NC | Tao Liang Group of West Lake University - TMPRSS2 "assists" virus infection and mediates the host invasion of Clostridium sothrix hemorrhagic toxin...

Clostridium soxii( Paeniclostridium sordellii )It is a spore-forming Gram-positive bacterium,Infects humans and other animals and causes tissue necrosis,Enterotoxemia,Severe diseases such as sepsis and toxic shock.The bacteria are commonly found in the environment,And there is a certain probability of colonization in the human intestine and female reproductive tract（约3-4%）.目前,The total number of acute infections caused by Clostridium soxii is small but deadly.约 74%An acute infection of Clostridium soxii occurs during childbirth or miscarriage in women,The fatality rate is close100%.

The virulence factors of Clostridium soxii consist mainly of lethal toxins TcsL and hemorrhagic toxins TcsH,Both toxins are classified as Clostridium macrotoxins（ Large Clostridial Toxin, LCT ）家族,Other members of this family include toxins produced by C. difficile TcdA 和 TcdB,Toxin of Clostridium perfringens TpeL and the toxin of Clostridium novii Tcnα 等.TcsH It is the only important member of the toxin family whose cellular receptor has not been elucidated,This largely hinders our research on its mechanism of action and pathogenesis.

Tao Liang's research group at the West Lake Laboratory Focuses on the effects and mechanisms of action of bacterial toxins on host cell function,Toxins have been discovered TcdA, Tcnα, TcdB multiple host cell receptors, etc,Some of the research results have been published in   Cell, Cell Research, Nature Microbiology 等期刊上. 近日,Tao Liang's research group has once again made new breakthroughs in this field.

北京时间2022年7月26日,团队在 Nature Communications 上在线发表题为 “Paeniclostridium sordellii hemorrhagic toxin targets TMPRSS2 to induce colonic epithelial lesions” 的研究论文,首次揭示 TMPRSS2 是 TcsH intestinal epithelial receptors,Filled up over the years against hemorrhagic toxins TcsH Little is known about the blanks in the field.

论文链接：

https://www.nature.com/articles/s41467-022-31994-x

Thanks to the reference about TcsH Previous studies of are relatively sparse,The team also encountered a lot of difficulties in the early days.One of the difficulties is the selection of suitable target cells to interact with the toxin.与其它 LCT 相比,TcsH The toxicity shown in many common cell lines is very weak.The research team realized the importance of screening for suitable cell lines,A large number of different tissue-derived cell lines were then tested,Most human cell line pairs TcsH Neither are sensitive,But two specific strains of cells were also found in the study,Human colon cancer cell line HT-29 and human colorectal cancer cell lines Caco-2,对 TcsH 高度敏感.基于这一发现,团队选取了 HT-29 cells for research TcsH model cells,And in order to increase the success rate,Select human sources from two different sources sgRNA 文库在 HT-29 Knockout libraries were constructed on cells.通过 TcsH 压力筛选,The researchers found that the two independent screening systems simultaneously enriched a set of common target genes,Including transmembrane serine proteases2（TMPRSS2 ）and fucosylation modification related genes GMDS、SLC35C1、FUT4（图1）.By constructing genetically mutated and complemented cells,The researchers demonstrated these gene pairs TcsH cell invasion plays an important role.

图1. CRISPR/Cas9mediated genome-wide knockout screen discovery withTcsHAction-related host genes

TMPRSS2 Due to extensive involvement including influenza、SARS、MERS Invading cells of various viruses, etc“臭名昭著”.TMPRSS2 The way it participates in viral invasion is by activating the virus's spike protein through its serine protease activity,thereby promoting membrane fusion.有意思的是,TMPRSS2 mediated by serine protease activity TcsH Invasion is completely irrelevant（图2）.另一方面,TMPRSS2 can be specifically associated with TcsH 进行结合,High affinity0.13 nM.说明与“助攻”The patterns of viral infection are different,TMPRSS2 It acts as a direct protein receptor to assist toxins TcsH Invasion of targeted cells.

图2. TMPRSS2 Loss of serine protease activity did not affect mediatedTcsH入侵

值得一提的是,TMPRSS2 Only a small number of cells have higher levels of expression,And its expression level in the cell is the same as that of the cell TcsH Sensitivity match,This also explains why previous studies found only a few cell line pairs TcsH 是敏感的（图3）.

图3. TMPRSS2 Cells with high expression exhibited pairsTcsHof high sensitivity

另一方面,Fucosylation modifications are widespread on many cell surfaces,Research has found that TcsH Binding at the cell surface is closely related.by further biochemical and cytological experiments,Includes construct knockout post-replenishment experiments、In vitro binding、Competitive antagonism, etc,The researchers proved that TMPRSS2 and fucosylation are independent of each other,Both can participate competitively TcsH cell identification（图4）.

图4. TMPRSS2 and fucosylated polysaccharides available asTcsHTwo types of receptors that are independent of each other

最后,为进一步探究 TMPRSS2 Physiological and pathological significance,The researchers used one Tmprss2 A full knockout mouse model.Found after intraperitoneal injection of toxin,野生型（Tmprss2+/+）and heterozygous（Tmprss2+/‒）小鼠对TcsHThe tolerance is significantly lower than that of the homozygous type（Tmprss2‒/‒）小鼠.Because the gut is one of the important sites for Clostridium soxellia colonization,And it is known that some intestinal epithelial cells have higher levels TMPRSS2 表达.Subsequent researchers used wild-type and  Tmprss2 Intestinal ligation model was constructed in knockout mice,结果显示 TcsH 处理后 Tmprss2 Colonic epithelial damage in knockout mice was significantly smaller than in wild-type mice（图5）.

图5. mouse model shownTMPRSS2 是TcsHimportant pathologically relevant receptors

Researcher Tao Liang from the West Lake Laboratory is the corresponding author of this article;Li Xingxing and He Liuqing, researchers in Tao Liang's research group, are the co-first authors;Other co-authors include Dr. Yanyan Li from Westlake University,Postdoctoral fellow Luo Jianhua,Doctoral student Zheng Yangling、周瑶、李丹阳,and research assistants Zhang Yuanyuan and Pan Zhenrui;In addition, we would like to thank Yongdeng Zhang, a researcher at the West Lake Laboratory, for his help in fluorescence photomicrography.本课题受到国家自然科学基金、西湖实验室（生命科学和生物医学浙江省实验室）及西湖教育基金会的资助;The implementation of the project is also supported and helped by the Biomedical Experiment Technology Center and the Laboratory Animal Center of West Lake University.

Microbial Host Interaction Laboratory（Laboratory home page link：https://tao.lab.westlake.

edu.cn/）致力于研究微生物-宿主之间的相互作用方式和内在规律,尤其是从分子、生化、细胞、生信、结构等方面研究重要病原细菌及其致病因子对宿主的影响及作用机制.

Tao Liang, a researcher at the West Lake Laboratory, has been the corresponding author in recent years Cell、Cell Research、Nature Microbiology、Nature Communications、PLOS Pathogens 等学科主流期刊上发表多篇学术论文.Tao Liang's laboratory has a number of positions for scientific researchers,方向包括且不限于分子微生物学、生物信息学和微生物组学、生物化学、免疫学、细胞生物学等,诚邀各方青年才俊加入、携手共同成长！

联系方式：

[email protected]

文章来源 / 陶亮团队

编辑 / Jin Xiaoye

校对 / 戴涵 朱逸青

审核 / 李凯娜

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