Behind enemy lines

Antibiotic resistance is what happens “when germs like bacteria or fungi develop the ability to defeat the drugs designed to kill them,” according to the CDC, and it’s one of the most pressing threats to human health. Each year, more and more patients succumb to infections because the available antibiotics don’t work.

In research unveiled earlier this year, a team helmed by UCSF School of Pharmacy faculty member Danica Fujimori, PhD, laid out a two-pronged strategy for staying one step ahead of the deadliest drug-resistant bacteria—all based on studying the evolution of antibiotic resistance in the laboratory.

In findings described in eLife, the group first uncovered how mutations to a bacterial enzyme, called Cfr, result in resistance to antibiotics. And in findings described in Nature Structural and Molecular Biology, in collaboration with fellow School faculty member James Fraser, PhD, the group used cryo-electron microscopy (cryo-EM), a powerful tool for determining the 3D structure of molecules, to observe exactly how a newer antibiotic is able to overcome this form of resistance at the molecular level.

Some of the mutations discovered in Fujimori’s lab, housed in the Department of Pharmaceutical Chemistry, are already being observed in patient cases, and the twin papers outline a path forward for drug developers.

“Cfr resistance is a massive clinical threat, affecting eight entire classes of antibiotics,” said Kaitlyn Tsai, PhD, first author on both papers, who was pursuing her doctorate in chemistry and chemical biology in the Fujimori lab at the time of the studies. “Hopefully, the knowledge generated from our work can be used to improve drugs that continue to be rendered ineffective by this type of resistance.”

To know one’s enemy

Cfr protects bacteria from antibiotics due to its impact on an important cellular structure, the ribosome—the protein factory of cells. Bacteria use ribosomes to grow and multiply, and many antibiotics are designed to gum up the gears of these ribosomes. Cfr is adept at making a chemical change in ribosomes, known as methylation, that physically blocks antibiotics from those molecular gears.

Danica Fujimori, PhD

To make headway into the problem of Cfr resistance, Tsai and Fujimori used a technique called directed evolution, which “mimics nature at the bench, but in more condensed timelines,” said Tsai.

With the help of collaborators from the Weizmann Institute (Israel), the team engineered bacteria with “mutagenized” Cfr—Cfr that had been given a slew of random mutations. They treated those bacteria with antibiotics. The surviving—and therefore most drug-resistant— bacteria were isolated, mutagenized further, and exposed to more antibiotics. It was evolution in a petri dish, but at warp speed.

After several rounds of this directed evolution, the team soon had the most antibiotic-resistant descendants of their initial bacterial strain. Tsai began to test these super-bacteria, aiming to understand why their Cfr enzymes were so adept at erecting molecular shields on bacterial ribosomes.

Tsai’s first experiments showed that these mutations to Cfr weren’t making the enzyme more catalytically efficient in modifying ribosomes—an initial “disappointment,” said Fujimori, given their early expectations that such directed evolution would lead to better enzymes.

Determined, Tsai buckled down and made a critical discovery.

“Rather than getting an enzyme that is a faster enzyme in methylating ribosomes, we got an enzyme that is more stable and accumulates in the cell better,” said Fujimori. “It provides more complete modification of the ribosome, thoroughly shielding it from antibiotics. This was surprising to us, and Kaitlyn was brave to stick with it.”

And perhaps most remarkably, the very mutations the group had found in the lab-grown bacteria were already present in samples from patients around the world.

“Our data suggests that laboratory evolution potentially mimics what has [actually] occurred in nature,” said Fujimori. “We have yet to test that, but it's really interesting that some of the predominant mutations in our library are also there, in environmental isolates.”

To overcome the antibiotic resistance caused by these mutations, however, Tsai and Fujimori would need the help of experts in cryo-EM, a technique that enables scientists to observe individual drug molecules interacting with bacterial enzymes.

A molecular workaround for antibiotic resistance

Elisabeth Fall

James Fraser, PhD

The team enlisted James Fraser, PhD, faculty member in the Department of Bioengineering and Therapeutic Sciences, to use cryo-EM to watch antibiotic-resistant bacteria square off against antibiotics. In particular, they were curious how certain antibiotics were thwarted by the Cfr-modified, drug-resistant ribosomes, while others remained potent.

“We really needed a high resolution structure to visualize that tiny methyl group that Cfr installs on ribosomes to figure out how everything fits,” said Tsai. “Thankfully, that was really championed by the cryo-EM experts here at UCSF.”

Tsai and Fujimori wanted to compare drugs with varying effectiveness against Cfr-resistant bacteria. Linezolid, an antibiotic that has been used for the last 20 years, has become less and less effective at treating infections by these bacteria. A related drug, radezolid, which is currently in clinical trials, is able to defeat these bacteria.

Antibiotic-resistant ribosomes were mixed with either drug and imaged under the penetrating power of cryo-EM, using “a really cool trick [that] stabilized antibiotic binding to the ribosome,” said Fujimori.

The cryo-EM snapshots revealed exactly how radezolid manages to gum up these protected ribosomes, while linezolid fails. The differences come down to subtleties in the structure of each drug, and are driven by additional physical contacts between the ribosome and radezolid. And importantly, these subtleties were likely to matter for the seven other classes of antibiotics with waning effectiveness against Cfr-resistant bacteria.

A figure from Tsai, Fujimori, Fraser and team’s Nature Structural and Molecular Biology paper, depicting the structures of the antibiotics, linezolid (LZD, yellow, left) and radezolid (RZD, green, right), interacting with an antibiotic-resistant bacterial ribosome (blue and purple). Only radezolid retains its ability to defeat bacteria with this form of drug resistance.

“We are very much just learning from a known molecule, radezolid, and providing this information as a set of guiding principles on how to design better antibiotics,” said Fujimori.

The two studies may have addressed just one of the many fronts in the ongoing fight against antibiotic resistance, but thanks to the success of their collaboration across the university and institutions worldwide, Fujimori and Tsai hope their strategy be extended more broadly.

“This work was definitely challenging but also extremely fun, especially because of the environment at UCSF, having so many people to talk to who had experimental insight and guidance, helping us get various aspects of these experiments off the ground,” said Tsai, who is now a scientist at Metagenomi, a biotech company.

And with cryo-EM structures of resistant ribosomes guiding the design of future drug therapies, the hope is that even the wiliest antibiotic-resistant bacteria won’t have a chance.