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Functional analysis of Salmonella intracellular survival and proliferation in macrophages via the type III secretion system encoded by Salmonella pathogenicity island 2

通过沙门氏菌致病性岛2编码的III型分泌系统对沙门氏菌在巨噬细胞中的细胞内存活和增殖进行功能分析

基本信息

批准号：
17590398
负责人：
OKADA Nobuhiko
金额：
$ 2.24万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Type III secretion system (T3SS) is a common virulence determinant in Gram-negative bacteria and the genetic information is often clustered in pathogenicity islands or on virulence plasmids. We have analyzed the T3SS encoded by Salmonella pathogenicity island 2 (SPI-2) that is indispensable for systemic disease of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mice. Since the low abundance of this secretion system restricted direct analysis by proteomic approaches, we have adopted the two-dimensional electrophoresis (2-DE) using agarose IEF for first dimension (agarose 2-DE). (1) Proteins prepared from supernatant grown the S. Typhimurium wild-type strain under SPI-2 inducing conditions were separated by agarose 2-DE and 17 protein spots were detected on agarose 2-DE gel stained with CBB. However, 16 out of 17 spots were originated from FliC, and remaining was PagC, an outer membrane protein of S. Typhimurium. (2)Next, to identify proteins whose expression is regulated by SsrA/SsrB two-component regulatory system encoded by SPI-2, we constructed S. Typhimurium strain carrying ssrB mutation. Whole-cell lysates prepared from S. Typhimurium wild-type and ssrB mutant strains, grown under SPI-2 inducing conditions were fractionated by using partial bacterial proteome extraction kit (Calbio Chem). and proteomic analysis was performed by agarose 2-DE. A total 107 proteins that were strongly reduced by an ssrB mutation have been identified. However, out of 107 proteins, only one protein was included as known SsrB-regulated protein. Thus, further analysis is required to reveal the ssrB regulon in S. Typhimurium. (3) We further characterize the protein-protein interaction among SPI-2 effectors, including SifA, SifB, SseF, SseG, and SseJ. Using bacterial two-hybrid system and pull-down assays, we have found the interaction between SifB and SseJ. In addition, the results indicate that SifB and SseJ form homodimer (or homoconcatemer).

III型分泌系统(T3SS)是革兰氏阴性菌常见的毒力决定因素，其遗传信息往往聚集在致病岛或毒力质粒上。我们分析了沙门氏菌致病岛2(SPI-2)编码的T3SS，它是鼠伤寒沙门氏菌全身性疾病所必需的。由于该分泌系统丰度低，限制了蛋白质组学方法的直接分析，我们采用了琼脂糖电泳法(2-DE)。(1)对SPI-2诱导条件下的鼠伤寒沙门氏菌野生型菌株培养上清液进行琼脂糖凝胶2-DE分离，在CBB染色的琼脂糖凝胶上检测到17个蛋白质斑点。在17个斑点中，有16个斑点来源于FLIC，其余为鼠伤寒沙门氏菌的外膜蛋白PagC。(2)构建了携带SSRB基因突变的鼠伤寒沙门氏菌，构建了SSRB基因突变的鼠伤寒沙门氏菌，以鉴定受SSRB/SSRB双组分调控系统调控表达的蛋白质。在SPI-2诱导条件下培养的鼠伤寒沙门氏菌野生型和SSRB突变株的全细胞裂解产物用部分细菌蛋白质组提取试剂盒(Calbio Chem)进行分离。用琼脂糖凝胶双向凝胶电泳法进行蛋白质组学分析。共鉴定出107种被SSRB突变强烈减少的蛋白质。然而，在107个蛋白质中，只有一个蛋白质被包括为已知的SSRB调节蛋白。因此，需要进一步的分析来揭示SSRB在鼠伤寒沙门氏菌中的调控。(3)进一步研究了SifA、SifB、SSEF、SseG和SseJ等SPI-2效应子之间的蛋白质相互作用。利用细菌双杂交系统和Pull-down实验，我们发现了SifB和SseJ之间的相互作用。此外，结果表明，SifB和SseJ形成了同源二聚体(或同链共聚体)。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation.

DOI：
10.1099/mic.0.29259-0
发表时间：
2007-02
期刊：
Microbiology
影响因子：
1.5
作者：
N. Okada;Yorie Oi;Mayuko Takeda-Shitaka;Kazuhiko Kanou;Hideaki Umeyama;Takeshi Haneda;T. Miki;Sachiko Hosoya;H. Danbara
通讯作者：
N. Okada;Yorie Oi;Mayuko Takeda-Shitaka;Kazuhiko Kanou;Hideaki Umeyama;Takeshi Haneda;T. Miki;Sachiko Hosoya;H. Danbara

Probiotic Lactobacillus casei activates innate immunity via NF-κB and p38 MAP kinase signaling pathways