More than half of the cells on or in our body are microbes. Germs help us digest food, affect our mood, and defend against invading pathogens.

For nearly 20 years, scientists have established how our complex communities of microbes, called microbiomes, are essential for health and how they relate to disease.

Now they want to probe exactly what the microbes are doing inside our bodies. Scientists are starting to understand that information about microbial function in a person’s gut, for example, could provide specific information about a patient’s health. Tailoring treatments based on personal markers of health or disease is the foundation for the future of medical care known as precision medicine.

Chemical biologists at the Pacific Northwest National Laboratory (PNNL), led by Aaron Wright, have developed custom chemical probes to study microbial function in human, animal, insect and environmental microbiomes.

“We can use the same chemical probe to measure the amount of activity in a mixture of microbial enzymes, in living cells, and in a collection of microbes in a community,” Wright said. The PNNL team has developed some of these molecular tools to be marketed for future clinical diagnostics.

Earlier this year, start-up Enzymetrics Bioscience, Inc. authorized two PNNL chemical probe platforms to develop tests for gut microbiome functions. This commercialization effort was also nominated for an award recognizing successful partnerships between federal laboratories and industry.

“One of the licensed chemical probe platforms has been designed to analyze human stool samples for specific gut microbiome activity that may impact cancer treatment outcomes,” said Jennifer Lee, manager. marketing to PNNL. “The information from the PNNL platforms could also help physicians refine their therapeutic orientations for gastrointestinal diseases and immunological disorders. “

Probe bait protein function

For any cell, proteins do the work that makes it function. This work often involves performing specific chemical transformations involved in metabolism or cell signaling. But just because a protein is present in a cell does not mean that it is active at some point. PNNL researchers have a way to label active proteins, called activity-based protein profiling.

“We first design a chemical probe to be a molecular bait for the active proteins that perform a given transformation,” Wright said. “Then we label the bait so that we can pull it out of the complexity of a microbial community. “

One of the recently licensed chemical probe platforms detects a microbial protein that causes side effects of certain drugs. Just as our own cells process the drugs we take to treat symptoms or illnesses, the microbes in our gut also process these drugs. When gut microbes produce the enzyme beta-glucuronidase, their metabolism can cause problems.

Here’s why: When human cells process a drug, they attach a particular sugar to it to mark it for elimination. Some intestinal bacteria produce beta-glucuronidase. When the sweet medicine reaches these bacteria, this enzyme cuts the sugar. The result is that a drug continues to circulate in the body for longer than expected, which can cause side effects.

For example, the drug irinotecan is a first-line drug for colorectal cancer because it is very effective. However, if a patient has increased levels of beta-glucuronidase from their gut microbiome, a side effect of this drug is severe diarrhea. It may take weeks of wasted treatment time to switch to other drugs that are not affected by beta-glucuronidase.

To build a chemical probe to detect beta-glucuronidase activity in a diagnostic test, PNNL scientists first developed a molecule that looks like the sugar that this enzyme recognizes. To detect where the probe has attached, scientists add a fluorescent label to the sample. This tag clicks on the probe bound to the enzyme and glows. Scientists then measure the amount of fluorescence in a sample to quantify the amount of active beta-glucuronidase.

The team refined their probes and sample preparation techniques to use this platform with human stool samples. The continued research and development through this license aims to transform the approach of laboratory experimentation into a reliable diagnostic test for the clinic.

Applications include environmental microbiomes and the science of exposure

The strength of PNNL’s chemical probe technology lies in the detection and quantification of the amount of protein activity in complex samples such as microbiomes. Measuring cell function at different scales provides molecular details of how living systems work together, whether it’s microbes in the human gut or microbes in the soil breaking down leaf litter.

Wright and his colleagues have already developed probes to identify enzymes in soil microbes that break down cellulose, the component of plants sought after for the production of biofuels. They also developed probes that mimic common B vitamins to help understand how these nutrients shape the makeup of environmental and human microbiomes.

For human health applications, PNNL researchers used these probes to track how fatty foods affect the metabolism of the liver or lungs in ways that could affect the way the body processes drugs. They tracked how key detoxifying enzymes in liver and lung tissue also change function with age or after exposure to environmental contaminants, street drugs, or dietary changes. And, they’re currently studying how antibiotics affect the functioning of the gut microbiome and how gut microbes might influence our body’s internal clock.

The team is currently working with researchers at the University of Delaware to study how gut microbial activity in mealworms could be harnessed to break down plastic. And, a team of PNNL researchers is exploring how to combine fluorescent chemical probes with automated analysis of microscopy images to detect pathogenic bacteria in soil.

Learn more about licensing chemical probe platforms for other applications that sort microbes by function.

This work was mainly supported by the research program of the Ministry of Energy, Bureau of Science, Biology and Environment; national institutes of health; and cooperative research and development agreements.

By Mélissa Fellet

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