Sandlin R C, Goldberg M B, Maurelli A T
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Mol Microbiol. 1996 Oct;22(1):63-73. doi: 10.1111/j.1365-2958.1996.tb02656.x.
IcsA of Shigella flexneri is required for intercellular spread and is located in the outer membrane at one pole of the bacterium, where it catalyses the polymerization of host-cell actin. The formation of the a tin tail provides the force to move the bacterium in a unidirectional manner through the host-cell cytoplasm. We have previously demonstrated that rough lipopolysaccharide (LPS) mutants of S. flexneri 2a are avirulent and cannot form plaques in tissue-culture monolayers. This inability to form plaques is associated with non-polar localization of IcsA and loss of host-cell membrane-protrusion formation ("fireworks'). To define the minimal LPS structure required for fireworks formation, we constructed a strain of S. flexneri (BS497) that contains a mutation in rfc, encoding the O side-chain polymerase, and a strain, BS520, that possesses a defective O side-chain ligase due to a mutation in rfaL. BS497 produces a LPS that consists of a core with one repeat unit of the O side-chain, while BS520 produces a LPS consisting of a complete core with no O side-chain. BS497 remained invasive but did not form fireworks or plaques in tissue-culture monolayers and was negative in the Serény test. BS520 was invasive, generated reduced numbers of short fireworks, and made tiny plaques, but it was negative in the Serény test. Analysis of BS497 with anti-IcsA antibody demonstrated that IcsA was distributed over the entire cell surface. The distribution of IcsA on the surface of BS520 was predominantly unipolar, with some trail-back of IcsA label along the sides of the bacterium. A similar pattern was seen when infected monolayers were stained for polymerized actin. These results suggest that both the presence and the length of the O side-chain are important in the proper localization or maintenance of IcsA at the pole which subsequently affects the ability to form actin tails and produce fireworks. This reduced ability to form actin tails and fireworks results in a decreased ability of Shigella to move into adjacent host cells. To determine if the sugar composition of the O side-chain is important in the ability to form fireworks, the rfb region of S. flexneri 2a was replaced with the rfb region from Escherichia coli serotype O8 or O25. Both hybrids were invasive, formed plaques, and gave positive Serény reactions. These results suggest that, unlike LPS length, the sugar composition of the O side-chain is not a critical requirement for the proper localization of IcsA and efficient intercellular movement.
福氏志贺菌的IcsA是细胞间传播所必需的，位于细菌一极的外膜中，在那里它催化宿主细胞肌动蛋白的聚合。肌动蛋白尾的形成提供了使细菌以单向方式在宿主细胞胞质中移动的力。我们先前已证明，福氏志贺菌2a的粗糙脂多糖（LPS）突变体无毒力，且不能在组织培养单层中形成噬菌斑。这种无法形成噬菌斑的情况与IcsA的非极性定位以及宿主细胞膜突出形成（“烟花”）的丧失有关。为了确定形成“烟花”所需的最小LPS结构，我们构建了一株福氏志贺菌（BS497），该菌株在编码O侧链聚合酶的rfc基因中有一个突变，以及一株BS520，由于rfaL基因中的突变，该菌株具有缺陷的O侧链连接酶。BS497产生的LPS由带有一个O侧链重复单元的核心组成，而BS520产生的LPS由没有O侧链的完整核心组成。BS497仍具有侵袭性，但在组织培养单层中不形成“烟花”或噬菌斑，并且在塞内试验中呈阴性。BS520具有侵袭性，产生的短“烟花”数量减少，形成微小的噬菌斑，但在塞内试验中呈阴性。用抗IcsA抗体对BS497进行分析表明，IcsA分布在整个细胞表面。IcsA在BS520表面的分布主要是单极的，沿着细菌侧面有一些IcsA标记的拖尾。当对感染的单层进行聚合肌动蛋白染色时，也观察到类似的模式。这些结果表明，O侧链的存在和长度对于IcsA在极区的正确定位或维持都很重要，这随后会影响形成肌动蛋白尾和产生“烟花”的能力。这种形成肌动蛋白尾和“烟花”的能力降低导致志贺菌进入相邻宿主细胞的能力下降。为了确定O侧链的糖组成在形成“烟花”的能力中是否重要，将福氏志贺菌2a的rfb区域替换为大肠杆菌血清型O8或O25的rfb区域。两种杂交菌株都具有侵袭性，形成噬菌斑，并在塞内试验中呈阳性反应。这些结果表明，与LPS长度不同，O侧链的糖组成不是IcsA正确定位和高效细胞间移动的关键要求。
