American Scientist

I look at the petri dish being held out to me and observe streaks of deep purple slime, carefully cultivated atop a cloudy gel that vaguely smells of sourdough bread. This eggplant-colored and largely harmless environmental bacteria may hold the key to uncovering one of the body’s most mysterious immune responses.

Ed Miao, an immunologist at Duke University in Durham, North Carolina, and his former graduate student Carissa Harvest, now a clinical fellow at the University of Washington in Seattle, worked to turn this colorful bacterium into a tool to understand the evolution of an immune response, as they recently described in Nature Communications. This common microbe, Chromobacterium violaceum—which aptly translates to “purple-colored bacteria” —is native to the soil and freshwater of tropical and subtropical regions around the world.

Most of us have never heard of it, because most of us have an intact immune system. C. violaceum is only a problem for people with impaired immunity, for whom it can be highly fatal. Our world contains an estimated 1 trillion microbes, but only about 1,400 of those are considered to be bona fide pathogens. Miao studies how our immune systems evolved with these microscopic organisms: How are we able to prevent so many of these microbes from causing us harm? And how do the ones that inflict damage evade the immune system?

Miao was studying pyroptosis, a form of programmed cell death that occurs during infection. “We were studying Salmonella, which is quite smart and can hide itself,” says Miao. “We went looking for a [similar] pathogen, but one not as sneaky and that would be able to be detected.” Cue C. violaceum. Miao and his team observed that mice infected with C. violaceum were able to fully clear the bacteria. What they didn’t expect was to see something truly remarkable under the microscope. When his team observed the livers collected from C. violaceum-infected mice, they saw huge, layered lesions dappled throughout the tissue.

This stage was when Harvest, a rotating graduate student in Miao’s lab at the time, was affectionately instructed to “figure it out.” And Harvest was ready for the challenge. She pored over countless microscope slides and consulted multiple expert pathologists from different universities. Then she finally got a name for the striking lesion she was seeing in her mice.

Granulomas are a cluster of immune cells that form within tissues in response to a pathogen, and can grow large enough to be seen on X-rays. The granuloma acts as a physical barrier to wall off the invader from the rest of the healthy tissue. However, granulomas are considered a bit of a double-edged sword. On the one hand, granulomas prevent the pathogen from spreading. On the other, when they are unresolved, which happens regularly in cases of tuberculosis, they can be a source of reinfection when the immune system weakens.

To better study granulomas, researchers would normally use an animal model, but few up to now have accurately mimicked human granulomas. With the mouse model being the most widely used in the laboratory setting, this new work with C. violaceum shows a lot of promise for the future of granuloma research. “The mouse took the textbook definition of a granuloma and made it,” Harvest stated.

Harvest and Miao showed that the C. violaceum granuloma depends on innate immune cells. Under the microscope, a granuloma looks like a cross-section through a layer cake. The center contains the pathogen and dying cells that are surrounded by a swarm of neutrophils, the type of white blood cell that are the first responders of the immune system. This layer is then encircled by a protective wall of macrophages. Both neutrophils and macrophages deploy a powerful free radical, nitric oxide, to neutralize pathogens. The researchers demonstrate that mice deficient for the gene that produces nitric oxide, Nos2, are incapable of forming a functional granuloma, resulting in the spread of C. violaceum that turns lethal. Similarly, they also show that the protein gasdermin D, an important factor in the cell death cascade that leads to the formation of pores in the cell membrane, is equally important for effective granuloma formation. Miao and Harvest thus illustrated that granuloma formation is not dependent on one single factor, but rather a careful orchestration of multiple factors at once to form a protective granuloma.

“Other people observed these lesions in mice, but no one ever took the chance,” said Harvest. Their work, she says, gives valuable insights into the power of basic science, the mechanism of granuloma formation, and how the immune system evolved to protect the body from pathogens. Adds Miao: “This project is a great example of how the freedom of basic science can lead you to discovery in ways you never expected.”