Iron-containing ferrosome granules have previously been discovered in Gram-negative environmental anaerobes, however, their presence in Gram-positive bacteria has not been previously documented. Now, a team of researchers has discovered that Clostridioides difficile (C. diff) produces ferrosomes and that these structures are important for infection in an animal model. In addition, the findings are a rare demonstration of a membrane-bound structure inside a pathogenic bacterium.

This work is published in Nature in the paper, “Clostridioides difficile ferrosome organelles combat nutritional immunity.

“The emerging idea that bacteria do compartmentalize biochemical processes in a way similar to eukaryotic cells really flips the field of microbiology on its head,” said Eric Skaar, PhD, professor of pathology and director of the Vanderbilt Institute for Infection, Immunology, and Inflammation.

“The best way to look for the accumulation of elements in a small space like a cell is with a method called STEM-EDS, which has not commonly been used for biological samples,” Skaar said. “We were fortunate to have access to a STEM-EDS instrument and collaborators at Vanderbilt Institute of Nanoscale Science and Engineering (VINSE) and we quickly proved that there was an accumulation of iron ‘dots’ within the bacterium.”

C. diff causes about 500,000 infections and more than 29,000 deaths in the United States each year, according to the Centers for Disease Control and Prevention, and treatment options are limited. People taking antibiotics that disrupt the healthy microbes in the gut are at increased risk for C. diff infection, which causes diarrhea and colitis. New strategies for treating this urgent public health threat are needed, Skaar said.

The researchers found that two genes (fezA and fezB), which are similar to those in environmental bacteria, were required for ferrosome formation. More specifically, they write, “Membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess.”

They showed that ferrosomes are required for C. diff to fully colonize and cause disease in an animal model and that ferrosomes were even more important for C. diff infection in a model of inflammatory bowel disease. These findings demonstrate that these iron-containing structures help the bacterium combat “nutritional immunity”—the host response of producing proteins to bind iron and attempt to starve the pathogen.

They then used cryogenic electron microscopy (cryo-EM) and cryo-tomography to show that the ferrosome structures were encased in a membrane, classifying them as organelles.

The results “establish ferrosome formation and all the factors involved in ferrosome formation as potential targets for new antibacterial drugs against an important infectious disease,” Skaar said. “Anytime we find new factors involved in host-pathogen interactions and show that they’re important for infection, that opens entirely new opportunities to make classes of antibacterial drugs that have not existed before. That is especially important in the face of rising antimicrobial resistance that we’re seeing globally.”

In future studies, the researchers plan to explore how ferrosomes are formed, whether other gut pathogens produce ferrosomes, and whether these structures might be shared in the gut as a source of iron. Skaar is also particularly interested in pursuing the emerging area of bacterial organelles.

“We think our study is a rare demonstration of an organelle in a pathogenic bacterium,” he said. “Now we want to know if there are other subcellular compartments in bacteria that we’re interested in that could teach us about how these cells perform various physiologic processes.”

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