This article is reprinted from WestlakeUniversity

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Scientific research is sometimes like Sherlock Holmes solving a case, facing an invisible and unseen opponent, requiring bold hypothesis, careful proof, plus meticulous observation.

"Clostridium difficile is one of the opponents of Dr. Tao Liang. It is an important pathogen causing diarrhea and enteritis in hospitals and communities, and has caused a series of outbreaks around the world in recent years, and is classified as a top threat drug-resistant pathogen by the CDC.

Tao Liang has been following this research for nine years. Since 2013, when he was a postdoctoral fellow at Harvard Medical School, he has been studying the interactions between pathogenic bacteria, including C. difficile, and their hosts. Simply put, it is to explain how C. difficile invades the human body and causes disease.

Now, they have made a new and important discovery.

Recently, Tao Liang's team in the School of Life Sciences at Westlake University, in collaboration with Yigong Shi's team, published online in Cell, entitled "TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile The latest research results, entitled "TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile", reveal for the first time the TFPI protein, the intestinal epithelial receptor of the hypervirulent branch of C. difficile toxin B.

This means that they have discovered a new "door" for Clostridium difficile toxin B to invade the human body.

Screenshot of △paper

Why focus on C. difficile?

Clostridium difficile is an anaerobic gram-positive bacterium that is difficult to isolate and culture, hence the name "difficile". It is commonly found in the environment, including air, water, soil, animal and human feces. The human intestine is also a suitable place for it to settle.

The misuse of antibiotics is to blame for why C. difficile infections are so serious. When long-term use of antibiotics, destroy the body's normal intestinal flora, Clostridium difficile will take advantage of the situation and multiply, causing pseudomembranous enteritis, diarrhea, intestinal perforation and many other disease symptoms, serious life threatening.

Previous studies have shown that C. difficile has five main evolutionary branches, including types I, II, III, IV and V. The type II branch, also known as the "super virulent branch" due to its strong pathogenicity, has been responsible for several outbreaks of infection in North America and Europe in recent years.

Clostridium difficile produces three toxins: toxin A, toxin B, and binary toxin. Among them, toxin B (TcdB) is the key "weapon" of C. difficile to cause disease, and different branches produce different subtypes of toxin B (labeled as TcdB1, TcdB2, etc.).

It invades the body through "another door"

Previously, the work of Tao Liang's team on the receptors of classical Clostridium difficile toxin B in intestinal epithelial cells has gained wide attention and recognition in the industry. They have identified the FZD protein, an important host cell receptor for TcdB1, TcdB3, and TcdB5 (note, the FZD protein is also a receptor for the important WNT signaling pathway). It can be simply understood that they have deciphered how some TcdB enter the intestinal epithelial cells through the "gate".

However, Clostridium difficile, the other major "king of virulence", exclusively expresses two variants (subtypes) of toxin B, TcdB2 and TcdB4, neither of which recognizes the FZD protein.

I wonder where the "gate" is and how these two toxin variants enter the cell?

△ Thesis Graphic Abstract

Tao Liang's team tried to knock down the FZD protein (classical receptor) in the cells, and the cells remained highly sensitive to TcdB4. They speculated that there must be "another door" - that is, some unknown receptor mediates the invasion of the variant's toxin B into the cells.

Using a knockout library screening technique called CRISPR/Cas9, Tao's team identified a series of relevant candidate factors, or "suspects". After analysis, they identified a protein called tissue factor pathway inhibitor (TFPI), which is most likely to be involved on the cell surface and is probably another door for the two toxin B variants to enter the cell. It is likely that this protein, which is present on the cell surface, is the most likely "other door" for the two toxin B variants to enter the cells.

TFPI is the new channel that has been hidden for a long time

In order to test this conjecture, the research team thoughtfully designed and conducted different types of experiments.

One of the key experiments was to knock out the "door" of TFPI to see if TcdB4 could still break into the cells.

TFPI exists in mammals as two major homodimers, α and β, and both forms have the ability to mediate the entry of TcdB4 into cells. Since full knockout of TFPI leads to mouse death, the research team constructed TFPI β knockout mice. The mice were then injected intraperitoneally with TcdB4 for toxin challenge experiments and found that more than 60 percent of the pure (TFPIβ-/-) mice survived. "TFPI was demonstrated to be a key receptor for TcdB4 and showed that the kidney is the major damaged organ under systemic systemic infection," said Tao Liang.

Three-dimensional structure of the complex formed by △TcdB4 and TFPI

Key experiment 2, "seeing" the binding of toxin and receptor directly from the molecular level.

In collaboration with researcher Yan-Yan Li from Yigong Shi's group, Tao Liang's team has resolved the 3D structure of the toxin-receptor complex (TcdB4-TFPI) with a resolution of 3.1 Å using cryo-electron microscopy single particle 3D reconstruction technique.

Subsequently, by comparison, the team categorized the pathways by which C. difficile toxin B enters cells.

• The first category, represented by TcdB1/3/5, enters through the "gate" of FZD.
• The second category contains mainly the "super toxic branch" of TcdB2 and TcdB4, which are selected to enter through the "gate" of TFPI.

(A) Structural comparison of the toxin-receptor complex TcdB1-FZD2 with TcdB4-TFPI. (B) Phylogenetic analysis revealed that TcdB can be divided into two major classes based on differences in receptor binding regions, recognizing FZD and TFPI, respectively.(C) TcdB of Clostridium difficile branches I, III, IV, and V recognize FZD as receptor, and TcdB of supertoxic branch II recognize TFPI as receptor.

The third key experiment was to construct a "TFPI decoy" to trap the toxin and see if it could protect the cells and experimental animals from harm.

The team constructed a "TFPI decoy" - a soluble protein containing part of the key region of TFPI - to confuse and trap the toxin molecule. It was found that these "TFPI decoys" effectively prevented the destruction of toxin B of Clostridium perfringens at both the cellular level and in mouse models, providing significant protection. "This not only further confirms that TFPI is the receptor for TcdB of C. difficile in the intestinal epithelium, but also predicts the potential application of soluble TFPI as a neutralizing protein in the prevention and treatment of C. difficile infection." Tao Liang explained.

As mentioned earlier, the characteristics of C. difficile make it less effective and relapses severely when treated with antibiotics.

In contrast, receptor-based targeted development, such as neutralizing antibodies, is a new class of strategy with a different route, with theoretically more precise targets and fewer negative effects on the human body and the environment.

This study is scientifically important for uncovering and understanding the pathogenesis of C. difficile infections and the evolution and workings of Clostridium macotoxins, and provides an important theoretical basis for the development of new methods for the prevention and treatment of C. difficile infections caused by super virulent branching strains.

Tao Liang's group

Researchers Liang Tao (Lead Contact) and Yan Yan Li (Yigong Shi's team) from the School of Life Sciences, Westlake University are co-corresponding authors of this paper.

Jianhua Luo, a postdoctoral student at Westlake University, Qi Yang, a postdoctoral student, Xiaofeng Zhang, a research assistant, Yuanyuan Zhang, and Wan Li, a postdoctoral student, are co-first authors.

Other co-authors include researchers Dr. Jing Huang and Dr. Ying Yan from Westlake University, postdoctoral student Chao Zhan, PhD students Yao Zhou, Liuqing He, and Danyang Li, and researcher Dr. Dazhi Jin from Hangzhou Medical College.

Special thanks also go to Professor Min Dong of Boston Children's Hospital/Harvard Medical School and Professor Dansheng Li of the School of Life Sciences at Westlake University for their support and discussions during the course of the project.

This project was funded by the National Natural Science Foundation of China, Zhejiang Provincial Natural Science Foundation, Westlake Laboratory (Zhejiang Laboratory of Life Sciences and Biomedicine) and Westlake Education Foundation; the project implementation was also supported and assisted by the Biomedical Experimental Technology Center, Experimental Animal Center and High Performance Computing Center of Westlake University.

The Microbial Host Interaction Laboratory at Westlake University is dedicated to studying the mode of microbial-host interactions and the intrinsic laws, especially the impact and mechanism of action of important pathogenic bacteria and their pathogenic factors on the host from molecular, biochemical, cellular, biochemical and structural aspects.

Tao Liang, a researcher in the School of Life Sciences at Westlake University, has published several academic papers in the mainstream journals of Cell, Nature, Science, Cell Research, Nature Microbiology, Nature Communications, PLOS Pathogens and other disciplines as first and corresponding author in recent years.

The laboratory currently has several postdoctoral, doctoral and research assistant positions, including and not limited to molecular microbiology, bioinformatics and microbiomics, microbial genetics, immunology, structural biology, biochemistry, cell biology, etc. We invite all young talents to join us and grow together!

Link to the paper.
https://authors.elsevier.com/a/1elpeL7PXingP
Lab Home
https://tao.lab.westlake.edu.cn/

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