Researchers at MIT have resolved that bacterial resistance should be overcome by hook or by hook. The hooks, in this case, are tail fibers, structures that bacteriophages use to snag bacteria and set them up for infection and, ultimately, death. Different tail fibers help phages target specific bacteria. If phages that brandish the right “hooks” are be found, they can be used to eliminate selected bacterial strains, treating infections while lessening the odds of provoking resistance.

The problem, however, is that finding and optimizing well-defined phages to use against a bacterial target is challenging. Fortunately, a solution is at hand. The right phages—that is, phages with the right tail fibers—may be engineered.

This solution has been demonstrated by a team of synthetic biologists led by MIT’s Timothy Lu, PhD, an associate professor of electrical engineering and computer science and of biological engineering. Lu and colleagues have developed a method to rapidly program bacteriophages to kill different strains of Escherichia coli by making mutations in a viral protein that binds to host cells.

“As we’re seeing in the news more and more now, bacterial resistance is continuing to evolve and is increasingly problematic for public health,” said Lu. “Phages represent a very different way of killing bacteria than antibiotics, which is complementary to antibiotics, rather than trying to replace them.”

The researchers created several engineered phages that could kill E. coli grown in the lab. One of the newly created phages was also able to eliminate two E. coli strains that are resistant to naturally occurring phages from a skin infection in mice.

Additional details of this work appeared October 3 in the journal Cell, in an article titled, “Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis.” The article describes how libraries of vastly diverse “phagebodies”—phages with different tail fibers—could be quickly generated by emulating antibody specificity engineering.

“Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis,” the article’s authors wrote. “This approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic ‘phagebodies.’”

This work builds on the Lu’s lab use of engineered viral “scaffolds” that can be easily repurposed to target different bacterial strains or different resistance mechanisms. “We think phages are a good toolkit for killing and knocking down bacteria levels inside a complex ecosystem, but in a targeted way,” Lu pointed out.

In 2015, the researchers used a phage from the T7 family, which naturally kills E.coli, and showed that they could program it to target other bacteria by swapping in different genes that code for tail fibers, the protein that bacteriophages use to latch onto receptors on the surfaces of host cells.

While that approach did work, the researchers wanted to find a way to speed up the process of tailoring phages to a particular type of bacteria. In their new study, they came up with a strategy that allows them to rapidly create and test a much greater number of tail fiber variants.

From previous studies of tail fiber structure, the researchers knew that the protein consists of segments called beta sheets that are connected by loops. They decided to try systematically mutating only the amino acids that form the loops, while preserving the beta sheet structure.

“We identified regions that we thought would have minimal effect on the protein structure but would be able to change its binding interaction with the bacteria,” explained Kevin Yehl, PhD, a researcher in Lu’s lab and a lead author of the current paper.

Vastly diverse libraries of phagebodies, or phages with different tail fibers, have been generated through natural evolution and structural modeling. Bacterial resistance to phagebodies was not observed across long timescales. [Cell]

The Lu team created phages with about 10,000,000 different tail fibers and tested them against several strains of E. coli that had evolved to be resistant to the nonengineered bacteriophage. One way that E. coli can become resistant to bacteriophages is by mutating lipopolysaccharide (LPS) receptors so that they are shortened or missing, but Lu and colleagues found that some of their engineered phages could kill even strains of E. coli with mutated or missing LPS receptors.

“We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model,” the authors of the Cell article concluded. “We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.”

Lu and Yehl now plan to apply the phagebody approach to targeting other resistance mechanisms used by E. coli, and they also hope to develop phages that can kill other types of harmful bacteria. “This is just the beginning, as there are many other viral scaffolds and bacteria to target,” Yehl said. The researchers are also interested in using bacteriophages as a tool to target specific strains of bacteria that live in the human gut and cause health problems.

“Being able to selectively hit those nonbeneficial strains could give us a lot of benefits in terms of human clinical outcomes,” Lu said.

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