EM) to visualize what Rip1 looks like and how it works.

Norris explains that cryo-EM works by taking snapshots of a protein, or group of proteins, from many possible anglse, allowing researchers to recreate the structure at near-atomic resolution. 

Their efforts revealed that Rip1 proteins pair up as inactive dimers when there is no active phage infection. During an infection, Rip1 recognizes the donut shape of its phage protein targets and uses it as a template to form its own Rip1 ring, which then inserts into the bacterial inner membrane. 

“It’s two rings stacked on top of one another,” says Maxwell. “There’s a channel down the centre that allows the contents to leak out from inside the bacterial cell, and that’s what kills the cell.” 

Based on their results, the researchers concluded that Rip1 provides anti-phage defense in two ways — first, by acting like a sponge to soak up the critical phage proteins needed to make more viruses and second, by punching holes in the bacterial inner membrane and killing the bacteria before the infecting phage has finished making its offspring.

Maxwell says that what makes Rip1 stand out is the integration of both phage sensing and defense into one small protein. In most immune systems, including our own, the tasks of first detecting a microbial invader and then neutralizing it falls to different proteins, some of which function as complex cellular machines.

“Phages are extremely sophisticated,” says Norris, whose research focuses on the structure and assembly of deadly RNA viruses like the measles, Nipah and Ebola virus.

“They pack a huge amount of functional diversity into very small genomes, and Rip1 shows how bacteria have evolved equally compact ways to respond.”

END