Trülzsch Konrad, Roggenkamp Andreas, Aepfelbacher Martin, Wilharm Gottfried, Ruckdeschel Klaus, Heesemann Jürgen
Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Germany.
Int J Med Microbiol. 2003 Jun;293(2-3):167-77. doi: 10.1078/1438-4221-00251.
We have constructed a mini-pYV plasmid (pTTSS) harboring the Yersinia type three secretion system (TTSS) and the adhesin yadA on a low-copy vector. Using this system we could demonstrate for the first time that YopO, YopP, YopM, and YopQ do not require any of the known or orphan chaperones for efficient secretion/translocation. Y. enterocolitica harboring pTTSS, (WA-C(pTTSS)) was able to secrete and translocate single Yop effector proteins in trans. WA-C(pTTSS) proved to be stable and secretion of Yops was Ca2+ and temperature dependent as is the case for the parental strain. This shows that all genes necessary for translocation and expression of the Ca(2+)-dependent phenotype are contained within the cloned region. In contrast to previously published multiple yop mutants which were constructed by sequential deletion of yops, our system which harbors only the TTSS region without yops, chaperones, and unknown ORFs can be sequentially complemented with yops and sycs of choice. WA-C(pTTSS) was able to translocate YopE, YopM and YopT into HeLa cells as demonstrated by Western blotting. Translocation of YopE and YopT was strictly dependent on the presence of their respective chaperones, whereas YopM did not require a chaperone for translocation. WA-C(pTTSS) harboring yopT and sycT was shown to translocate active YopT by demonstrating modification of the small GTP-binding protein RhoA. This shows for the first time that RhoA modification is strictly dependent on YopT and does not require additional effector Yops. WA-C(pTTSS) harboring YopP was shown to induce apoptosis. This system is ideal to study chaperone-dependent Yop secretion/ translocation without the background of other effector Yops (YopE, YopM, YopO, YopP, YopT, YopH), chaperones (SycE, SycH, SycT) and unknown ORFs. In addition this system can secrete heterologous proteins fused to the N-terminal secretion/translocation domain of YopE.
我们构建了一个微型pYV质粒（pTTSS），该质粒在低拷贝载体上携带耶尔森氏菌三型分泌系统（TTSS）和粘附素yadA。利用该系统，我们首次证明YopO、YopP、YopM和YopQ在高效分泌/转运过程中不需要任何已知或孤儿伴侣蛋白。携带pTTSS的小肠结肠炎耶尔森氏菌（WA-C(pTTSS)）能够反式分泌和转运单个Yop效应蛋白。事实证明，WA-C(pTTSS)是稳定的，Yops的分泌如亲本菌株一样依赖于Ca2+和温度。这表明克隆区域内包含了转运和表达Ca(2+)依赖性表型所需的所有基因。与之前通过依次缺失yops构建的多个yop突变体不同，我们的系统仅包含TTSS区域，没有yops、伴侣蛋白和未知开放阅读框（ORF），可以依次用选定的yops和sycs进行互补。如蛋白质免疫印迹法所示，WA-C(pTTSS)能够将YopE、YopM和YopT转运到HeLa细胞中。YopE和YopT的转运严格依赖于它们各自伴侣蛋白的存在，而YopM的转运不需要伴侣蛋白。通过证明小GTP结合蛋白RhoA的修饰，携带yopT和sycT的WA-C(pTTSS)被证明能够转运活性YopT。这首次表明RhoA修饰严格依赖于YopT，不需要其他效应Yops。携带YopP的WA-C(pTTSS)被证明可诱导细胞凋亡。该系统是研究伴侣蛋白依赖性Yop分泌/转运的理想系统，不存在其他效应Yops（YopE、YopM、YopO、YopP、YopT、YopH）、伴侣蛋白（SycE、SycH、SycT）和未知ORF的背景干扰。此外，该系统能够分泌与YopE的N端分泌/转结构域融合的异源蛋白。
