CEL

Researchers found that certain enzymes within a class known as bile salt hydrolases (BSHs) can restrict Clostridioides difficile (C. diff.) colonization by both altering existing bile acids and by creating a new class of bile acids within the gut's microbial environment. The work could lead to 'designer' probiotics that protect against disease by introducing specific BSHs to the gut after antibiotic treatment. Not all probiotics are created equal. In a new study, researchers found that certain enzymes within a class known as bile salt hydrolases (BSHs) can restrict Clostridioides difficile (C. diff.)colonization by both altering existing bile acids and by creating a new class of bile acids within the gut's microbial environment. The work could lead to "designer" probiotics that protect against disease by introducing specific BSHs to the gut after antibiotic treatment. Selecting the right suite of BSH-producing bacteria is critical, because the study found that interactions between BSHs and bile acids differ depending upon the type of bacteria the BSHs come from. Certain bacteria within the gut microbiota contain BSH enzymes, which chemically modify bile acids. Bile acids are made in the liver and play an important role in modulating cholesterol levels, regulating fat absorption, shaping the immune system, and affecting which bacteria can colonize the gut. Although researchers had long suspected a connection between BSHs from beneficial bacteria, the bile acid pool, gut microbial composition and host health, until now relatively little was known about how BSHs function and their potential impacts on host health. "The old dogma -- that BSHs are needed for gut colonization because they render toxic bile acids non-toxic -- oversimplified what's actually happening," says Casey Theriot, associate professor of infectious disease at North Carolina State University and co-corresponding author of the study. "The reality is that BSHs' interactions are context-dependent, meaning they're affected by the type of bacteria they come from," Theriot says. "And they don't just interact with bile acids produced by the host. BSHs in the microbiota can create and interact with a new class of bile acids called microbial conjugated bile acids (MCBAs) -- bile acids that we didn't even know existed until recently." In the new study, Theriot led a collaborative research team that included microbiologists, chemists, biochemists, and clinicians from NC State, the University of North Carolina at Chapel Hill, and the University of California, San Diego on a deep dive into BSHs. Specifically, they looked at hundreds of BSHs from different Lactobacillaceae bacteria (which houses most probiotic strains) and then included BSHs from the gut microbiota (nearly 1,000 unique BSHs in total). Matthew Redinbo, Kenan Distinguished Professor of Chemistry in UNC-Chapel Hill's College of Arts and Sciences, and his departmental colleagues (led by then graduate student Morgan Walker) were instrumental in determining the structure of BSHs and how they "choose" to interact with bile acids, by either adding or taking away certain amino acids. "We found the tiny molecular fingerprint that defined whether a BSH would 'turn left' or 'turn right' in terms of what they processed," Redinbo says. "Knowing that allowed Casey's team to steer the bile acid pool in whatever direction they wanted." The researchers used a cocktail of Lactobacillus BSHs to figure out if they could change the bile acid pool enough to alter C. diff colonization in both human stool samples collected from patients susceptible to C. diff infection (CDI) and in a mouse model of CDI. In both human stool samples and mice, the researchers saw that pre-treatment with BSH cocktails impacted C. diff colonization. Interestingly, the researchers noted elevated levels of MCBAs in the gut microbiota of the BSH-treated mice. To determine whether the MCBAs were also involved in inhibiting C. diff germination and growth, they tested the MCBAs against C. diff in vitro. In most cases, the presence of MCBAs inhibited multiple steps of the C. diff life cycle. "This is more evidence that BSHs are driving changes in the bile acid pool -- including making MCBAs -- that could serve to inhibit C. diff," Theriot says. "We've uncovered a new function for BSH enzymes." "This work highlights the importance of BSHs as key intestinal enzymes and promising new therapeutics," says Matt Foley, research scholar at NC State and co-first author of the study. "Using BSHs in combination with other strategies may offer a new approach to treat C. diff." The researchers see the work as the first step toward potential probiotics that could be customized to protect against a variety of bacterial infections and intestinal diseases. But first, more work must be done to determine how and why the BSHs decide which MCBAs to produce and/or target. "This is an important illustration of how deciphering the biochemical and genetic basis for probiotic functionality both leads to a better understanding of how we can combat gut disease with novel modalities, and also practically design and formulate next-generation commercial probiotics," says Rodolphe Barrangou, the Todd R. Klaenhammer Distinguished Professor in Probiotics Research at NC State and co-corresponding author of the study. The work appears in Nature Microbiology and was supported by the National Institutes of Health, the National Science Foundation, IFF Corporation and the U.S. Environmental Protection Agency. The MCBA detection work was done by Erin Baker, formerly of NC State and currently at UNC-Chapel Hill, Allison Stewart of NC State, and Emily Gentry and Pieter Dorrestein from UCSD.

Scientists have long tried to introduce genetically engineered bacteria into the gut to treat diseases. In the past, these attempts have focused on engineering common lab strains of E. coli, which cannot compete with the native gut bacteria that are well adapted to their host. Now, a group of researchers successfully engineered E. coli collected from both human and mice gut microbiomes and showed that they have the potential to treat diseases such as diabetes. Scientists have long tried to introduce genetically engineered bacteria into the gut to treat diseases. In the past, these attempts have focused on engineering common lab strains of E. coli, which cannot compete with the native gut bacteria that are well adapted to their host. Now, a group of researchers from the University of California, San Diego, successfully engineered E. coli collected from both human and mice gut microbiomes and showed that they have the potential to treat diseases such as diabetes. Their finds are publishing in the journal Cell on August 4. "All I can say to the non-native bacteria is good luck. The gut microbiome is very dynamic and is constantly changing, making things even harder for the non-native bacteria," says Amir Zarrinpar, a gastroenterologist at UC San Diego Health and the senior researcher of the paper. "It is challenging for bacteria that have never lived inside of a mammal before to now go into the gut microbiome jungle with all of these hostile conditions that are geared towards preventing bacterial invaders from taking hold." The group devised a solution to this problem by directly engineering E. coli collected from the hosts. "Bacteria in our body are adapted to each one of us specifically: the kind of foods we eat, the common stresses our body experiences or induces, and our genetic background," says Zarrinpar. "This constantly fluctuating environment is their normal." This is a big advantage for native bacteria and makes them ideal candidates for engineering. "We have engineered these bacteria to become factories that can live in our microbiome and potentially produce medicines," says Zarrinpar. "We know that E. coli can pick up pathogenic genes and cause disease, and now we're just realizing that if we put a beneficial gene in, it can help us to treat chronic diseases, maybe even cure some of them." The team first collected stool samples from the host and extracted E. coli for further modifications. "We say to the bacteria: Hey, we will give you a new superpower, which you may not even benefit from, but we will put you right back into the environment that you thrive in," says Zarrinpar. The superpower that the team gave to these specific bacteria is a protein called bile salt hydrolase (BSH). After a single treatment in mice, E. coli with BSH were found throughout the entire gut of the mice and they retained their BSH activity for the entire lifetime of the host. The group also show that the BSH activity was able to positively influence diabetes progression in mice. This is a significant improvement over similar treatments with non-native laboratory strains of engineered bacteria, where more than one treatment is often required. And these engineered bacteria do not stay in the host's gut for nearly as long as, or as consistently as, the native E. coli method identified by Professor Zarrinpar's team. In addition to successfully influencing diabetes in mice, the group was also able to make a similar modification to E. coli extracted from human gut. While they have demonstrated substantial results, engineering native bacteria comes with another set of challenges. "Native bacteria are very resistant to modifications; it is part of their innate defense mechanism," says Zarrinpar. Their data suggest that inserting a gene into a native bacterium has about 100-fold lower success rate than doing so with lab strain bacteria, but Zarrinpar and his team are optimizing this process. "There are many new genetic engineering tools available now that will allow us to engineer these bacteria more effectively," says Zarrinpar. The group is planning on using this technology to find ways to treat more diseases. "We are dreaming big," says Zarrinpar. "This technology is something that can potentially open up the application of the microbiome therapy to influence so many different chronic and genetic diseases."