New tools for critical research

It has no facial recognition software, no fingerprinting system, no ID cards to scan and verify. But the human immune system is constantly evaluating the identity of the bacterial cells it encounters. Are they the friendly type — the ones that help digest food, for example? Or are these enemy agents, likely to create trouble and misery wherever they go?

These first impressions are critical to the immune response and scientists are eager to understand more about the processes behind that response. With that kind of knowledge, new therapies and precise medical approaches can be used to arrest or prevent disorders of the immune system.

Millions around the world suffer from chronic inflammatory disorders caused by inadequate responses to the microbiome, the huge population of microorganisms that share our bodies. Enhanced immune responses or delayed healing create something like a civil war within the body, prompting tissue damage and organ dysfunction. Inflammatory bowel diseases such as Crohn’s Disease and ulcerative colitis are examples of such disorders.

Now the labs of University of Delaware chemical biologist Catherine Leimkuhler Grimes and immunologist Hans-Christian Reinecker of the University of Texas Southwestern Medical Center have produced potent new tools that offer researchers precise, new perspectives on how the immune system is triggered and what happens in the cascade of responses that follows.

Grimes’ research has focused on just this kind of work. She and her team of students study what happens when the human immune system first encounters the cell walls of bacteria, whether the bacteria are commensal (bringing benefits to the body) or pathogenic (causing illness). To learn about those first encounters, they have studied the outermost shell of bacterial cells by studying the bacterial peptidoglycan (PG) fragments that bacterial cell walls are made of.

Grimes’ lab has looked at many angles of peptidoglycan. In a recent publication of the American Chemical Society’s journal Chemical Biology, for example, her team, led by graduate student Ashley Brown, builds on their metabolic engineering work, prompting bacteria to produce cell walls with modified building blocks. This allows fundamental features of these barricades to be understood. Fragments of these cell walls eventually are sloughed off, but now they contain something like hooks that capture physiologically relevant bits.

Grimes said she and her team are somewhat obsessed with these fragments because they hold great potential for cancer therapies and immune system modulation.

The fragments used in traditional immunological experiments are muramyl dipeptides (MDP) — readily available “off-the-shelf” minimal PG fragments that have provided much insight into innate immune responses to bacteria.

Grimes and her team have built on this work in collaboration with the Reinecker lab. The teams have shown that more biologically relevant, complex PG fragments — exactly the kind the immune system would see — can be synthesized in the lab. They also have demonstrated that these biologically inspired synthetic fragments reveal more complex signaling than was previously recognized.

In this project, Grimes’ students synthesized four variations of PG fragments to see what kind of immune responses would occur with each. For one, they were inspired by the bacterial breakdown products found in cultures of Lactobacillus acidophillus, a bacterium normally found in your gut or the yogurt you eat. The team found that each of the four derivatives had unique genetic signatures and produced unique responses, all of them different than MDP would have produced. The product from the human gut bacteria produced the most potent response.​

Earlier this year, the researchers published their findings in ACS Central Science, a journal of the American Chemical Society, and now are working to develop a library of relevant PG fragments in a project supported by a $1.9 million grant from the National Institute of General Medical Sciences (NIGMS). The new funding will yield data that will be critical in future development of antibiotics and treatments for inflammatory disorders and is complementary to the funding Grimes and her team received to metabolically engineer bacterial cell walls.

"These are the small molecules we should be using,” Grimes said. “And if these things truly become immune modulators, they could be used in all kinds of front-line work.”

As an example of the potential gains these new tools provide, Reinecker said they identified more than 30 genes related to inflammatory bowel disease that were activated in response to the gut bacteria fragment the Grimes Lab synthesized.

“It all starts with having sufficient research tools,” he said. “These components are now in our hands and that is critical for getting at these pathways…. Once we have the mechanisms, we can interfere with them. We can block them or enhance them. Now we have components that will allow us to more precisely understand how the immune system recognizes bacteria.”

Grimes’ collaboration with Reinecker stems from her days as a postdoctoral researcher at Harvard with Dr. Daniel K. Podolsky. Reinecker was establishing his lab at Massachusetts General Hospital at the time and the two would often talk in the hallway about the missing tools that immunologists desperately needed.

“This is a beautiful collaboration — chemistry with immunology,” Reinecker said.

Grimes’ students produced the chemical fragments Reinecker’s immunology lab needed for experiments that simply couldn’t be done with the simpler “off-the-shelf” MDP probes. Reinecker’s immunology lab, in turn, gave Grimes’ students insight into how their fundamental science can provide powerful new possibilities for medical research.

Lead authors Kristen DeMeester and Klare Bersch both earned their doctorates at UD during this work. DeMeester now is working at Scripps Research in California and Bersch works as a medicinal chemist at Prelude Therapeutics in Wilmington, Delaware.

“I’m a synthetic chemist, but I hadn’t done any biological assays prior to this experience,” DeMeester said. “Now I do a lot of it. Infecting cells is all I do.”

Both had key roles in this work. DeMeester helped identify the fragments needed for the work in collaboration with Kimberly Wodzanowski. Bersch synthesized and characterized them and established the process for others to use in the lab so future testing can continue.

“You can’t get these from a vendor,” Bersch said. “You have to have a synthetic chemist.”

Once they had synthesized the compounds they wanted to use, they took them to Reinecker’s lab in Boston (before he moved to Dallas) and worked with Rachid Zagani there to treat macrophage cells (the cells that provide immune defense), extract genetic material and test it.

They found exciting things.

“Each fragment we tested had a different signature of genes,” Bersch said. “They were unique responses.”

The immunologists in Reinecker’s lab also got new insights from the project.

“As chemists, we look at the structure of molecules, bonds and atoms,” DeMeester said. “We’re looking at atomic networks. Immunologists look at cells in that complex way, in those types of networks. But they’re looking at cellular protein networks.”

Grimes said making these new molecular probes available will expand the capacity of researchers to pursue new questions and will surely lead to new findings.

Having just one primary kind of probe restricted the insight researchers could gain.

Grimes said it reminded her of her daughter’s fondness for the color pink. It is her “go-to color” in the crayon box. Everything winds up pink.

“Just as I’m trying to teach my daughter that there is more than one color in the box to choose and how much more could be represented on the paper with all of the colors, Christian and I are trying to teach the immunologists that there is more than one peptidoglycan fragment and it’s important to use more than one,” Grimes said. “We need to widen our palette when considering the depth of immunological response around these fragments.”

DeMeester said it is exciting to be part of such work.

“This really shows the power of collaboration and how great it can be,” DeMeester said. “The best part of the whole thing for me was seeing something for the first time that the world hasn’t seen before. I wouldn’t be the scientist I am without my mentor Catherine Grimes.”

Also participating in the collaborative research were Siavash Mashayekh and Kimmie Wodzanowski of UD’s Department of Chemistry and Biochemistry and Shuyuan Chen and Shuzhen Liu, both of the University of Texas Southwestern Medical Center.

The work was supported by the Delaware COBRE (Centers of Biomedical Research Excellence) Program with a grant from the National Institute of General Medical Sciences, by the National Science Foundation and by grants from the National Institutes of Health.​

​Catherine Grimes is professor of chemistry and biochemistry at the University of Delaware and co-director of the Chemical-Biology Interface Graduate Program at UD. She earned her bachelor’s degree in chemistry at Villanova University, her master’s at Princeton University and her doctorate at Harvard University, then did a postdoctoral research fellowship at Harvard and Massachusetts General Hospital before joining the UD faculty in 2011. She has been recognized with many awards and honors and has been named an Alfred P. Sloan Research Fellow and a Pew Biomedical Scholar. She was recently selected for the David Gin Young Investigator Award by the American Chemical Society. She is most proud of the students that she mentors through her research and teaching.​

​ Article by Beth Miller; Photo illustration by Jeffrey C. Chase; Photos by Evan Krape and courtesy of Catherine Grimes 

Published December 02, 2021​