Chen Chen, Kawamoto Jun, Kawai Soichiro, Tame Akihiro, Kato Chiaki, Imai Tomoya, Kurihara Tatsuo
Institute for Chemical Research, Kyoto University, Uji, Japan.
Marine Works Japan, Ltd., Yokosuka, Japan.
Front Microbiol. 2020 Jan 14;10:3001. doi: 10.3389/fmicb.2019.03001. eCollection 2019.
Extracellular membrane vesicles (EMVs) play an important role in various bacterial activities. EMVs have potential for use as vaccines, drug-delivery vehicles, platforms for extracellular production of recombinant proteins, and so on. In this study, we newly isolated a cold-adapted bacterium, HM13, which abundantly produces EMVs, characterized them, and analyzed their cargo transport mechanism. HM13, isolated from the intestine of a horse mackerel as a prospective host for a low-temperature secretory protein expression system, produced a single major secretory protein, P49, of unknown function in the culture supernatant. Analysis using sucrose density gradient ultracentrifugation indicated that P49 is a cargo protein carried by EMVs. . HM13 displayed extensive blebbing on the surface of the outer membrane, and the size of blebs was comparable to that of EMVs. These blebs are thought to be precursors of the EMVs. Disruption of the P49 gene resulted in only a marginal decrease in the EMV production, indicating that the EMVs are produced even in the absence of the major cargo protein. Whole genome sequencing of . HM13 revealed that this bacterium has a gene cluster coding for a non-canonical type II protein secretion system (T2SS) homolog in addition to a gene cluster coding for canonical T2SS. The P49 gene was located downstream of the former gene cluster. To examine the role of the putative non-canonical T2SS-like translocon, we disrupted the gene coding for a putative outer membrane channel of the translocon, named GspD2. The disruption lead to disappearance of P49 in the EMV fraction, whereas the production of EMVs was not significantly affected by this mutation. These results are indicative that the T2SS-like machinery functions as a novel type of protein translocon responsible for selective cargo loading to the EMVs. We also found that GFP fused to the C-terminus of P49 expressed in HM13 was transported to EMVs, indicating that P49 is useful as a carrier to deliver the fusion partner to EMVs. These findings deepen our understanding of the mechanism of biogenesis of EMVs and facilitate their applications.
细胞外膜泡（EMVs）在各种细菌活动中发挥着重要作用。EMVs有潜力用作疫苗、药物递送载体、重组蛋白胞外生产平台等。在本研究中，我们新分离出一种产EMVs丰富的冷适应细菌HM13，对其进行了表征，并分析了其货物运输机制。HM13是从竹荚鱼肠道中分离出来的，作为低温分泌蛋白表达系统的潜在宿主，它在培养上清液中产生了一种单一的主要分泌蛋白P49，其功能未知。使用蔗糖密度梯度超速离心分析表明，P49是EMVs携带的一种货物蛋白。HM13在外膜表面呈现广泛的起泡现象，泡的大小与EMVs相当。这些泡被认为是EMVs的前体。P49基因的破坏仅导致EMV产量略有下降，表明即使在没有主要货物蛋白的情况下也能产生EMVs。HM13的全基因组测序显示，除了一个编码典型Ⅱ型分泌系统（T2SS）的基因簇外，该细菌还有一个编码非典型Ⅱ型蛋白分泌系统（T2SS）同源物的基因簇。P49基因位于前一个基因簇的下游。为了研究假定的非典型T2SS样转运体的作用，我们破坏了编码该转运体假定外膜通道的基因，命名为GspD2。这种破坏导致EMV组分中P49消失，而EMV的产生并未受到该突变的显著影响。这些结果表明，T2SS样机制作为一种新型的蛋白转运体，负责将选择性货物装载到EMVs中。我们还发现，在HM13中表达的与P49 C末端融合的绿色荧光蛋白（GFP）被转运到EMVs中，表明P49可用作将融合伴侣递送至EMVs的载体。这些发现加深了我们对EMVs生物发生机制的理解，并促进了它们的应用。
