Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion
Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion
复制标题
DOI：
10.1128/iai.64.6.2019-2023.1996
复制
发表时间：
1996-06-01
影响因子：
3.1
通讯作者：
Guiney, DG
中科院分区：
文献类型：
--
作者：
Cirillo, DM;Heffernan, EJ;Guiney, DG
Rck编码于鼠伤寒沙门氏菌毒力质粒上，是肠杆菌科相关的17- 19 kda外膜蛋白家族的成员，包括Ail（小肠结肠炎耶尔森菌）和PagC（鼠伤寒沙门氏菌）。这些蛋白质的结构模型预测了八个跨膜结构域与亲水性内环和外环交替。当在大肠杆菌中表达时，Rck和Ail，而不是PagC，赋予对补体杀菌活性的高水平抗性，以及粘附和侵入哺乳动物细胞系的能力。为了确定Rck的功能域，我们制作并筛选了Rck中降低生物活性的随机突变。我们发现，在假定的第三外环中，一个单一的氨基酸取代（甘氨酸到天冬氨酸）大大降低了rck介导的血清抗性和真核细胞侵袭。然后我们在Rck和PagC之间构建了两个嵌合蛋白。用含有第三和第四外环的相应PagC片段替代Rck的c端一半，可消除Rck介导的血清抗性和侵袭表型。用含有第四个外环的较小的PagC c端部分替代Rck不影响侵袭性表型或血清抗性。这些数据表明，第三个假定的外膜环区对Rck蛋白的毒力相关特性很重要，并表明血清耐药机制和涉及相同Rck结构域的上皮细胞侵袭机制之间存在相似性。
Rck is encoded on the Salmonella typhimurium virulence plasmid and is a member of a family of related 17- to 19-kDa outer membrane proteins of Enterobacteriaceae, including Ail (Yersinia enterocolitica) and PagC (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and outer loops. When expressed in Escherichia coli, Rck and Ail, but not PagC, confer high-level resistance to the bactericidal activity of complement as well as the ability to adhere to and invade mammalian cell lines. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single amino acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryotic cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresponding PagC fragment containing both the third and the fourth outer loops abolishes the Rck-mediated serum resistance and invasion phenotypes. Substitution of Rck with a smaller C-terminal portion of PagC containing the fourth outer loop did not affect the invasive phenotype or serum resistance. These data reveal that the third putative outer membrane loop region is important for the virulence-associated properties of the Rck protein and suggest a similarity between the mechanism of serum resistance and epithelial cell invasion involving the same domain of Rck.