Multidrug-resistant pathogen's ferocity found to stem from targeting RNA of competitor bacteria

It’s a jungle out there, even for tiny microbes. Bacteria are constantly fighting for survival and resources, duking it out not only with host immune cells but with each other. Perhaps the most formidable warrior of all is Pseudomonas aeruginosa, the multidrug-resistant bacteria associated with hospital-acquired infections like sepsis and pneumonia.

Now, scientists from McMaster University in Ontario have discovered that P. aeruginosa’s ferocity stems in part from its ability to target its fellow bacteria with an RNA-damaging toxin that kills them. The team believes the findings they reported on Tuesday in Molecular Cell will lead to a better understanding of bacterial physiology and perhaps could open a door to developing new antibiotics.

“From a bacterial physiology standpoint, it’s really cool, because RNA is involved with everything,” Nathan Bullen, first author and graduate student in Dr. John Whitney's lab at McMaster’s Michael G. DeGroote Institute for Infectious Disease Research, said in an interview. “It’s a very interesting way to kill a competing cell by attacking all these processes at once.”

For Bullen and the team, which also included colleagues at Imperial College London and the University of Manitoba, the decision to study P. aeruginosa stemmed from a simple question: Why was it able to grow virtually anywhere?

“It’s really good at competing with the native bacteria and establishing its own environmental niche. It takes over and propagates,” Bullen said. “We got interested in the mechanism behind that.”

The researchers first focused on a protein complex called the type VI secretion system, or T6SS. T6SS sits across the cell envelope of Gram-negative bacteria and secretes bacterial effectors, or proteins that enable bacteria to invade host cells in addition to helping them survive, grow and replicate. In the case of P. aeruginosa, findings from other labs’ studies had shown that the effectors of one T6SS complex, H2-T6SS, were especially potent against other bacteria. To figure out which effectors contributed most to H2-T6SS’s antibacterial activity, Bullen's team generated knockout strains of the gene clusters encoding each one and ran competition assays against E. coli.

The results homed in on the enzyme RhsP2. Unlike the other effectors the team assessed, its function had not yet been characterized. When the team ran X-ray crystallography to figure out its structure, they found that it looked similar to a well-known class of molecules called APD-ribosyltransferases, or ARTs. ARTs have long been implicated in the toxicity of bacteria like cholera and diphtheria, the latter of which RhsP2 closely resembled.

While scientists have known about ART involvement in human disease for some time, this finding was among the first examples where one appeared to be a weapon against other bacteria.

“The role of ARTs in bacterial competition is really new,” Bullen said. Another antibacterial ART, Tre1, was only characterized in 2018.

Even more novel was the team’s next finding: that RhsP2 wiped out the competition not by targeting proteins but RNA. Most ARTs, including Tre1, exact their toxicity by attacking proteins. In contrast, RhsP2 modifies the 2'-hydroxyl groups of transfer RNA, preventing protein synthesis downstream. It also appears to modify many other types of non-coding RNA across a broad range of functions, hitting the bacteria it’s targeting on multiple fronts.

“It’s relatively promiscuous as an enzyme with regards to substrate recognition,” Bullen said. “We think most RNAs that it comes across, it's going to modify.”

This could present a challenge for developing future applications. Most antibiotics work by tackling bacteria-specific proteins that aren’t found in eukaryotes. But in this case, it’s not yet clear whether the enzyme’s RNA recognition mechanism is unique to bacterial RNA.

This is the question Bullen plans to tackle in his next phase of research, when he and other researchers on the team will undertake structural studies to see exactly how the enzyme is interacting with RNA. Modeling this interaction is a significant endpoint in itself.

Better understanding how RhsP2 recognizes RNA could open the door to biomedical engineering applications, such as making it so it can be engineered to recognize bacteria more specifically.

And if the work never directly leads to new therapies? Bullen still won’t be disappointed, because someone else will likely use it to conceive of something he never even considered.

“That’s the beauty of the collaborative nature of science,” he said. “Even if a new antibiotic doesn’t come out of this, people will have lots of ideas.”

Editor's Note: A previous version of this article named Nathan Bullen a co-corresponding author of the study. It has been updated to reflect that he is first author and is a graduate student in Dr. John Whitney's lab, and that their team included researchers at Imperial College London and the University of Manitoba.