How the Sugars In Spit Tame the Body’s Unruly Fungi

Katharina Ribbeck’s lab collects mucus—the often gooey substance present in places like the mouth, gut, reproductive tract, and intestines. While the slimy goop may not be pretty from the get-go, a purification process can brighten it up. “Once you remove particulates and microbes, it’s a beautiful, beautiful clear gel—like egg white,” says Ribbeck, a professor of bioengineering at the Massachusetts Institute of Technology. “It’s really gorgeous.”

Ribbeck cares about spit because she’s trying to deconstruct how glycans, tiny sugar molecules hidden inside mucus, work to keep a particular organism healthy. Scientists already know that mucus is important in maintaining human health and supporting the microbiome. The glycans’ job, according to Ribbeck and others’ work, is critical. They specialize in managing microorganisms that can be beneficial—assisting in food digestion, regulating immunity, and protecting against germs—but that can be harmful if they outcompete one another or become virulent, potentially leading to infection. Like microscopic conductors, glycans ensure that each section of the microbial orchestra is playing in harmony.

In a study published this month in Nature Chemical Biology, Ribbeck and her collaborators showed how glycans keep a fungus called Candida albicans (C. albicans) from becoming problematic. The line between friend and foe is nebulously drawn in the case of C. albicans. The fungus is polymorphic, meaning it can take on different shapes: a rounded, yeast-like structure (generally considered normal) can turn into a filamented, thread-like shape associated with virulence. While the fungus can contribute to immunity, it can also lead to yeast infections or, even more seriously, a systemic infection of the bloodstream.

Sing Sing Way, a physician-scientist at Cincinnati Children’s Hospital Medical Center who was not involved in this study, has researched the ways that shapeshifting Candida can be beneficial for human health. “Complex microbes like Candida have co-evolved with not just humans, but other mammalian hosts, for a long, long time,” Way says. “They’ve developed strategies where it’s good for both.” He thinks that if we understand why and how the fungi change form, we can exploit this relationship to keep them on good behavior.

Ribbeck’s group had done previous work establishing how mucus stops other microbes from becoming dangerous. In this new set of experiments, the scientists wanted to know exactly how it works in the case of C. albicans.

But first, they needed a lot of goo. “It’s surprisingly hard to collect larger volumes of mucus,” Ribbeck says. “It’s a really precious material.” The team collected three kinds of mucus using different methods: aspirating human spit (similar to the way a dentist uses a suction tube to suck saliva from under a patient’s tongue), as well as scraping the insides of pig intestines and stomachs. Then, they incubated the purified mucus with C. albicans inside a well plate—a clear rectangular dish, punctuated with 96 beehive-like holes containing small volumes of fungi.

They discovered that all three types of mucus stopped the fungi from adhering to the plate, compared to a negative control. C. albicans also appeared rounder when the mucus was present, as opposed to the elongated version associated with filamentation. This, the researchers thought, indicated that the mucus could stop the fungus from sticking to bodily surfaces or forming biofilms, which are stringy, intertwined layers of the fungi that are associated with infections.

At the same time, they tested this effect in lab mice. Ribbeck’s team made small puncture wounds on the backs of the mice, then infected them with C. albicans and treated them topically with purified mucus. This significantly reduced the number of viable fungal colonies. The mucus didn’t directly kill the fungi, but the scientists hypothesized that by decreasing its virulence, it allowed the immune system to swoop in and clear the microbes out of the wound. Ribbeck compares it to pacifying an angry kid by giving them a lollipop—instead of squelching bad behavior with force, it persuades the troublemaker to be more pliant.

Now the team knew the mucus worked, but figuring out exactly what inside of it provided these protective properties required a bit of complicated biochemistry. Specifically, they wanted to know which glycans were doing the job. The scientists used a technique called non-reductive alkaline beta elimination—stripping glycans from the mucus proteins while preserving their individual structures. With this pool of 100 or so isolated glycans, they could run mass spectrometry to identify which varieties occurred in all three types of mucus and were probably the most important microbe-wranglers.

Then it was time to generate the most promising of these individual glycans from scratch in order to see if they could stop C. albicans from going bad. That task fell to Rachel Hevey, a research associate at the University of Basel and one of the senior authors of the study. Glycans are hard to make artificially because they consist of approximately the same molecules—a bunch of hydroxyl, or oxygen-hydrogen, groups connected to a carbon backbone. Figuring out how to orient each molecule in the correct position to make each distinct glycan takes a lot of time and expertise. “It’s a bit of a puzzle,” Hevey says.

To solve this sugary puzzle, Hevey and others developed step-by-step procedures to make sure that each chemical group was correctly attached to the chain. The scientists were particularly interested in O-glycans, ones that attach to things through an oxygen molecule, as those were among the most abundant and were common among the three mucus types.

Hevey says the final glycan product is akin to a bristle on a brush. When they added them to a plate of C. albicans, the scientists found that certain O-glycans could stop the fungus from becoming virulent—all by themselves. These specific glycans, which fall under a category called core 1 and core 2 based on their unique molecular building blocks, could stop the fungi from filamenting and downregulate the expression of virulence-related genes.

For Ribbeck, figuring out that single glycans can do the job was a “gamechanger.” “Something as common as mucus has all these tools at play,” she says.

“I think it’s definitely an advance,” says David Perlin, a professor at the Hackensack Meridian School of Medicine who was not involved in the study. “Understanding how O-linked glycans, which are the main components of mucus, contribute to controlling Candida, keeping it at bay, and trying to dampen down its pathogenic properties is quite interesting.”

Ribbeck’s team now has a whole host of future directions to explore. One option is to study translation, or how to turn this knowledge into new therapies. Building drug molecules that can replace helpful missing glycans could aid in developing medications to keep microbe populations in check.

More study into how artificial glycans act in a living mouse, rather than in a petri dish, would be important for future therapeutic work, says Way: “We would also then be interested to know if these types of things impact [C. albicans’] friendliness.”

Another direction involves understanding the role mucus and glycans play as conductors of the entire microbiome, helping C. albicans and its neighbors peacefully coexist. The scientists also found that a lack of mucus can disrupt this coexistence and introduce intense competition. In a way, the glycans protect C. albicans from getting overrun by other microbes—such as the bacteria Pseudomonas aeruginosa—ultimately throwing a person’s microbiome out of balance. “It’s like putting your kids in separate rooms,” Ribbeck says. “They don’t team up anymore.”

Figuring out how to keep a vast array of microorganisms in a friendly state will take quite a bit of work. But according to Ribbeck, harnessing the powers of this sugary slime may be a potent peacekeeping strategy. “Over millions of years, mucus has evolved strategies to keep those pathogens in check,” she said. “And—this is important—it doesn’t kill them. It tames them.”

How the Sugars In Spit Tame the Body’s Unruly Fungi

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