Report on the Study of Bacterial Toxins Inducing Non-Canonical Migracytosis and Aggravating Acute Inflammation

Background and Research Objectives

In recent years, the discovery of migrasomes, a new type of membrane structure, has gained significant attention in the scientific community for its role in cell migration and various biological functions. Migrasomes are formed and released on retraction fibers during cell migration, and are believed to play key roles in intercellular signaling, mitochondrial quality control, and organ morphogenesis. The process of forming and releasing migrasomes is known as “migracytosis,” which is regulated by various proteins and enzymes.

However, migracytosis is typically triggered by cell migration. Researchers from Zhejiang University, Zhejiang Province Key Laboratory of Multi-Omics in Infection and Immunity, and other institutions recently published a paper titled “Bacterial Toxins Induce Non-Canonical Migracytosis to Aggravate Acute Inflammation” in the journal Cell Discovery. This study reports for the first time a non-canonical form of migracytosis induced by bacterial toxins, expanding the potential significance of migrasomes in biological processes. The findings demonstrate that bacterial toxins can trigger migrasome formation in static cells, thereby contributing to acute inflammatory responses and providing new insights into how the immune system senses and responds to microbial infections.

Research Source

This study was conducted by Zhejiang University College of Life Sciences, Westlake University College of Life Sciences, Westlake Institute for Advanced Study, and the Zhejiang Provincial Key Laboratory of Multi-Omics in Infection and Immunity. The research team includes Diyin Li, Qi Yang, Jianhua Luo, Yangyushuang Xu, Jingqing Li, and Liang Tao, and was formally published in 2024 in Cell Discovery.

Research Design and Methods

1. Study Process and Experimental Design

The study consisted of multiple stages to systematically investigate the mechanism of non-canonical migracytosis induced by toxins, changes in cell structure, biochemical characteristics, in vivo expression, and its impact on acute inflammation. The specific steps are as follows:

• Preliminary Experiments on Toxin-Induced Migrasome Formation: The research team first applied Clostridioides difficile toxin B (Tcdb3) to various cultured cells, including U2OS, SH-SY5Y, L929, and others. They observed the formation of a large number of migrasomes in cells treated with Tcdb3. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that these migrasomes were vesicles ranging from 200 nm to 1 µm in diameter with typical migrasome morphology.

• Biochemical Characterization of Migrasomes: Using liquid chromatography-mass spectrometry, the study identified biomarkers, such as tspan family proteins and Rab35, enriched in Tcdb3-induced migrasomes, confirming the biochemical properties of migrasomes. Tspan4 overexpression and knockout experiments further verified that Tcdb3-induced migrasome formation was dependent on specific tspan proteins.

• Dynamic Imaging of Migracytosis: Real-time live-cell imaging showed that Tcdb3-induced migracytosis is a rapid but unsustainable process, where migrasomes are generated in large quantities and eventually burst, releasing cellular components.

• Selective Induction of Migracytosis by Specific Bacterial Toxins and Effectors: The study also investigated other bacterial toxins and effectors, such as Tcdb1, Tcda, etc., and found that only Tcsl and Tpel could induce migracytosis in a manner similar to Tcdb3.

• Verification of Migrasome Formation In Vivo: By injecting Tcdb3 into mice, migrasomes were observed to form in liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), demonstrating that migracytosis also occurs during acute inflammation in vivo.

2. Data Analysis and Control of Biochemical Reactions

• Relationship Between GTPase Activity and Migrasome Formation: The study found that toxin-induced migrasome formation depends on Rho GTPases, particularly the enrichment of RhoA in migrasomes. Pretreatment with the microtubule dynamics stabilizer nocodazole inhibited Tcdb3-induced migrasome formation, highlighting the importance of Rho signaling and local microtubule stability.

• Gene Knockdown Experiments and Survival Assay: Using tspan9 knockout mice (with defective migracytosis), the study showed that approximately 50% of the tspan9−/− mice survived after injection with Tcdb3, while all wild-type mice died within 12 to 36 hours. The data indicate that mice with defective migracytosis were more resistant to the toxin.

• Gene Expression Changes Related to Immunity and Inflammation: Transcriptome analysis of liver tissue revealed significantly lower expression of chemotaxis- and inflammation-related genes in tspan9−/− mice compared to wild-type mice, supporting the role of non-canonical migracytosis in exacerbating inflammatory responses.

Research Results

1. Bacterial Toxins Can Induce Non-Canonical Migracytosis: The study observed that Tcdb3 toxin can induce migrasome formation even in static cells, defining this as “non-canonical migracytosis.”

2. Non-Canonical Migracytosis Contributes to Acute Inflammatory Responses In Vivo: In the mouse model, Tcdb3 injection led to migrasome formation in LSECs and KCs. These migrasomes contained cytokines and chemokines, such as CXCL10, CCL5, VEGFA, and IL-10, indicating their role in acute inflammation and intercellular communication.

3. Rho GTPase Plays a Critical Role in Migrasome Formation: The study found that Rho GTPases, especially RhoA, are essential for migrasome formation. It also demonstrated the cooperative role of microtubule stability and Rho signaling in toxin-induced migracytosis.

4. Migracytosis Aggravates Early Inflammation: Knockout experiments showed that tspan9 deficiency resulted in reduced inflammation and higher survival rates, suggesting that toxin-induced migracytosis plays a key role in exacerbating inflammation, potentially contributing to rapid progression of sepsis.

Research Significance and Value

This study reveals, for the first time, a mechanism by which bacterial toxins can induce migracytosis in non-migrating cells, shedding new light on the role of migrasomes in acute inflammation and immune responses. The significance of the findings is reflected in several areas:

• Expanding the Biological Function of Migrasomes: Migrasomes were previously thought to form only during cell migration. This study demonstrates that static cells can also generate migrasomes under bacterial toxin stimulation, broadening the biological applications of migrasomes.

• Explaining the Mechanism of Bacterial Toxins: By studying the interactions between Tcdb3 and small GTPases, the study provides further insights into how bacterial toxins disrupt host cell signaling pathways to activate migracytosis and exacerbate immune responses.

• Providing New Insights for Anti-Infection Therapy: The role of migracytosis in acute inflammation suggests that inhibiting migrasome formation could have therapeutic potential in bacterial toxin-induced diseases, such as sepsis, thus providing new targets and strategies for drug development.

• Gender Differences in Migracytosis: The study found that male mice were more sensitive to Tcdb3-induced migrasome-mediated inflammation, suggesting that the biological function of migrasomes may exhibit gender differences, providing new clues for future research on sex-based differences in immune system responses.

Study Highlights

The study systematically analyzed toxin-induced non-canonical migracytosis through a variety of experimental methods, from in vitro cell experiments to in vivo mouse model experiments, demonstrating how bacterial toxins can trigger migracytosis and aggravate acute inflammation. Major highlights include: - Discovery of a New Mechanism: The study revealed, for the first time, that bacterial toxins can induce migrasome formation in non-migrating cells, defining this process as non-canonical migracytosis. - Key Role of Rho GTPase: Rho GTPases, especially RhoA, were shown to be crucial in migrasome formation. - Proposing New Therapeutic Targets: Migrasome’s role in acute inflammation presents new potential therapeutic targets, particularly in the context of bacterial infection-induced inflammation.

Conclusion

This study provides new mechanistic insights into how bacterial infections trigger acute inflammation by emphasizing the key role of migrasomes in immune responses.