Scientists find strange bacterial immune system that works backward

Researchers at Gladstone Institutes have uncovered an extraordinarily unusual bacterial immune system that operates in reverse of known defenses.

The discovery, published in Molecular Cell, details the mechanics of "type VI retrons"-a uniquely counterintuitive class of bacterial defenses. More than just a biological curiosity, decoding these complex systems provides a molecular blueprint that scientists can adapt to engineer more efficient DNA-editing tools for human health and therapies.

This is, by far, the strangest of the retron systems that we or anyone else has come acrossThese retrons operate with Rube Goldberg-level complexity, using molecular mechanisms that we would never have thought to invent, but that we'll use in future technologies."

Seth Shipman, PhD, Gladstone Investigator, senior author of the new study

Bacteria have long evolved specialized defenses to destroy phages, which are viruses that target bacterial cells. A particularly powerful line of defense, known as retrons, recognize viral attacks and trigger self-destruction to protect the broader bacterial colony.

Because retrons can also efficiently modify and deliver single-stranded DNA, researchers including Shipman and his team are working to harness them to rewrite disease-causing mutations inside human cells with unprecedented precision.

Inverted expectations

Many types of retron systems exist, but they all follow the same basic principle: they sense when a specific strain of phage has attacked the bacterial cell, and they then unleash a toxic protein to kill the cell. This self-destruction stops the phage before it has a chance to multiply and spread to neighboring cells in the same colony.

Decades of research have led to the general understanding that all retrons produce a toxin that, in uninfected cells, is constantly present but deliberately repressed by small, single-stranded DNA molecules. When a phage invades, it perturbs the DNA and causes it to release the toxin.

"The way we typically detect retrons inside uninfected cells is by looking for this single-stranded DNA," says Karen Zhang, a PhD student in Shipman's lab and lead author of the study. "But type VI retrons caught our attention because they didn't appear to have this signature. It was very odd, and we wondered if they might be broken."

But when the scientists tested certain bacteria containing type VI retrons, they found that the retrons were still able to successfully defend their cells against phages.

"That kicked off a long molecular odyssey," Shipman says. "Whenever we tried the experiments we normally use to study retrons, they would fail, so we kept having to think of new ways to figure out how these retrons work."

The experimental saga ultimately revealed that, unlike other retrons, type VI retrons do not produce noticeable levels of single-stranded DNA nor toxin in uninfected cells. Instead, the entry of a phage into a cell is needed to trigger the retron to start producing single-stranded DNA.

Once it builds up, the DNA then interacts with an RNA that has been blocking production of the toxin. This interaction releases the block, allowing toxin production to ramp up.

The toxin appears to be a tiny protein that embeds itself into the membrane surrounding the cell, which destabilizes the cells and kills it.

"It's almost the mirror-image version of how we traditionally view retrons, because the DNA switches on toxin production instead of inhibiting an already-produced toxin," Zhang says. "It would be fascinating to explore how bacterial evolution could have resulted in such a complex molecular system-whether type VI retrons are older or newer than other retrons, or whether they're a stepping stone between different types."

Rethinking retron research

To decipher the unique machinery of type VI retrons, the team had to concoct novel experiments and repurpose existing methods in new ways. One early breakthrough, for instance, arose from the repeated, mysterious failure of standard methods to detect DNA made by type VI retrons in uninfected cells.

The researchers turned to a different tool called Spacer-seq previously developed in Shipman's lab. This tool harnesses the molecular components of another kind of bacterial immune system, CRISPR-Cas, to create a record of gene activity inside a cell.

"We had just about given up hope that any DNA was being made when this approach finally revealed a very, very low level of production in uninfected cells," Zhang says.

The finding motivated the team to keep digging, and further experiments showed that the DNA only ramps up to defensive levels after the phage enters.

Nature-inspired tools and treatments

The unique machinery of type VI retrons is already inspiring the development of novel tools in Shipman's lab, including new synthetic sensors and switches that can control gene activity.

"This system represents a unique way to potentially build a molecular sensor and switch encoded in DNA and RNA, that no one has thought of before," Zhang says. "Synthetic sensors and switches have many possible applications, both in treating disease and in designing experiments to better understand how certain diseases operate."

The tiny, highly potent toxins produced by the type VI retrons could also prove useful in a medical context. The scientists are working on ways to incorporate these toxins into new types of antimicrobial therapies, which could target and kill bacteria that cause human disease.

"This study highlights why it's so important for a molecular technology development lab like ours to keep looking to nature for inspiration," Shipman says. "We can use what evolution has given us as starting points to build complex machines that would have been tremendously hard to even conceptualize."