Developed to Stop Bacteria, Designed to Stabilize

The new proteins, created by cooperating over long distances, may reverse antibiotic resistance in some bacteria

Communication is crucial. Bacteria may be more dangerous when in contact – they band together to build fortress-like structures known as biofilms that give them antibiotic resistance. But the biomolecular scientist in Israel and the microbiologist in California have forged their own links that could lead to new protocols for blocking colonies protected by biofilms. Their research was published in Proceedings of the National Academy of Sciences (PNAS), Uses.

This interdisciplinary collaboration began with a lecture given at the Weizmann Institute of Science at the Life Sciences Symposium. Professor Diane Newman of the California Institute of Technology was the speaker, and Institute professor Sarrell Fleischmann of the Department of Biomolecular Sciences decided to attend, although the lecture had no apparent impact on his own research. Newman described an enzyme she discovered that can interrupt the metabolism of the biofilm-building bacteria, Pseudomonas aeruginosa. The enzyme interferes with the action of the molecule (biocyanin) that the bacteria generate when it reaches a high cell density and begins to deplete of oxygen, and thus it is responsible for helping the bacteria deep within the biofilm to survive as well as better tolerate conventional antibiotics. However, this molecule is a double-edged sword: it could also be toxic to P. aeruginosa in the outer layers of the biofilm, where oxygen is present. Because biocyanin affects both biofilm development and antibiotic tolerance, Newman’s lab focused on identifying ways to disrupt its activities. Newman’s only problem, she said, was that the enzyme blocking the newly discovered biocyanin was unstable and produced in small quantities, and so far, standard laboratory methods for growing such proteins have not been successful.

Pseudomonas aeruginosa is an opportunistic bacterium that causes disease mainly in those with underlying conditions: in the lungs of patients with cystic fibrosis, peripheral wounds of patients with diabetes and on various medical devices implanted in hospital patients. Biofilms that are difficult to remove may help re-infection even after treatment, which contributes to increased antibiotic resistance, especially in hospital-acquired strains.

After the lecture, Fleischmann suggested to Newman that they try a new method for producing larger amounts of the enzyme. His lab specializes in computational protein design, and some of their recent work has included redesigning vaccine proteins to make them more stable.

A research student in his lab, Rosalie Lipsch-Sokolik, together with Dr. Olga Khersonky, Research Adjunct, took up the challenge of designing an improved, more stable enzyme to break down biofilms. But the enzyme was different from any enzyme Fleischmann’s lab had worked with before, and would require them to develop a new methodology: It was a pruner – three identical copies of an assembled protein “like barrels tied together,” says Fleischmann. That is to say, in addition to the protein’s structure. Singles, they will need to understand how the whole package fits together.

The group’s first step was to map the enzyme down to its atomic structure. This gave them a detailed picture of the forces that bind the protein together. When they added the resulting models from the three copies together to understand the formation of the shear, they noticed that the contact areas between the copies were poorly packed, atomically speaking, and they believed that these weaknesses would be a good place to start in designing a more stable structure.

But even after narrowing down the potential sites of modification, the number of design possibilities for such a protein compound was huge. Lebesh Sokolik ended up adopting a two-pronged approach. The first was to look at proteins made by other similar but slightly different bacteria, to see what they could borrow. The second was a kind of “hidden” atomic design approach, identifying only dozens or so points on the enzyme that could be modified and experimenting with different combinations of amino acids at only those points.

The beauty of the computer design methods developed in Fleischmann’s lab is not only the fact that they can produce hundreds of thousands of different possible protein designs in a very short time, but they also rank them from most likely to work to unlikely. To work at all. However, the only way to know if your hypothesis is correct – about which areas need a boost or the enzyme’s ability to keep working despite changes in the protein’s sequence – is to make those proteins and test them in real biological systems. Enter Dr. Chelsey M. VanDrisse, a postdoctoral fellow at Newman’s Laboratory, who led all experimental tests of Fleishman’s lab designs.

Fleischmann admits his team was nervous when VanDrisse and Newman told them that they could run experiments with only ten of Trimer’s enzymes designed due to the extremely challenging nature of the experiments. Their challenges were not limited to creating these new proteins, but rather included discovering how to purify them in sufficient quantities in the laboratory and then test them on an actual biofilm to build Pseudomonas aeruginosa with standard antibiotic treatment. The question was, could the team not only produce a more active protein, but determine if its application could facilitate the control of biofilms and begin to understand the mechanisms underlying the enzyme’s effects?

“Both teams were over the moon when the results came out,” Fleischmann says: Eight out of ten designer enzymes were produced in greater quantities than usual in Newman’s lab, without compromising their ability to fight biofilms. VanDrisse joked that she could now produce so much protein that she “could have put in my pills every morning!” “This showed that our hypothesis about the contact areas was correct,” says Fleischmann. One of the enzymes seemed particularly powerful and was produced in large quantities, so Vandress and Newman went above and beyond: they set out to check whether this type of enzyme could, at least in the laboratory, work in conjunction with a commonly used antibiotic for eradication. Biofilms.

In fact, they found that the enzyme, in combination with this antibiotic, worked much better than they had expected. Further analysis suggested that the enzyme first helps the antibiotic kill the bacteria in the oxygenated outer regions of the biofilm in a manner not previously seen, which leads, in a short time, to a significant reduction in the total number of viable biofilm cells.

Fleischmann adds that with deepening collaboration between two groups of scientists who typically read different journals, attend different conferences, and try very different methods at different levels – until VanDrisse arrived at Weizmann’s lab just before the first COVID-19 shutdown – he realized what started for him as a test of protein design methods. NSAIDs in his lab now have a very real chance of finding a cure for some of the most aggressive bacterial infections. It all comes down to making the right connections.


Professor Sariel Jacob Fleischmann’s research is supported by the Dr. Barry Sherman Institute for Medicinal Chemistry. Yeda Sila Basic Research Center; Schwartz / Reisman Collaborative Science Program; The Sam Usher Switzer Charitable Foundation; Dianne and Irving Kipnes Foundation, Caroline Hewitt and Anne Christopoulos, in memory of Sam Switzer; The Milner Foundation and the Ben B. and Joyce E. Eisenberg Foundation.

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