Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli.
Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli.
复制标题
通过兔肠致病性大肠杆菌的减毒 ler 突变体通过溶血素或自转运体系统递送异源蛋白抗原。
DOI：
10.1016/j.vaccine.2005.07.024
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Boedeker,EdgarC
中科院分区：
文献类型：
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作者：
Zhu,Chengru;Ruiz-Perez,Fernando;Yang,Zhuolu;Mao,Ying;Hackethal,VeronicaL;Greco,KarlaM;Choy,Wendy;Davis,Katherine;Butterton,JoanR;Boedeker,EdgarC
在本报告中，我们描述了使用兔肠致病性大肠杆菌 (rEPEC) 的减毒调节突变体作为活疫苗载体，使用两个专用运输系统（沙门氏菌自转运器和大肠杆菌溶血素装置）递送异源蛋白抗原。我们之前报道过 rEPEC O103:H2 的同基因 ler（LEE 编码调节因子）突变体在兔子中具有减毒作用并具有免疫原性。我们首先评估了沙门氏菌自转运蛋白 MisL，其含有来自恶性疟原虫的环子孢子蛋白 (NANP)8 的免疫显性 B 细胞表位，该表位在组成型 Tac17 启动子的控制下与 C 端易位蛋白结构域融合。 rEPEC ler 突变体能够表达(NANP) 8 过客肽并将其易位至细菌表面。接下来，我们研究了 rEPEC ler 突变体通过 MisL 自转运蛋白或大肠杆菌溶血素分泌装置从人肠出血性大肠杆菌中传递志贺毒素 B 亚基 (Stx1B)。自转运蛋白和溶血素质粒表达的 Stx1B 水平相似（30-40ng/ml/OD600）。自转运蛋白上清液中仅发现 6% 的 Stx1B；其余部分与细胞相关，通过免疫荧光测定，一小部分 Stx1B 表面暴露。相比之下，88%的Stx1B通过溶血素分泌系统分泌到培养物上清液中。在一项体内研究中，在用 RDEC-H19A（含有 Stx 转换噬菌体的原型 rEPEC）进行实验攻击后，接种带有 Stx1B 自转运蛋白质粒的 ler 突变体的兔子没有观察到明显的保护作用。相反，与接种亲本 ler 突变体的兔子相比，接种含有 Stx1B-溶血素融合体的 rEPEC ler 突变体的兔子部分免受 RDEC-H19A 感染，体重减轻减少 (p<0.008)。我们的结果表明减毒的rEPEC能够作为疫苗载体从不同的细胞位置表达异源蛋白抗原并将这些抗原递送至肠粘膜。有了这个系统，分泌蛋白在产生保护方面可能比细胞相关抗原更有效。
In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30–40ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p<0.008) when compared to rabbits inoculated with the parent ler mutant. Our results suggest that attenuated rEPEC are capable of serving as vaccine vectors to express heterologous protein antigens from different cellular locations and deliver these antigens to the intestinal mucosa. With this system, secreted proteins may be more effective than cell-associated antigens in generating protection.