Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium, Shewanella vesiculosa HM13
Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium, Shewanella vesiculosa HM13
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DOI：
10.1101/2023.04.25.538355
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发表时间：
2023-04
期刊：
影响因子：
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通讯作者：
Kouhei Kamasaka;J. Kawamoto;Taiku Tsudzuki;Yuying Liu;T. Imai;T. Ogawa;T. Kurihara
中科院分区：
文献类型：
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作者：
Kouhei Kamasaka;J. Kawamoto;Taiku Tsudzuki;Yuying Liu;T. Imai;T. Ogawa;T. Kurihara
细菌细胞外膜囊泡（EMV）在货物蛋白的介导下发挥着重要的生理功能。然而，我们对EMV装载货物的分子机制的理解是有限的。在这项研究中，我们分析了货物蛋白装载到EMV从鱼肠道革兰氏阴性菌，希瓦氏囊泡HM13的机制。该菌株分泌携带主要货物蛋白P49的EMV。在P49基因附近，我们发现与蛋白质分泌和表面多糖链合成相关的基因具有同源性。其中，编码参与细菌胞外多糖合成的翻转酶（HM 3343）、磷酸乙醇胺转移酶（HM 3344）和甘油磷酸二酯磷酸二酯酶（HM 3345）的同源物的基因的缺失导致EMV的荚膜多糖（CPS）的损失。我们进行了体外P49负载到无P49的EMV上的测定，以检查P49是否通过其与EMV的CPS的相互作用负载到EMV上。我们发现纯化的P49在体外被加载到携带CPS的EMV上，而由于HM 3343、HM 3344和HM 3345的丢失，它不能加载到来自缺乏CPS产生的突变体的EMV上。在体外和体内加载有P49的EMV的透射电子显微镜显示在EMV周围的球形纳米颗粒，而对于没有加载P49的EMV没有观察到这样的颗粒，这意味着P49构成EMV表面上的那些颗粒。这些结果表明，P49通过与EMV的CPS相互作用而负载到EMV上。阐明细菌细胞外膜囊泡（EMV）的负载机制对于理解其生物起源和开发其应用具有重要意义。在这里，我们表明，从鱼肠道革兰氏阴性菌，希瓦氏囊泡HM13，EMV的主要货物蛋白，通过其与EMV的荚膜多糖（CPS）的相互作用加载到EMV上。还鉴定了参与CPS合成的基因。据我们所知，还没有报道描述货物蛋白负载机制，其中CPS作为EMV的蛋白结合支架。因此，这项研究代表了一种新的蛋白质加载到EMV上的模式。研究结果加深了我们对EMV货物装载的理解，并将有助于其应用的发展。
Bacterial extracellular membrane vesicles (EMVs) play various physiologically important roles mediated by cargo proteins. However, our understanding of the molecular mechanism underlying cargo loading onto EMVs is limited. In this study, we analyzed the mechanism of cargo protein loading onto EMVs from a fish intestinal Gram-negative bacterium, Shewanella vesiculosa HM13. This strain secretes EMVs carrying a major cargo protein, P49. Near the P49 gene, we found genes having homology to genes involved in protein secretion and surface polysaccharide-chain synthesis. Among them, the deletion of genes encoding homologs of a flippase involved in bacterial extracellular polysaccharide synthesis (HM3343), phosphoethanolamine transferase (HM3344), and glycerophosphodiester phosphodiesterase (HM3345) resulted in the loss of capsular polysaccharide (CPS) of EMVs. We conducted an in vitro P49 loading assay onto P49-free EMVs to examine whether P49 was loaded onto the EMVs via its interaction with the CPS of the EMVs. We found that purified P49 was loaded onto EMVs harboring CPS in vitro, whereas it was not loaded onto EMVs from the mutants lacking CPS production due to the loss of HM3343, HM3344, and HM3345. Transmission electron microscopy of EMVs loaded with P49 in vitro and in vivo showed spherical nanoparticles around the EMVs, whereas such particles were not observed for EMVs without loaded P49, implying that P49 constitutes those particles on the surface of EMVs. These results indicate that P49 is loaded onto EMVs via its interaction with the CPS of EMVs. IMPORTANCE Elucidating the mechanisms of cargo loading onto bacterial extracellular membrane vesicles (EMVs) is important to understand their biogenesis and to develop their applications. Here, we show that the major cargo protein of EMVs from a fish intestinal Gram-negative bacterium, Shewanella vesiculosa HM13, is loaded onto EMVs through its interaction with capsular polysaccharide (CPS) of EMVs. Genes involved in CPS synthesis were also identified. To our knowledge, there have been no reports describing the cargo protein-loading mechanism in which CPS serves as the protein-binding scaffold for EMVs. Thus, this study represents a new mode of protein loading onto EMVs. The results deepen our understanding of cargo loading onto EMVs and would contribute to development of their applications.