Studies by researchers at Howard Hughes Medical Institute (HHMI) and colleagues have demonstrated how some gut bacteria possess what they describe as the “spooky superpower” of being able to reanimate dormant viruses hiding in other microbes. This viral awakening, the research found, is triggered by a molecule called colibactin, which can summon killer viruses from their slumber, to unleash full-blown infections that destroy the virus-carrying cells.

Lead investigator Emily Balskus, PhD, and colleagues, reported on their studies in Nature, in a paper titled, “The bacterial toxin colibactin triggers prophage induction.” In their report the team concluded, “…as links between the human gut virome and diseases continue to be established, our findings set the stage for further investigations of how gut bacterial metabolite production modulates phage behaviors and may influence human disease.”

Microbes often generate noxious compounds to attack one another within the close confines of the gut. But among these chemical weapons, colibactin appears unusual, suggested Balskus, a chemical biologist at Harvard University. “It doesn’t directly kill the target organisms, which is what we normally think of bacterial toxins doing within microbial communities.” Instead, the new research shows that colibactin tweaks microbial cells, to activate latent—and lethal—viruses tucked away in some bacteria’s genomes.

In 2006, a French team reported that mammalian cells that encountered the gut bacteria E. coli suffered fatal damage to their DNA. The researchers linked this damage to a cluster of E. coli genes encoding machinery for building a complex molecule. Dubbed colibactin, the molecule was extraordinarily difficult to study. After many tries, researchers simply couldn’t isolate it from the E. coli making it.

Colibactin is one of many ephemeral compounds that scientists suspect microbes make. Like invisible particles of dark matter in space, this “chemical dark matter” requires creative means to study. And as the authors further noted, “Despite its important biological activity, colibactin has eluded traditional isolation and structural elucidation. Information regarding its chemical structure has largely been derived from bioinformatic analyses and biochemical characterization.”

Scientists have known for years that colibactin can wreak havoc on human cells. Research by Balskus and many others has shown that the compound damages DNA, which can lead to colorectal cancer. As part of her exploration of the gut’s microbial chemistry, Balskus uses indirect approaches to examine these elusive molecules. Over the past 10 years, her team has probed colibactin by studying the microbial machinery that manufactures it. She and her colleagues have pieced together colibactin’s structure and determined that it damages DNA by forming errant connections within the double helix.

Building off this work, scientists elsewhere uncovered a definitive link to cancer: the molecule’s distinctive fingerprints appear in genes known to drive colorectal tumor growth. “Mechanistic studies have revealed that colibactin induces inter-strand DNA cross-links in vitro, causes cell-cycle arrest in eukaryotic cell culture, and affects tumor formation in mouse models of colorectal cancer,” the team noted. “ … studies have identified colibactin-associated mutational signatures in cancer genomes, predominantly from colorectal cancer.” Balskus’s most recent colibactin study got its start with the effects on the lab of another disease, COVID-19. Like many others, the Balskus lab had to undergo some rearrangements to reduce physical contact among the researchers. As part of this reshuffling, postdoc Justin Silpe, PhD, and graduate student Joel Wong ended up working near one another for the first time. Their conversations led them and Balskus to wonder how colibactin affected other microbes in a crowded gut. As the authors acknowledged in their paper, “In contrast to its effects on eukaryotic organisms, the effect of colibactin on the surrounding microbial community remains largely unknown.”

Early on, they found that exposing colibactin-producing bacteria to non-producers had little effect, suggesting that, on its own, the molecule isn’t particularly deadly. So, while previous studies had shown that colibactin production caused shifts in the composition of the gut microbial community in mice and inhibited the growth of specific staphylococci, they noted, “exposure to colibactin did not affect the growth of the vast majority (97%) of bacterial species tested …”

Silpe and Wong weren’t sure if colibactin, a large, unstable molecule, could even enter bacterial cells to damage their DNA. They then wondered if a third party—bacteria-infecting viruses—might be involved. Comprising little more than bits of genetic information, these viruses can slip into bacteria’s DNA and lie quietly in wait. Then, once triggered, they cause an infection that blows up the cell like a landmine.

When the researchers grew colibactin producers alongside bacteria carrying such latent viruses, they saw the number of viral particles spike, and the growth of many virus-containing bacteria drop. That suggested the molecule sparked a surge in active, cell-killing infections. Colibactin does indeed enter bacteria and damage DNA, the team showed. That damage sounds a cellular wake-up bell that rouses the viruses. “Although other functions of colibactin may exist, our discovery that it induces prophages provides one mechanism by which production of and immunity to this natural product might confer a competitive advantage over other microorganisms,” the team further noted.

Many microbes appeared equipped to protect themselves against colibactin. The Balskus lab identified a resistance gene encoding a protein that neutralizes the compound in a wide variety of bacteria. “… we identify bacteria that have colibactin resistance genes but lack colibactin biosynthetic genes,” the scientists wrote. “Many of these bacteria are infected with predicted prophages, and we show that the expression of their ClbS homologues provides immunity from colibactin-triggered induction.”

As the authors concluded, “By uncovering the phage-inducing activity of colibactin-producing bacteria, our findings reveal a previously unrecognized mechanism by which colibactin and potentially other DNA-damaging natural products may shape microbial communities.” And, while colibactin clearly has a dangerous side, it may serve as more than just a lethal weapon, Balskus suggested. For example, both DNA damage and awakened viruses can also induce genetic changes, rather than death, in neighboring bacteria, potentially benefiting colibactin producers.

The new discoveries indicate that cancer may be collateral damage caused by whatever else colibactin-producing bacteria are doing. “We always suspected that bacteria made this toxin to target other bacteria in some way,” she said. “It didn’t make sense from an evolutionary perspective that they acquired it to target human cells.”

Balskus next plans to investigate how the compound alters the community of microbes in the gut—which ones disappear and which thrive after exposure to colibactin. “The key to preventing cancer may be understanding the effects colibactin has on the microbe community and how its production is controlled,” she said.

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