Sugar! How C. difficile toxin A enters intestinal cells

Clostridiodes difficult the infection has become a major cause of severe, sometimes fatal diarrheal disease. It's in hospitals and long-term care facilities where patients follow long-term antibiotic treatment as the product grows best, but it's also a growing problem in the community. Much of the damage caused by C. diff is caused by the toxins produced by the bacteria, which damage the intestinal lining.

Now with the new research published today in Microbiology of naturewe finally know how C. diffThe two main toxins, A and B, enter the intestinal cells, the first step towards a possible treatment without antibiotics.

"The disease is purely due to toxins," said Min Dong, Ph.D., who conducts research on bacterial toxins in the urology department of Boston Children's Hospital. "It's hard toxin-free strains can colonize the intestine, but they are not a problem. "

Screening for genetic vulnerabilities

In 2016, Dong and his colleagues revealed the port of entry of toxin B. With the help of gene editing technology CRISPR / Cas9, they examined all human cell genes to determine their potential role in toxin binding and entry into cells. When they mutated the gene of a receptor called Frizzled, the toxin was unable to enter the cells and the intestinal tissues became less sensitive to it.

Using the same approach, the team has now identified the port of entry for toxin A. More importantly, she discovered that the activity of this toxin could be blocked by molecules under development for various medical indications.

CRISPR / Cas 9 toxin A assays were conducted by Liang Tao, Ph.D., Songhai Tian, ​​Ph.D., and Jie Zhang, Ph.D. in Dong's laboratory. They discovered that many genes involved in the synthesis of certain sugar molecules were necessary for the binding of toxin A to cells. These sugars, called sulfated glycans, are very abundant on the surface of cells. Viruses, natural growth factors and signaling molecules often use them to enter or communicate with cells.

"When we saw the potential role of sulfated glycans, we asked the help of our virology colleagues, doctors Zhuoming Liu and Lindsey Robinson-McCarthy at Dr. Sean Whelan's lab at Harvard Medical School," Dong explains. , principal investigator of the study. "They know these glycans as factors in attachment to various viruses."

A convenient entrance

Together, the researchers determined that toxin A uses a wide range of sulfated glycans to bind to the cell surface. But it was not all history.

"It's not enough to bond with cells," says Dong. "The toxin has to find a way to get into the cells efficiently."

And that has. The main result of their screening was a gene encoding the low density lipoprotein receptor (LDLR). This receiver is constantly trafficked back and forth between the surface of the cell and its interior to bring the lipoproteins into the cells.

"Toxin A diverts LDLR for it to effectively enter cells," says Dong. "By combining the initial recognition of glycans with the recruitment of LDLR, the toxin maximizes its chances of landing on the surface of cells and can rapidly enter the cells."

Biologically speaking, intestinal cells shed a convenient welcome mat for the toxin. But it's a biology that can be relatively easy to thwart, Dong thinks, simply by preventing the toxin from binding to sugar.

Diversion with a lure

Commercial interests are already seeking to develop sulfated compounds that prevent various molecules from binding to the sulfated glycan receptor. These compounds act as decoys, binding molecules that would otherwise bind to cells on the intestinal surface.

"The toxin has a preference for sugars on the surface of cells," Dong said. "Artificial sugars could block it."

A decoy under development is a sugar-like molecule called GM-1111, whose structure is similar to that of anticoagulant heparin. It was originally designed to suppress the immune response in inflammatory states without the anticoagulant effects of heparin, which can lead to bleeding. Its manufacturer, GlycoMira, supplied GM-1111 to Dong's laboratory, which reassigned it to fight toxin A.

In a mouse model, GM-1111 reduced toxin-induced fluid accumulation and tissue damage in the colon, an encouraging sign.

The next scientific question of the lab is how toxins A and B work together to contribute to C. diff disease. "Most infections involve both A and B," Dong said. "Now that we have ways to block both toxins, we can try a combined approach."